Homocitrulline, also known as L-Homocitrulline, N(6)-carbamoyl-L-lysine, N(6)-(amino-carbonyl)-L-lysine, (2S)-2-amino-6-(carbamoyl-amino) hexanoic acid, has the molecular formula C₇H₁₅N₃O₃, and a molecular weight of 189.2123 daltons. Molecular models for homocitrulline are shown below.

The amino acid homocitrulline is a metabolite of ornithine in human metabolism and mammalian. The amino acid can be detected in larger amounts in the urine of individuals with urea cycle disorders. At presence, it is thought that the depletion of the ornithine supply causes the accumulation of carbamyl-phosphate in the urea cycle which may be responsible for the enhanced synthesis of homocitrulline and homoarginine. Both amino acids can be detected in urine. Amino acid analysis allows for the quantitative analysis of these amino acid metabolites in biological fluids such as urine or blood.

Homocitrulline is one methylene group longer than citrulline, but similar in structure. The metabolite is generated from a lysine residue after lysine reacts with cyanate. Cyanate is present in the human body in equilibrium with urea. Under physiological conditions the urea concentration may be too low to allow extensive carbamylation. However, the conversion process leading to the formation of homocitrulline from lysine in proteins is known to occur in vivo. During renal failure conditions, the urea concentration increases and carbamylation of many proteins can occur, which can be detected. It is believed that most carbamylation takes place during inflammation when the enzyme myeloperoxidase is released from neutrophils. This enzyme converts thiocyanate to cyanate. Increased levels of cyanate can now carbamylate lysine residues.

Homocitrulline has been identified as an antigen specific to rheumatoid arthritis as a target of anti-citrulline protein/peptide antibodies. More recently, it has been shown that homocitrulline-containing proteins are present in rheumatoid arthritis (RA) joints of rodents and that they may affect T-cell triggering and possibly autoantibody formation, and possibly also in humans.  

In another metabolic disorder, in the hyperornithinemia-hyperammonemia-homocitrullinuria (HHH) syndrome, first described in 1969, ornithine levels maybe elevated five to ten times in comparison to normal levels. In addition, in this syndrome, levels of alanine, orotic acid and homocitrulline may be elevated as well. In people with hyperammonemia orotic acid and homocitrulline appear to be chronically elevated after a high protein diet, but may be normal when fasting.

The metabolic disorder, lysinuric protein intolerance is caused by the body's inability to digest and use certain protein building blocks or amino acids. These are lysine, arginine, and ornithine. These amino acids are found in many protein-rich foods. Since in this disorder the human body cannot effectively break down these amino acids people typically experience nausea and vomiting after ingesting protein rich foods. Associated features of this protein intolerance may include an enlarged liver and spleen, short stature, muscle weakness, impaired immune function, and progressively brittle bones that are prone to fracture and a lung disorder called pulmonary alveolar proteinosis may also develop. In addition, the accumulation of amino acids in the kidneys can cause end-stage renal disease (ESRD).  In ESRD the kidneys are no longer able to filter fluids and waste products from the body effectively.

Reference

Dionisi Vici C, Bachmann C, Gambarara M, Colombo JP, Sabetta G: Hyperornithinemia-hyperammonemia-homocitrullinuria syndrome: low creatine excretion and effect of citrulline, arginine, or ornithine supplement. Pediatr Res. 1987 Sep;22(3):364-7.

Evered DF, Vadgama JV: Absorption of homocitrulline from the gastrointestinal tract. Br J Nutr. 1983 Jan;49(1):35-42.

http://pubchem.ncbi.nlm.nih.gov/rest/chemical/homocitrulline

http://www.perkinelmer.com/industries/healthcare/newborntestingservices/clinician-information/hypermethioninemia.xhtml

Hommes FA, Roesel RA, Metoki K, Hartlage PL, Dyken PR: Studies on a case of HHH-syndrome (hyperammonemia, hyperornithinemia, homocitrullinuria). Neuropediatrics. 1986 Feb;17(1):48-52.

Kato T, Sano M, Mizutani N.; Homocitrullinuria and homoargininuria in lysinuric protein intolerance. J Inherit Metab Dis. 1989;12(2):157-61.

Kato T, Sano M, Mizutani N: Inhibitory effect of intravenous lysine infusion on urea cycle metabolism. Eur J Pediatr. 1987 Jan;146(1):56-8. Pubmed: 3107993.

Kato T, Sano M, Mizutani N, Hayakawa C: Homocitrullinuria and homoargininuria in hyperargininaemia. J Inherit Metab Dis. 1988;11(3):261-5.

Kato T, Sano M: Effect of ammonium chloride on homocitrulline and homoarginine synthesis from lysine. J Inherit Metab Dis. 1993;16(5):906-7.

Kato T, Sano M, Mizutani N: Homocitrullinuria and homoargininuria in lysinuric protein intolerance. J Inherit Metab Dis. 1989;12(2):157-61.

Koshiishi I, Kobori Y, Imanari T: Determination of citrulline and homocitrulline by high-performance liquid chromatography with post-column derivatization. J Chromatogr. 1990 Oct 26;532(1):37-43.

Kraus LM, Gaber L, Handorf CR, Marti HP, Kraus AP Jr: Carbamoylation of glomerular and tubular proteins in patients with kidney failure: a potential mechanism of ongoing renal damage. Swiss Med Wkly. 2001 Mar 24;131(11-12):139-4.

Kraus LM, Elberger AJ, Handorf CR, Pabst MJ, Kraus AP Jr: Urea-derived cyanate forms epsilon-amino-carbamoyl-lysine (homocitrulline) in leukocyte proteins in patients with end-stage renal disease on peritoneal dialysis. J Lab Clin Med. 1994 Jun;123(6):882-91.

Rajantie J, Simell O, Perheentupa J: Oral administration of epsilon N-acetyllysine and homocitrulline in lysinuric protein intolerance. J Pediatr. 1983 Mar;102(3):388-90.

Shi J, Knevel R, Suwannalai P, van der Linden MP, Janssen GM, van Veelen PA, Levarht NE, van der Helm-van Mil AH, Cerami A, Huizinga TW, Toes RE, Trouw LA.;Autoantibodies recognizing carbamylated proteins are present in sera of patients with rheumatoid arthritis and predict joint damage. Proc Natl Acad Sci U S A. 2011 Oct 18;108(42):17372-7.

Simell O, Mackenzie S, Clow CL, Scriver CR: Ornithine loading did not prevent induced hyperammonemia in a patient with hyperornithinemia-hyperammonemia-homocitrullinuria syndrome. Pediatr Res. 1985 Dec;19(12):1283-7.

Tuchman M, Knopman DS, Shih VE: Episodic hyperammonemia in adult siblings with hyperornithinemia, hyperammonemia, and homocitrullinuria syndrome. Arch Neurol. 1990 Oct;47(10):1134-7.

Zammarchi E, Donati MA, Filippi L, Resti M: Cryptogenic hepatitis masking the diagnosis of ornithine transcarbamylase deficiency. J Pediatr Gastroenterol Nutr. 1996 May;22(4):380-3. 

Homocitrulline with the molecular formula C₇H₁₅N₃O₃, and a molecular weight of 189.2123 daltons, can be analyzed using amino acid analysis. Molecular models for homocitrulline are shown below.

Figure 1: Structural models for homocitrulline. A: Chemical structure; B: Stick model, energy minimized; C: Space filling model.

The amino acid homocitrulline is a metabolite of ornithine in human and mammalian metabolism. The amino acid can be detected in larger amounts in the urine of individuals with urea cycle disorders. Both amino acids can be detected in urine. Amino acid analysis allows for the quantitative analysis of these amino acid metabolites in biological fluids such as urine, blood, plasma or proteins.

Figure 2: Chromatograph of a homocitrulline standard using AQC chemistry.  

Figure 3: Chromatograph of an amino acid standard including homocitrulline using AQC chemistry.

Figure 4: Chromatograph of an amino acid standard including homocitrulline. An aliquot of 100 pimoles each were injected into the amino acid analyzer column. The zoomed in region from 12 to 40 minutes of the chromatogram is shown.

MOG or myelin oligodendrocyte glycoprotein is a key central nervous system (CNS)-specific autoantigen for primary demyelination in multiple sclerosis. The gene product is a membrane protein expressed on the oligodendrocyte cell surface and the outermost surface of myelin sheaths, in the brain and spinal cord. Due to this localization, it is a primary target antigen involved in immune-mediated demyelination. This protein is thought to be involved in completion and maintenance of the myelin sheath and in cell-cell communication. In addition, alternatively spliced transcript variants encoding different isoforms have been identified. MOG is a transmembrane protein that belongs to the immunoglobulin superfamily. The protein contains an Ig-like domain and has two potential membrane-spanning regions. Even thought the disease-inducing role of MOG has been established, its precise function in the CNS is still unknown.

Three possible functions for MOG have been suggested in the past:

(1) The protein may function as a cellular adhesive molecule.

(2) The protein may function as a regulator of oligodendrocyte microtubule stability.

(3) The protein may function as a mediator of interactions between myelin and the immune system, as well as the complement cascade.

Amor et al. in 1996 used myelin basic protein (MBP) and synthetic MBP peptides to screen for their ability to induce experimental allergic encephalomyelitis in Biozzi ABH (H-2Ag7) mice. Their data suggest the presence of a peptide core between MBP 21-26 (HARHGF). This peptide motif contains similar elements to the previously defined encephalitogenic MOG 1-22 and PLP 56-70 peptides. The authors further investigated the fine specificity of these epitopes using frame-shifted peptides, which indicated cores between MOG 9-15 (GYPIRAL) and PLP 62-68 (NVIHAFQ). Based on these pathogenic peptides, a putative H-2Ag7 binding motif was suggested that contains a series of hydrophobic, basic, small, and large hydrophobic residues within a 6 to 7 amino acid core. The authors suggest that these findings may have relevance in the design of strategies in the treatment of experimental autoimmune diseases in animals that express this haplotype.

Figure 1: Crystal structures of the extracellular domain of MOG (MOGIgd) at 1.45-A resolution and the complex of MOGIgd with the antigen-binding fragment (Fab) of the MOG-specific demyelinating monoclonal antibody 8-18C5 at 3.0-A resolution.

The demyelination in multiple sclerosis involves an autoantibody response to myelin oligodendrocyte glycoprotein. Breithaupt et al. in 2003 reported the crystal structures of the extracellular domain of MOG (MOGIgd) at 1.45-A resolution and the complex of MOG-Igd with the antigen-binding fragment (Fab) of the MOG-specific demyelinating monoclonal antibody 8-18C5 at 3.0-A resolution. The structures showed that MOG-Igd adopts an IgV like fold with the A'GFCC'C" sheet harboring a cavity similar to the one used by the costimulatory molecule B7-2 to bind its ligand CTLA4. The antibody 8-18C5 binds to three loops located at the membrane-distal side of MOG. Dominant contribution to these interactions are made by MOG residues 101-108 containing a strained loop that forms the upper edge of the putative ligand binding site. The sequence R₁₀₁DHSYQEE₁₀₈ is unique for MOG. However, large parts of the remaining sequence are conserved in MOG homologues that potentially produce immunological tolerance which are expressed outside the immuno-privileged environment of the CNS.

Figure 1: Crystal structure of myelin oligodendrocyte glycoprotein, a key autoantigen in multiple sclerosis.

To gain new insights into the physiological and immunopathological role of MOG, Clements et al. also in 2003 determined the 1.8-A crystal structure of the MOG extracellular domain (MOGED). MOGED adopts a classical Ig (Ig variable domain) fold that was observed to form an antiparallel head-to-tail dimer. The dimeric form of native MOG was also observed. MOGED was also shown to dimerize in solution. This observation is consistent with the view of MOG acting as a homophilic adhesion receptor. The MOG35-55 peptide, a major encephalitogenic determinant recognized by both T cells and demyelinating autoantibodies, is partly occluded within the dimer interface.

MOG peptides can be synthesized using automated solid phase peptide synthesis (SPPS) which are useful tools for the study of protein-protein or protein-peptide interactions to help elucidate the role of MOG and/or similar proteins. 

Reference

Amor S, O'Neill JK, Morris MM, Smith RM, Wraith DC, Groome N, Travers PJ, Baker D,; Encephalitogenic epitopes of myelin basic protein, proteolipid protein, myelin oligodendrocyte glycoprotein for experimental allergic encephalomyelitis induction in Biozzi ABH (H-2Ag7) mice share an amino acid motif. J Immunol. 1996 Apr 15;156(8):3000-8.

Breithaupt C, Schubart A, Zander H, Skerra A, Huber R, Linington C, Jacob U; Structural insights into the antigenicity of myelin oligodendrocyte glycoprotein.Proc.Natl.Acad.Sci.Usa (2003) 100 p.9446

Clements CS, Reid HH, Beddoe T, Tynan FE, Perugini MA, Johns TG, Bernard CC, Rossjohn J.; The crystal structure of myelin oligodendrocyte glycoprotein, a key autoantigen in multiple sclerosis.Proc.Natl.Acad.Sci.Usa (2003) 100 p.11059

http://www.genecards.org/cgi-bin/carddisp.pl?gene=MOG&search=8df341b292f81c6f4b6083f744e16c37

http://www.ncbi.nlm.nih.gov/structure/?term=myelin+oligodendrocyte+glycoprotein

https://www.wikigenes.org/e/gene/e/17441.html

By Klaus D. Linse

The approval of rituximab, a chimeric monoclonal antibody (mAB) that recognizes the CD20 protein, in 1997 by the U.S. Food and Drug Administration to treat B-cell non-Hodgkin lymphomas resistant to other chemotherapies ushered in an area in which monoclonal antibodies became important components of therapeutic regimens in oncology. To circumvent obstacles encountered with earlier drugs, showing off-target effects such as the targeting of healthy dividing cells as well as other severe side effects, researchers turned to ADCs to selectively deliver toxic compounds to diseased tissue to be used as “Magic Bullets”. A “Magic Bullet” is a drug or concept first proposed in the early 1900s by PaulEhrlich, a German physician and scientist, that allows the selective targeting of diseased tissue cells.

The mAB rituximab is used to treat cancers of the white blood system such as leukemias and lymphomas. Monoclonal antibodies now have become a successful class of therapeutic molecules. This success was a result of great progress made in molecular biology and biotechnology which contributed to our understanding of the inner works of the human cell as well of the key players in the immune system. According to the antibody society 35 monoclonal antibodies (mABs) have been approved as therapeutic agents and close to 7 are pending approval (July 2014). 

Therapeutic mABs have had a substantial effect on medical care for a wide range of diseases in the past two decades. This is because of the high specificity and ability of mABs to bind target antigens marking these for removal by methods such as complement-dependent cytotoxicity or antibody-dependent cell-mediated cytotoxicity. In addition, mABs can also have therapeutic benefits by binding and inhibiting the function of target antigens. Unfortunately, as medical scientists have found out, antibodies against tumor-specific antigens often lack therapeutic activities.

In recent years it has been found that the conjugation of cytotoxic drugs or radionuclides can expand the utility of mABS. Antibody-drug-conjugates (ADCs) with improved potency and effectiveness are now used as a means to target and deliver a toxic payload to the selected diseased tissue. In recent years this approach has become a major focus for therapeutic research. In the past decade antibodies have been conjugated to a number of cytotoxic drugs using various linker chemistries. Already two such drugs are marketed in the United States. These are ado-trastuzumab emtansine (Kadcyla®) and brentuximab vedotin (Adcetris®), and over 30 ADCs are currently undergoing clinical studies. This makes it likely that more conjugates may be approved in the near future.

Figure 1: A model of an ADC where a mAb is conjugated to DM1 illustrating the conjugation approach used for Ado-Trastuzumab Emtansine. Ado-trastuzumab emtansine, formerly called Trastuzumab-DM1 (T-DM1), is a HER2 antibody drug conjugate (ADC). In this ADC the trastuzumab antibody is linked to the cell-killing agent, DM1. T-DM1 combines two strategies— anti-HER2 activity and targeted intracellular delivery of the potent anti-microtubule agent, DM1 (a maytansine derivative)—to produce cell cycle arrest and apoptosis. Ado-trastuzumab emtansine is marketed under the brand name Kadcyla and is indicated for use in HER2-positive, metastatic breast cancer patients who have already used taxane and/or trastuzumab for metastatic disease or had their cancer recur within 6 months of adjuvant treatment.

The ADC brentuximag vedotin or Adcetris® combines an anti-CD30 antibody and the drug monomethyl auristatin E (MMAE). It is an anti-neoplastic agent used in the treatment of Hodgkin lymphoma and systemic anaplastic large cell lymphoma and was approved in 2011. In January 2012, the drug label was revised to include a boxed warning of progressive multifocal leukoencephalopathy and death following JC virus infection. The structure of the cAC10-vcMMAE system used for the production of brentuximag vedotin is illustrated in figure 2.

Figure 2: Structure of the cAC10-vcMMAE system used for the production of brentuximag vedotin. Francisco et al. 2003 report how this ADC was prepared. A controlled partial reduction of internal cAC10 disulfides with DTT, followed by addition of the maleimide-vc-linker-MMAE was used. Stable thioether-linked ADCs were formed by the addition of free sulfhydryl groups on the mAbs to the maleimides present on the drugs. cAC10-vcMMAE and the cIgG-vcMMAE used in the report contained approximately 8 drugs/mAb.

The development of ADCs in the past decade has evolved from the use of murine antibodies to conjugate standard chemotherapeutics drugs to fully human antibodies conjugated to highly potent cytotoxic drugs. Critical factors required for the successful development of ADCs, as scientists have learned over the past decade, include target antigen selection, as well as the selection of the best antibody, the correct linker and payload. Typically, ADCs are complex biomolecules composed of an antibody or antibody fragment linked to a biological active cytotoxic or anti-cancerous payload using stable chemical linkers with labile bonds. Other terms used for this type of molecules are the terms “bioconjugate” and “immune-conjugates”. The reasoning for the combination of the unique targeting abilities of antibodies with cancer-killing drugs to create unique ADCs is the potential these drugs can have to treat cancer that are hard to treat with standard classical chemotherapies.

One area of research important for the development and design of ADCs is that of conjugation chemistry. One recent improvement is the implementation of site-specific conjugation methods. Here, the conjugation only occurs at engineered cysteine residues or unnatural amino acids. The result is a homogeneous ADC production and improved ADC pharmacokinetic (PK) properties. The following figure illustrates critical factors that influence the performance of ADC therapeutics.

Figure 3: Critical factors that influence ADC therapeutics. ADCs are designed and produced by conjugating a cytotoxic drug to a monoclonal antibody via a selected linker. All components of an ADC affect the performance of the molecule. The optimization of all molecular parts is essential for the development of successful ADCs.

In the beginning years of ADC development many obstacles had to be overcome. Early ADCs showed little or no therapeutic effect. The primary reason may have been pour target selection. In addition, the use of either chimeric or murine (mouse) antibodies, which can elicit an immunogenic response, and the use of lower potency drugs can also be factors that limit potency. Ultimately, researches learned from their failures and incorporated what they had learned in the next generations of ADCs. Knowledge gained from the development of ADCs has led to a better understanding of the ways in which ADCs function and their clinical performance.
  

Design of ADCs

Many papers reporting the use of conjugates have been published since the 1970s. A PubMed search using the terms “Antibody and Drug and Conjugates” retrieved a list of over 2,300 papers covering this subject indicating that this has now become an active research field. Antibody-based therapeutics against cancer are now highly successful in clinical trials and are currently recognized for their potential.

Hundreds of mAbs including bispecific mAbs and multispecific fusion proteins, mAbs conjugated with small molecule drugs and mAbs with optimized pharmacokinetics have already been produced and are in clinical trials. However, many challenges still remain and a deeper understanding of mechanisms for how ADCs work is needed to overcome encountered problems including resistance to therapy, access to

molecular targets, as well as to understand the complexity of biological systems and individual variations and how these drug conjugates interact with them.

Unlike conventional chemotherapeutic drugs which do not selectively localize to tumors, antibody-drug conjugates can bind specifically to cells that express the targeted antigen. Therefore these molecules are considered to be ideal vehicles for applications that require delivery of drugs. With the help of various linker strategies antibodies can be conjugated to a variety of drugs or “payloads”. 

Ongoing research is investigating new strategies how to conjugate proteins such as IgGs to cytotoxic molecules without altering the natural functions of the proteins.

Antibody or protein vehicles

Although whole IgGs already have shown to have many benefits, for certain applications the use of smaller antibody fragments such as monomeric or dimeric fragments, sometimes called diabodies, single-chain variable fragments (scFv) or other small proteins maybe of advantage. This may allow tailoring the delivery time, as indicated by the half-life of the ADC, more precisely. For example, when delivering cytokines to the extracellular space of a tumor, a longer half-life may cause unwanted inflammation.

Linkers

A variety of linkers have now been investigated to connect an antibody or the protein of choice to the drug of choice. The most commonly used linkers are based on amide bond, also called peptide linkers, disulfide bonds or hydrazones as illustrated in the next figure. The selection of the linker determines what happens to the ADC once it is inside a cell. Most linkers are selected so that they can be cleaved by specific mechanisms. Depending on their sequence peptide linkers can be site-specifically cleaved by proteases that recognize the selected peptide sequence motif, or by statistical proteolysis. Disulfide bonds can be broken by the reducing environment of the cytosol and hydrazones can be cleaved by acid-mediated hydrolysis. In addition, ADCs containing proteins such as cytokines maybe produced as fusion proteins. 

Payloads

Very potent cytotoxic agents can be used to ensure that the tumor killing can be mediated with acceptably low doses of the ADCs employed. ADCs containing monomethyl auristin E (MMAE) or the maytansioniod DM1 kill tumor cells by inhibiting microtubule polymerization. However, alternative payloads may be used as well.  Examples are DNA damaging drugs or cytokines.

The next figure illustrates some building blocks that are used for the design and production of ADCs.

Figure 4: Structures of some building blogs and linkers used in ADC therapeutics. Linkers with an amide bond, a disulfide bond, and a hydrazone bond are illustrated in the upper panel. Structures of drug-SPDP-mAB, drug-SPP-mAB, drug-SSNPP-mAB, and drug-SMCC-mAB are listed in order of stability, least to greatest. The drug-SMCC-mAB conjugate contains a nonreducible linker.

To summarize, ADCs are sophisticated delivery systems for antitumor cytotoxic drugs. Monoclonal antibodies are used to guide the toxin precursor to the target cancer cell. When the target is reached the prodrug can be converted chemically or enzymatically to the parent drug and unfold its activity to kill the cancer. On its journey from the blood vessels to the molecular target in the tumor tissue the ADC is exposed to different conditions.

• During circulation:The ADC must behave like a naked antibody when circulating in the plasma. The linkers used must be stable in the blood stream. The goal is to limit the damage to healthy tissue since decomposition or decay would relase the cytotoxin before being delivered to the target.

• Antigen Binding: The conjugated mAB needs to retain high immunoaffinity thus the attachment of the cytotoxic drug must not disturb the binding specificity.

• Internalization: For the ADC to work well, a sufficient intracellular concentration of the drug must be achieved. This maybe one of the biggest challenges since antigen targets on cell sufaces are often present in limited numbers. In addition, the internalization process for antigen-antibody complexes is often inefficient.

• Drug Release: Once inside the cell, the ADC needs to efficiently release the selected cytotoxic drug in its active form inside the tumor cell.

• Drug Action: The potency of the selected drug must be sufficient to kill the tumor cells, often even at low concentration. To achieve this the use of very potent drugs is necessary. Compounds that are too toxic when tested as a stand-alone chemotherapy appear to be quite suitable candidates to be used as ADC payloads.  

The insight gained during the development of ADCs in the past decade have resulted in a number of new strategies and drugs. This is indicated by the flood of publications in this research field.

ADC poster: http://www.nature.com/nbt/extra/adc/index.html

Franco Dosio, Paola Brusa and Luigi Cattel; Immunotoxins and Anticancer Drug Conjugate Assemblies: The Role of the Linkage between Components. Toxins 2011, 3, 848-883; doi:10.3390/toxins3070848

Ducry L, Stump B.; Antibody-drug conjugates: linking cytotoxic payloads to monoclonal antibodies. Bioconjug Chem. 2010 Jan;21(1):5-13. doi: 10.1021/bc9002019.

Joseph A. Francisco, Charles G. Cerveny, Damon L. Meyer, Bruce J. Mixan, Kerry Klussman, Dana F. Chace, Starr X. Rejniak, Kristine A. Gordon, Ron DeBlanc, Brian E. Toki, Che-Leung Law, Svetlana O. Doronina, Clay B. Siegall, Peter D. Senter, and Alan F. Wahl; cAC10-vcMMAE, an anti-CD30–monomethyl auristatin E conjugate with potent and selective antitumor activity. August 15, 2003; Blood: 102 (4).

SEAN L KITSON, DEREK J QUINN, THOMAS S MOODY, DAVID SPEED, WILLIAM WATTERS, DAVID ROZZELL; Antibody-Drug Conjugates (ADCs) – Biotherapeutic bullets. Chemistry Today - vol. 31(4) July/August 2013.

Nilvebrant J, Åstrand M, Georgieva-Kotseva M, Björnmalm M, Löfblom J, Hober, S.; (2014) Engineering of Bispecific Affinity Proteins with High Affinity for ERBB2 and Adaptable Binding to Albumin. PLoS ONE 9(8): e103094. doi:10.1371/journal.pone.0103094.

Lewis Phillips GD, Li G, Dugger DL, Crocker LM, Parsons KL, Mai E, Blättler WA, Lambert JM, Chari RV, Lutz RJ, Wong WL, Jacobson FS, Koeppen H, Schwall RH, Kenkare-Mitra SR, Spencer SD, Sliwkowski MX; Targeting HER2-positive breast cancer with trastuzumab-DM1, an antibody-cytotoxic drug conjugate. Cancer Res. 2008 Nov 15;68(22):9280-90. doi: 10.1158/0008-5472.CAN-08-1776.

Panowksi S, Bhakta S, Raab H, Polakis P, Junutula JR.; Site-specific antibody drug conjugates for cancer therapy. MAbs. 2014 Jan-Feb;6(1):34-45. doi: 10.4161/mabs.27022.

Sassoon I, Blanc V.; Antibody-drug conjugate (ADC) clinical pipeline: a review. Methods Mol Biol. 2013;1045:1-27. doi: 10.1007/978-1-62703-541-5_1.

Feng Tiana, Yingchun Lua, Anthony Manibusana, Aaron Sellersa, Hon Trana, Ying Suna, Trung Phuonga, Richard Barnetta, Brad Hehlia, Frank Songa, Michael J. DeGuzmanb, Semsi Ensarib, Jason K. Pinkstaffc, Lorraine M. Sullivanc, Sandra L. Birocc, Ho Chod, Peter G. Schultze, John DiJoseph, Maureen Dougher, Dangshe Ma, Russell Dushing, Mauricio Lealh, Lioudmila Tchistiakovai, Eric Feyfanti, Hans-Peter Gerber, and Puja Sapra; A general approach to site-specific antibody drug conjugates. (2014) PNAS 111, 5, 1766-1771. 

Biotin- a water-soluble Vitamin

Figure 1:  Structural models for biotin.

(A) Chemical structure;
(B) Stick model of the energy minimized structure;
(C) Structure of biotin as observed in a crystal of a deglycosylated avidin in  complex with biotin published 1993 by Livnah et al.  [2AVI];
(D) Space filling model of biotin.

Biotin was discovered during nutritional experiments that revealed it as a factor in many foods. Biotin was found to be capable of curing scaly dermatitis, hair loss, and neurologic signs induced in rats when fed dried egg whites. Deficiency in biotin causes dermatitis and loss of hair. Biotin is a water-soluble, coenzyme present in small amounts in every living being. It occurs mainly bound to proteins or polypeptides. However, biotin is abundant in liver, kidney, pancreas, yeast, and milk. In humans biotin is a coenzyme for five carboxylases: propionyl-CoA carboxylase, methylcrotonyl-CoA carboxylase, pyruvate carboxylase, and 2 forms of acetyl-CoA carboxylase. This fact makes biotin essential for amino acid catabolism, gluconeogenesis, and fatty acid metabolism.

The enzyme biotin holocarboxylase synthetase (EC 6.3.4.10) catalyzes the attachment of biotin to the apocarboxylases by an amide bond to a specific lysine residue–producing holocarboxylases. Biotinylated proteins are normally turned over by proteolytic degradation to biocytin (biotinyl-lysine) and biotinylated oligopeptides that are subsequently cleaved by biotinidase (EC 3.5.1.12). These reactions recycle biotin. In addition, biotin is found covalently attached to histones and appears to be necessary for gene stability, repression of transposable elements and some genes.

Normal dietary intake of foods usually supplies enough biotin to prevent a biotin deficiency. However, deficiency can be caused by eating too many raw egg whites for a prolonged time period. Egg whites contain the protein avidin in large amounts. This protein can bind biotin strongly and prevent it from being ingested. Biotin in the body is regulated by dietary intake, the biotin transporters monocarboxylate transporter 1 and sodium-dependent multivitamin transporter, peptidyl hydrolase biotinidase (BTD), and the protein ligase holocarboxylase synthetase. Inhibiting any of these enzymes can cause a biotin deficiency.

Biotin Deficiencies

Nutritional biotin deficiency and inherited enzymatic deficiencies of the biotin-dependent carboxylases cause abnormally increased urinary excretion of characteristic organic acids. 
 

Structures of observed metabolites

  

Figure 2: Structures of metabolites observed in urine for biotin deficiencies. 

Biotin as a coenzyme is a catalyst for carboxylation reactions. One example is the reaction catalyzed by propionyl-coenzyme A (CoA) carboxylase illustrated in figure 3. 

Figure 3: Carboxylation reaction catalyzed by propionyl-CoA carboxylase.

In the reaction catalyzed by propionyl-CoA carboxylase, acidic hydrogen on carbon is removed as a proton and is replaced by the electrophilic acyl carbon of bicarbonate in an acyl exchange reaction. Here, water is the formal leaving group. The energy-yielding reaction for biotin catalyzed carboxylation reactions usually is the hydrolysis of MgATP to MgADP which is coupled to the carboxylation reaction. Biotin is covalently attached to the active site of an enzyme by an amide between the carboxylate of the coenzyme and a lysine from the protein.

Figure 4: Biotin covalently attached to the active site of an enzyme. The biotin moiety is attached by an amide between the carboxylate of the coenzyme and a lysine from the protein.

Figure 5: Biotin-Avidin Complex.The crystal structures of a deglycosylated form of the egg-white glycoprotein avidin and the avidin-biotin complex have been determined to 2.6 and 3.0 Å by Livnah et al. in 1993. The structures revealed the amino acid residues critical for the stabilization of the tetrameric protein complex assembly as well as for the very tight binding of biotin. Each avidin monomer folds into a eight-stranded antiparallel beta-barrel structure. This structure is quite similar to that of the genetically distinct bacterial analog streptavidin. Similar to streptavidin, binding of biotin involves a highly stabilized network of polar and hydrophobic interactions. Different views of the tetramer and monomer are illustrated. The red arrow points to the biotin binding pocket.

The valeric acid side chain of biotin molecules contains a carbonic acid group which can be readily conjugated to various reactive groups. This allows the attachment of biotin to molecules such as proteins, peptides or oligonucleotides and others. Any molecule attached to biotin can be captured for detection, immobilization or affinity purification using conjugates or supports conjugates to avidin or streptavidin proteins. These proteins bind strongly and specifically to the biotin group.

A wide variety of native and recombinant derivatives of avidin and streptavidin proteins are now readily commercially available in modified, labeled and immobilized forms. This "avidin-biotin system" has now been used in many research applications, for example, the detection or purification of target molecules. However, since the avidin-biotin affinity interaction is very strong, it is usually impractical to elute biotinylated targets that have been captured to immobilized avidin or streptavidin. For this reason, modified biotin labeling reagents have been developed. For example, cleavable biotin, iminobiotin and desthiobiotin provide reversible interactions with streptavidin and have now become useful tools for soft-release applications.

To conclude, optimized modern conjugation reactions allow for the design and synthesis of many different versions of biotinylated reagents or conjugates. 

Reference

http://lpi.oregonstate.edu/infocenter/vitamins/biotin/

Lanska DJ.; The discovery of niacin, biotin, and pantothenic acid.Ann Nutr Metab. 2012;61(3):246-53. doi: 10.1159/000343115. Epub 2012 Nov 26.

Livnah O, Bayer EA, Wilchek M, Sussman JL; Three-dimensional structures of avidin and the avidin-biotin complex.Proc. Natl. Acad. Sci. U.S.A. (1993) 90 p.5076-5080.

Rebecca Mardach, Janos Zempleni, Barry Wolf, Martin J. Cannon, Michael L. Jennings, Sally Cress, Jane Boylan, Susan Roth, Stephen Cederbaum, Donald M. Mock; Biotin dependency due to a defect in biotin transport. J Clin Invest. 2002 June 15; 109(12): 1617–1623. doi: 10.1172/JCI13138

Hamid M Said; Biotin: the forgotten vitamin. Am J Clin Nutr 2002; 75:179–80.

Wolf, B. 2001. Disorders of biotin metabolism. In The Metabolic and Molecular Basis of Inherited Disease. C.R. Scriver, A.L. Beaudet, W.S. Sly, and D. Valle, editors. McGraw-Hill Inc. New York, New York, USA. 3151–3177.

Human growth hormone or hGH 

Human growth hormone (hGH) is a naturally occurring peptide or protein hormone secreted by the pituitary gland. Growth hormone (GH or HGH) is also known as somatotropin or somatropin. It is a peptide or protein hormone that stimulates growth, cell reproduction and regeneration in humans. In the body the hormone is rather heterogeneous. The major isoform of the human growth hormone is a protein of 191 amino acids and a molecular weight of 22,124 daltons.

3D models of HGH  {Source PDB: 1HGU}

The three-dimensional structure is stabilized by two disulfide bridges four helical structures. The position of the helices and the overall three-dimensional structure of this hormone are important for receptor binding. The hormone shares structural homologies with prolactin and human chorionic somatomammotropin (hCS). HCS is a growth hormone variant synthesized exclusively in the placenta. There is a cluster of five genes from which these polypeptide hormones may be synthesized but normally only one gene expressed tissue-specifically. Binding of the tissue-specific transcription factor Pit-1 to the promoter region of the growth hormone gene results in only one form of growth hormone that are secreted by the anterior pituitary gland.

Before recombinant technology was available, the only source of hGH was human cadavers, but the contamination that led to Creutzfeldt–Jakob disease made this form of treatment obsolete. During the late 1980s, recombinant hGH (rhGH) was developed through genetic engineering. Recombinant hGH has been used with good results in the treatment of patients with hGH deficiency allowing bone growth and impacting on the patient’s final stature. This form of hGH has a sequence identical to the naturally occurring 22 kDa hormone.

Some athletes and bodybuilders appear to have used rhGH and claim that it increases lean body mass and decreases fat mass. Besides its anabolic properties hGH also effects carbohydrate and fat metabolism. During sport doping investigations rhGH has been found in swimmers and also in players taking part in other major sports events. International federations and the International Olympic Committee have hGH now on the list of forbidden compounds since 1989.

Human growth hormone is secreted from somatotropic cells in the anterior pituitary gland in a pulsating fashion. Two hypothalamic peptides, growth hormone releasing hormone, which stimulates hGH secretion, and somatostatin, which inhibits hGH secretion by back regulation, regulate its secretion.

hGH binds to specific receptors present throughout the whole body and exerts its biological effects on target cells. The secretion of hGH is slightly higher in women than in men. The highest levels are observed at puberty. Secretion decreases with age by approximatelly 14 % per decade. In addition, secretion of the hormone varies with normal physiological and pathological conditions and hGH levels are higher during slow wave sleep and are increased by exercise, stress, fever, fasting and, with increased levels of some amino acids (leucine and arginine). Drugs, such as clonidine, L-dopa and c-hydroxybutyrate, increase its secretion, as do androgens and estrogens. hGH binds to specific membrane receptors found in abundance throughout the body. It has both direct and indirect effects on the tissues. Indirect effects are mediated by IGF-1, generated in the liver in response to GH.

References

Chantalat L, Jones ND, Korber F, Navaza J, Pavlovsky AG; The crystal-structure of wild-type growth-hormone at 2.5 angstrom resolution. Protein Pept.Lett. (1995) 2 p.333.

M Saugy, N Robinson, C Saudan, N Baume, L Avois, P Mangin; Human growth hormone doping in sport. Br J Sports Med 2006;40(Suppl I):i35–i39. doi: 10.1136/bjsm.2006.027573.

Molecules with the same atoms and functional groups are called stereoisomers.  Stereoisomers are compounds made up of the same atoms and bonded by the same sequence of bonds, but having different three dimensional (3D) structures. The different 3D structures are called configurations and are not interchangeable. Two stereoisomers cannot superimpose. Thus, even when two molecules contain the same functional groups but are stereoisomers organisms can usually distinguish between them.

Figure 1 illustrates the concepts of chirality, enantiomers, handedness, isomers and stereoisomers. The amino acid alanine is used here as an example.     

Enantiomers have identical physical and chemical properties except in two important properties:

• They rotate the plane of polarized light in opposite directions, however in equal amounts. The isomer or enantiomer that rotates the plane counterclockwise or to the left is called the levo isomer and is designated (-). The other isomer rotates the plane clockwise or to the right and is therefore called the dextro isomer and is designated (+). They are also called optical antipodes.
• They react at different rates with other chiral compounds. However, the reaction rates may be so close together that a distinction is not always possible, or they may be so far apart that one isomer reacts much faster than the other or not at all. This explains why many compounds are biologically active while their enantiomers are not. However, enantiomers react at the same rate with achiral compounds.

In addition, the amount of rotation α is not constant for a given enantiomer. The amount of rotation depends on the length of the sample vessel, the temperature, the solvent and concentration (in the case of solutions), the pressure (in the case of gases), and the wavelength of light.

The specific rotation [α] is defined by the formula [α] = α / lc, for solutions, and [α] = α / ld for pure compounds, where α is the observed rotation, l is the length of the cell in decimeters, c is the concentration in grams per milliliter, and d is the density in the same units. The specific rotation is usually reported along with the temperature and wavelength, for examples as [α]25546. [α]D indicated that the rotation was measured with sodium D light at λ= 589 nm. The molecular rotation [M]tλ is the specific rotation times the molecular weight divided by 100.

The reporting structure is important since changes in conditions can change not only the amount of rotation but sometimes also the direction of rotation. For example, one of the enantiomers of aspartic acid, when dissolved in water, has [α]D equal to  +4.36° at 20 °C and -1.86° at 90 °C, although the molecular structure is unchanged.

In 1891, Emil Fisher, invented the Fisher projections, a method of how to represent tetrahedral carbons on paper. By this convention, the model is held so that the two bonds in front of the papers are horizontal and those behind the paper are vertical. The ability of these models is limited but they are useful for a quick test if the molecules in question are chiral. In any case, 3D models are much better for the determination of the nature of enantiomers or stereoisomers.  

The DL system has been widely used in the past but it is not without faults. Therefore it is only used nowadays for certain groups of molecules, such as carbohydrates and amino acids. The DL system has been replaced by the Cahn-Ingold-Prelog system in which the four groups on an asymmetric carbon are ranked according to a set of sequence rules.

Reference

Any school or college book covering biochemistry and molecular biology including handbooks for amino acids and organic molecule may be reviewed.

http://goldbook.iupac.org/C00772.html

IUPAC. Compendium of Chemical Terminology, 2nd ed. (the "Gold Book"). Compiled by A. D. McNaught and A. Wilkinson. Blackwell Scientific Publications, Oxford (1997). XML on-line corrected version: http://goldbook.iupac.org (2006-) created by M. Nic, J. Jirat, B. Kosata; updates compiled by A. Jenkins. ISBN 0-9678550-9-8.

March’s Advanced Organic Chemistry; Reactions, Mechanisms, and Structure. 6th editions. M.B. Smith and J. March. 2007. Wiley & Sons. Hoboken, NJ.

Oligonucleotide conjugation is predominantly carried out by use of a nucleophilic group on an oligonucleotide to react with an electrophilic group on a reporter molecule or a solid support. This is predominant approach because the common oligonucleotide deprotection is performed by base treatment e.g. ammonia, primary alkylamines or their combinations which are inherently nucleophilic. However, there are many situations when researchers need to introduce an electrophilic group into oligonucleotides and use it in the attachment method towards of a nucleophilic moiety.

In the case, when oligonucleotides have been used in that type of the bio-conjugation the activated carboxylate have been generated post-synthetically.2 Free carboxyl modified oligonucleotides can be activated by EDC in situ in the organic or aqueous conditions and subsequently conjugated to aminated counterpart (Fig. 1). However, in order to generate free carboxylate attached oligonucleotide using commercially available building blocks it requires cleavage of the ester bond with alkaline base4,5 before oligonucleotide base deprotection in concentrated ammonia, otherwise the ester will beconverted into corresponding amide.




Figure 1. Bio-conjugation between carboxyl modified oligonucleotide and alkylamino moiety via EDC reagent.

Recently Prof. Brown’s group discovered that the neutral heteroaromatic “click” backbone, when it introduced instead of natural phosphodiester bond is acceptable for Taq polymerase and can be used for most polymerase dependent proceses.¹³

Cooper dependent “click chemistry” often limits that type of attachment chemistry due to cytotoxicity. Recently developed azadibenzocyclooctyne doesn’t require any catalysts and it is highly reactive towards aliphatic and aromatic azides.¹⁴

1. http://www.biosyn.com/Bioconjugation.aspx
2. E. Jablonski, E. W.Moomaw, R. H.Tullis and J. L.Ruth Nucleic Acid Res., 1986, 14, 6115-6128.
3. J. D. Kahl and M. M. Greenberg J. Org. Chem., 1999, 64 (2), 507–510
4. A.V. Kachalova, T. S. Zatsepin, E. A. Romanova, D. A. Strelenko, M. J. Gait, T. S. Oretskaya Nucleosides, Nucleotides Nucleic Acids. 2000, 19, 1693-1707
5. T. P. Prakash, A. M. Kawasaki, E. A. Lesnik, S. R. Owens, M. Manoharan Org. Lett. 2003, 5, 403-406.
6. M. A. Podyminogin, E. A. Lukhtanov and M. W. Reed Nucleic Acid Res., 2001, 29, 5090-5098.
7. S. Raddatz, J. Mueller-Ibeler, J. Kluge, L. Wäß, G. Burdinski, J. R. Havens, T. J. Onofrey, D. Wang, and M Schweitzer Nucleic Acid Res., 2002, 30, 4793-4802.
8. E. N. Timofeev, A. D. Mirzabekov, S. V. Kochetkova and V. L. Florentiev Nucleic Acid Res., 1996, 24, 3142-3148.
9. V. V. Rostovtsev, L.G. Green, V. V. Fokin, K.B. Sharpless, Agnew. Chem. Int. Ed., 2002, 41, 2596-2599.
10. A.V. Ustinov, et al, Tetrahedron, 2007, 64, 1467-1473.
11. Agnew, B. et al., US Patent application 20080050731/A1.
12. X. Ming, P. Leonard, D. Heindle and F. Seela, Nucleic Acid Symposium Series No. 52, 471-472, 2008.
13. A. H. El-Saghner, and T. Brown, Accounts of Chemical Research, 2012, 45 (8), 1258-67.
14. M. F. Debets, S. S. van Berkel, S. Schoffelen, F. P. J. T. Rutjes, J. C. M. van Hest and F. L. van Delft, Chem. Commun., 2010,46, 97-99.
15. V. Marcha ´n, S. Ortega, D. Pulido, E. Pedroso and A. Grandas, Nucleic Acid Res., 2006, 34, e24

By Klaus D. Linse

Protein precipitation methods are used for the concentration of diluted proteins in solution. The goal is to purify and concentrate contaminated proteins or proteins dissolved in various matrices, buffers, detergents or from natural sources, such as blood, urine or other biofluids. The mechanism of precipitation for proteins is to alter the solvation potential of the solvent. The solubility of the solute is lowered by adding a specific reagent to manipulate the repulsive electrostaci forces between proteins.

The following is list of precipitation methods collected over several years. 

Trichloroacetic Acid (TCA) Precipitation = TCA Precipitation

• Add 1/2 volume 50% (w/v) TCA (0 ^oC) to the aqueous protein solution.

·                optional: (0.1% b-mercaptoethanol or 20 mM dithiothreitol)

• Equilibrate (0 ^oC) for 10-20 min.
• Centrifuge (~5000-13,000 x g cold) to pellet protein.
• Resuspend in appropriate solution (may require neutralization).

Acetone Precipitation

·        Add acetone (-20 ^oC) at a 4:1 ratio to the aqueous protein solution.

·                optional: (0.1% b-mercaptoethanol or 20 mM dithiothreitol)

·        Equilibrate (-20 ^oC) for 60 min.

·        Centrifuge (~5000-13,000 x g cold) to pellet protein.

·        Resuspend in appropriate solution.

The precipitate is often difficult to resuspend in aqueous solution.  Sonication, detergents, acetonitrile, methanol, salts, TFA (acetic or formic acid) are often used to assist in the process.  Be careful of what effects these processes have on the downstream processes.

TCA-Acetone Precipitation

·        Add 15% TCA in acetone (-20 ^oC) at a 4:1 ratio to the aqueous protein solution.

·                optional: (0.1% b-mercaptoethanol or 20 mM dithiothreitol)

·        Equilibrate (-20 ^oC) for 20-60 min.

·        Centrifuge (~5000-13,000 x g cold) to pellet protein.

·        Resuspend in appropriate solution.

This technique is somewhat superior in overall recovery to TCA and Acetone separately.  The precipitate is often difficult to resuspend in aqueous solution.  Sonication, detergents, acetonitrile, methanol, salts, TFA (acetic or formic acid) are often used to assist in the process.  Be careful of what effects these processes have on the downstream processes.

Phenol, Ammonium Acetate/Methanol Precipitation

 “It’s a little tricky but I like this the best” 

            Hurkman and Tanaka, 1986, Plant Physiology 81:802-806.

• Add 1 volume of buffered (pH~8.5-9.0) phenol (e.g. 0.1 M Tris-HCl pH 8.8, 10 mM EDTA(~3 basic), 0.2% 2-mercaptoethanol/20 mM dithiothreitol, 900 mM sucrose).
• Mix for 30 min and centrifuge (~5000-13,000 x g cold) to phase separate.
• Re-extract aqueous phase (top) with 1 volume each of co-equilibrated Phenol and aqueous extraction buffers.
• Mix for 30 min, and then centrifuge (~5000-13,000 x g cold) to phase separate.
• Combine phenol phase.
• Precipitate proteins by adding 5 volumes of 0.1 M Ammonium Acetate in Methanol (-20 ^oC, 1 hr-overnight).
• Pellet protein by centrifugation (20 min 20-30 min, 14,000-20,000 x g, 4 ^oC).
• Wash pellet (2x) with 0.1 M Ammoniuim Acetate in Methanol (Resuspend 15 min, -20 ^oC; 10 min >14,000 x g, 4 ^oC).
• Wash pellet (2x) with Acetone (Resuspend 15 min, -20 ^oC; 10 min >14,000 x g, 4 ^oC).
• Wash pellet with 70 % Ethanol (Resuspend 15 min, -20 ^oC; 10 min >14,000 x g, 4 ^oC).
• Dry pellet under vacuum and store under Ar/N₂ (-20 ^oC)

This technique is superior to the other methods, particularly for plant tissues, however it is very time consuming.  The precipitate is often difficult to resuspend in aqueous solution.  Sonication, detergents, acetonitrile, methanol, salts, TFA (acetic or formic acid) are often used to assist in the process.  Be careful of what effects these processes have on the downstream processes.

Methanol Chloroform   (A)

        Wessel and Fluegge, 1984;   Anal. Biochem. 138,141-143.

·        Add 4 volumes methanol.

·        Add 3 volumes chloroform.

·        Add 3-4 volumes H₂O

·                optional: (0.1% b-mercaptoethanol or 20 mM dithiothreitol)

·        Vortex.

·        Centrifuge (~9,000 x g 10-60 min) to pellet protein between phases.

·        Discard upper phase (aqueous).

·        Add 3 volumes (original volumes) methanol

·        Centrifuge (~9,000 x g 10-20 min) to pellet protein.

·        Discard supernatant

·        Dry sample (N₂/Ar or using vacuum).

·        Resuspend in appropriate solution.

In my hands, this technique is superior to the other methods. Minimal amounts of protein (1 µg or less) are distributed over a rather large area and many proteins are, subsequently, insoluble in buffers lacking SDS. Only small quantities of chemicals or solutions are needed. Use the best grade solvents (highest purity available) for this method. 

For amino acid analysis and protein sequencing: The dried pellet can be dissolved either using pure TFA, TFA/water, heptafluoracetone (HPFA)/TFA or heptafluorpropanol (HPFA)/TFA for direct spotting on to the sample support used in the protein sequencer.

Methanol Chloroform  (B)      Modified for speed !

• In a 1.5 ml micro centrifuge tube add to 100 µl of your sample solution
  
  400 µl Methanol,
  300 µl Chloroform and
  300 to 400 µl H2O  
  

• Vortex the solution intensively and centrifuge for 10 to 20 (sometimes even 30 to 60 minutes), at about 9000 rpm (table centrifuge). Separation into two phases should be visible. The protein/peptide is found between the two phases. Discard the upper phase carefully (or keep it for further investigations or distribution studies).

• Add another 300 µl Methanol, vortex and centrifuge for 10 - 20 minutes.

• Discard the liquid and dry the precipitated pellet (with a stream of air or, better, N2 or argon, or by evaporating the remaining liquid by applying a vacuum in a speed-vac centrifuge or equivalent).

• Dissolve the sample in a buffer suitable for your next separation method and proceed.

Desalting/Lyophylization

• Desalt sample by dialysis, ultrafiltration or a desalting column.
  
• Freeze sample with N_{2 liq}, Dry ice-Methanol or freezer and lyophylize sample.
  

Click Chemistry - A Review

Click chemistry refers to a modular chemical approach that utilizes the copper (I) catalyzed 1,2,3-triazole formation from azides and terminal acetylenes as a powerful linking reaction to produce unique useful and versatile new biological compounds. This copper (I) catalyzed coupling of azides to terminal acetylenes is the premier reaction in click chemistry and creates 1,4-disubstitued 1,2,3-triazole linkages. These linkages share useful topological and electronic features with ubiquitous amide connectors that are not susceptible to cleavage.

The beauty of click reactions is that click chemistry employs chemical reactions that are high yielding, cover a wide scope of reactions, create only byproducts that can be removed without chromatography, are stereospecific, simple to perform, and can be conducted in easily removable solvents. Since its introduction by K. B. Sharpless in 2001 click chemistry has enabled modular approaches for the generation of novel pharmacophores via a collection of reliable chemical reactions. The power of click chemistry enables the production of stereoselective products in high yields. The resulting products contain inoffensive byproducts, are insensitive to oxygen and water, utilize readily available starting materials, and have a thermodynamic driving force of at least 20 kcal mol-1. According to Rostovtsev et al. (2002) by simply stirring in water, organic azides and terminal alkynes are readily and cleanly converted into 1,4-disubstituted 1,2,3-triazoles through a highly efficient and regioselective copper(I)-catalyzed process.

The dipolar structure of azides was first recognized by Linus Pauling in 1933. Pauling published a paper in 1933 describing the investigation of the structures of methyl azide, CH₃-N₃, and carbon suboxide, C₃O₂, by electron-diffraction. The deduced structure for methyl azide as determined by Pauling is illustrated in figure 1.  

Figure 3: Reaction schemes of the 1,3-dipolar addition to yield triazolines. A. The addition of molecules with the sequence a-b-c to double bonds is shown. The cycloaddition of phenyl azide is used as an example on the right. B.  The addition of molecules with the sequence a-b-c to triple bonds is shown. The reaction of phenyl azide is used as an example on the right. These type of reactions were intensively studied by Rolf Huisgen and are known as 1,3-dipolar Huisgen cycloaddition reaction. C. The copper (I) catalyzed cycloaddition to a triple bond is illustrated here. This chemistry is known as “click chemistry” and yields exclusively the 1,4-disubstituted 1,2,3-triazole.

As pointed out by Barry Sharpless’s group (Kolb et al. 2001), the characteristic of click reactions is the high thermodynamic driving force which is usually greater than 20 kcal per mol. The reactions quickly proceed to completion and tend to be highly selective for a single product. Carbon-heteroatom bond forming reactions are the most common examples.

Click chemistry includes the following classes of chemical transformations:

• Cycloadditions of unsaturated species. Including 1,3-dipolar cycloaddition reactions and Diels-Alder transformations.

• Nucleophilic substitution chemistries. For example, ring-opening reactions of strained heterocyclic electrophiles such as epoxides, aziridines, aziridinium ions, and episulfonium ions.

• Carbonyl chemistry of the “non-aldol” type, such as formation of ureas, thioureas, aromatic heterocycles, oxime ethers, hydrazones, and amides.

• Additions to carbon-carbon multiple bonds. For example, epoxidation, dihydroxylation, azirdination, sulfenyl halide addition and Michael additions of Nu-H reactants.

References

Franck Amblard, Jong Hyun Cho, and Raymond F. Schinazi; The Cu(I)-catalyzed Huisgen azide-alkyne 1,3-dipolar cycloaddition reaction in nucleoside, nucleotide and oligonucleotide chemistry. Chem Rev. 2009 September ; 109(9): 4207–4220. doi:10.1021/cr9001462.

Bohm, T.; Webber, A.; Sauer, J. "Nonstereospecific 1,3-Dipolar Cycloadditions of Azomethine Ylides and Enamines." Tetrahedron1999, 55, 9535-9558.

Z. P. Demko and K. B. Sharpless, An Expedient Route to the Tetrazole Analogs of α–Amino Acids, Org. Lett., 4, 2525 (2002).

R. Huisgen, R. Grashey, J. Sauer in Chemistry of Alkenes, Interscience, New York, 1964, 806-877.

IUPAC Nomenclature: http://goldbook.iupac.org/

Hartmuth C. Kolb, M. G. Finn, and K. Barry Sharpless; Click Chemistry: Diverse Chemical Function from a Few Good Reactions. Angew. Chem. Int. Ed. 2001, 40, 2004 ± 2021.

Jerry March: Advanced Organic Chemistry: Reactions, Mechanisms, and Structure. 2^nd Edition McGRAW-HILL International Book Company.

March’s: Advanced Organic Chemistry: Reactions, Mechanisms, and Structure. 6^nd Edition, M.B. Smith and J. March. WILEY-INTERSCIENCE. WILEY.COM.

Sarah A McCarthy,Gemma-Louise Davies & Yurii K Gun'ko; Preparation of multifunctional nanoparticles and their assemblies. Nature Protocols 7, 1677–1693 (2012). doi:10.1038/nprot.2012.082.

Vsevolod V. Rostovtsev, Luke G. Green, Valery V. Fokin and K. Barry Sharpless; A Stepwise Huisgen Cycloaddition Process: Copper(I)-Catalyzed Regioselective “Ligation” of Azides and Terminal Alkynes. Article first published online: 15 JUL 2002. DOI: 10.1002/1521-3773(20020715)41:14<2596::AID-ANIE2596>3.0.CO;2-4. © 2002 WILEY-VCH Verlag GmbH, Weinheim, Fed. Rep. of Germany. Angewandte Chemie International Edition. Volume 41, Issue 14, pages 2596–2599, July 15, 2002.

Publications of the Sharpless Lab: http://www.scripps.edu/sharpless/pubs.html

  ! Contact BSI for more info on click chemistry based conjuagtion !

or

Click here ! 

Gene Expression

Gene expression is the most fundamental process that organisms undergo to perform the cellular functions required for life.  Gene expression can also be manipulated in the lab to take genes wanted from one organism and express that gene in another organism of interest.   Genes are expressed in a two-step process called transcription and translation.

The first step towards gene expression is transcription.  A gene is located within the genomic DNA of a cell.  The genomic DNA is similar to a blueprint in that it contains all the information needed to build each components of the cell.  These components are all proteins.  Transcription begins with RNA polymerase making a copy of the template strand of the gene of interest to form a single stranded RNA called messenger RNA.  Once messenger RNA is made, it proceeds to the next step called translation. 

In the last few years, rapid progress has been made in mapping every gene of every organism due to high throughput sequencing techniques.  As a result of this, any kind of gene encoding for any type of protein can be found online in a gene databank.  In the lab the gene of interest can be obtained by looking at the computer database instead of having proteins scan genomic DNA like it does in a cell.  While a cell is limited to take a certain portion of its own genomic DNA and convert it into mRNA for gene expression, scientists are able to synthetically synthesize genes from any organism and insert it into a vector for gene expression to take place.  All of this is only possible due to advances in gene sequencing and gene synthesis. 

The last step toward gene expression is translation.  Weather the gene of interest was obtained by the cell or by laboratory manipulation; translation occurs naturally using cellular proteins called ribosomes.  Once mRNA is made, it is tagged with a poly-A tail which guides the mRNA outside the nuclear pores of the nucleus to the ribosomes of the cell.  Within the ribosomes, mRNA is finally expressed into genes.  Gene expression is defined as the portion of genomic DNA (a gene) which can be expressed into a protein. 

In summary, gene expression is a complex process that occurs within the cell to express proteins of interest.  Due to advancements in technology, genes from any type of organism can be synthetically made and expressed into a wide variety of hosts.  These advancements in gene expression techniques will and have led to discoveries in therapeutic research.  

Oligonucleotide Fluorescent Labeling

By Andrei Laikhter

Optimally labeled nucleic acids are used as molecular probes and are very useful for a variety of nucleic acid based applications such as in antisense technology, biochemistry, biology, chemistry, cell biology, DNA sequencing, forensic science, genetic analysis, medicinal chemistry, molecular diagnostics, neuroscience, pharmacology, RT-PCR based molecular detection, and many others. However, major applications of fluorescent or fluorogenic oligonucleotides appear to be sequencing, forensic and genetic analysis.

Fluorophores, fluorescent chemical compounds or molecules that can re-emit light upon light excitation, have been and are used for the labeling of many biomolecules. Oligonucleotides can be be used as reporter molecules and typically contain covalently linked functional modifications. However, most non-radioactive labels incorporated into nucleotides are not stable during chemical synthesis of oligonucleotides. Therefore, blocked nucleophilic groups such as alkyl-sulfhydral or -amines are incorporated during the oligonucleotide synthesis procedure. These groups can then be used to direct the incorporation of nucleophile-specific labeling reagents. Oligonucleotides labeled in this way have a wide variety of applications. Among them are DNA and RNA probes (1,2), micro-arrays, molecular diagnostic probes, automated sequencing(3), electron microscopy, fluorescence microscopy (4) and hybridization affinity chromatography (5).

Structures and spectral properties of fluorescent dyes

Several groups of chromophores consisting of conjugated unsaturated hydrocarbons and hetero-aromatic molecules have strong fluorescent properties. The most common fluorophores employed in fluorescent assays are derived from fluorescein, rhodamine, coumarin or cyanine type of chromophores which structures are illustrated in figure 1.  

Figure 1. Structures of the most common fluorophores. Where X is a linker, R is an oligonucleotide.

Each of these molecules has a characteristic absorbance spectrum and a characteristic emission spectrum. The specific wavelength at which one of these molecules will most efficiently absorb energy is called the absorbance peak and the wavelength at which it will most efficiently emit energy is called the emission peak as illustrated in figure 2.  

Table 1. Fluorescent properties of commonly used dyes.

 
The conjugation or addition of electron withdrawing groups (EWG) to a basic fluorophore moiety usually leads to a red shift resulting in a shift of the absorbance and emission peaks to longer wavelengths or lower energies.      

Methods of incorporation

The most common and convenient method for the attachment of a fluorescent dye to an oligonucleotide is the phosphoramidite method. This method makes it possible to use commercially available fluorescent phosphoramidites for the conjugation or incorporation of one or more fluorophores into or to both, the 5' and/or 3' end, of the oligonucleotide. However, if the fluorophore is not stable in basic conditions needed for the oligonucleotide base deprotection step, the attachment to an oligonucleotide has to be done using a post-synthetic method after the base deprotection step is completed. In this situation, it is best that the oligonucleotide contains a functional group that will react with a reactive moiety on the selected fluorophore resulting in a stable covalent bond between the fluorophore and the oligonucleotide.

Several chemo-selective methods are available that can be used for the post-synthetic oligonucleotide labeling. One of the commonly chemo-selective labeling method used employs amino modified oligonucleotides together with the corresponding NHS esters or similar amine reactive synthons as illustrated in figure 3.

Figure 3.  Oligonucleotide labeling using TAMRA NHS ester.

More recently “click chemistry” was successfully employed to label oligonucleotides with various fluorescent reporter molecules (7-9).  This chemical process is outlined in figure 4.

Figure 4.  Huisgen’s 1,3-dipolar cycloaddition between an alkyne modified oligonucleotide (1) and an azide modified reporter molecule (2).  Where R1 and R2 are a hydrogen atom (H) or an extension of the oligonucleotide chain, X is a linker, and Y is a reporter molecule.

The advantage of this chemistry is that it is completely orthogonal to any other attachment method. This chemistry can also be used in addition to any type of Michael-Addition reaction or chemistry as well as any other active esters that are reactive towards alkylamino modified oligonucleotides.

Polymerase dependent polynucleotide labeling using fluorescently labeled deoxynucleoside-5’-triphosphates (NTP) can be considered to be the major method for cDNA labeling (10). This method uses DNA polymerase or terminal deoxynucleotidyl transferase in order to incorporate fluorescently labeled nucleobases with the help of the corresponding NTPs into polynucleotides. These types of oligonucleotides may be used further in any type of micro-array applications.

Dark quenchers suitable for ultrasensitive probes

In recent years Dabcyl, TAMRA and other fluorescent acceptor molecules used in qPCR probes, have been replaced with one or more of the growing family of dark quencher molecules. For this reason, fluorophore-quencher dual-labeled probes have become a standard in kinetic qPCR assay. The properties of dark quencher dyes are provided in table 2.

Table 2.  Characteristic properties of quencher dyes.

However, even the most efficient quencher dyes show a narrow and limited range of quenching that is predetermined by their narrow absorbance spectra. Therefore, each of the quencher dyes requires a fluorophore within a certain fluorescence emission spectrum range in order to have an efficient energy transfer between the two dyes or chromophors. The broad absorbance spectrum of our new generation of quencher dyes, for Instant for the Quencher dyes (IQ4), makes these probes suitable for multiplexing (11). Their highly efficient quenching characteristics lead to a higher sensitivity expressed by the probe. These significantly improved novel quencher dyes, also showing improved Ct values, now allow for the design of new linear highly sensitive probes. A comparison of the UV spectral properties for standard mono-labeled oligonucleotides are illustrated in the figure 4.  

Figure 4.  UV Spectra of standard mono-labeled decamer oligonucleotides labeled with the leading quencher dyes.

Biosynthesis, Inc.  now offers all types of fluorescently labeled oligonucleotides including their conjugates with peptides proteins and various nanoparticles. 

References:

1. Zimmerman, J.; Voss, H.; Schwanger, C.; Stegemann, J.; Erfle, H.; Stucky, K.; Kristensen, T.; Ansorge, W., Nucleic Acids Res., 1990, 18,  1067.
2. Agrawal, S.; Zamecnik, P. C.,. Nucleic Acids Res., 1990, 18, 5419.
3. a) Landgraf, A.; Reckmann, B.; Pingoud, A., Anal. Biochem., 1991, 193, 231. b) Lee, L. G.; Connell, C. R. and Bloch, W. Nucleic Acids Res., 1993, 21, 3761. c) Tyagi, S.; Kramer, F. R., Nature Biotechnology, 1996, 14, 303.
4. Fisher, T. L.; Terhorst, T.; Cao, X.; Wagner, R. W., Nucleic Acids Res., 1993, 21,  3857.
5. Urdea, M. S.; Warner, B. D.; Running, J. A.; Stempien, M.; Clyne, J.; Horn, T., Nucleic Acids Res., 1988, 16,  4937.
6. http://www.biosyn.com/oligonucleotide-modification-services.aspx
7. A.V. Ustinov, et al, Tetrahedron, 2007, 64, 1467-1473.
8. Agnew, B. et al., US Patent application 20080050731/A1.
9. X. Ming, P. Leonard, D. Heindle and F. Seela, Nucleic Acid Symposium Series No. 52, 471-472, 2008.
10. a) Hessner, M.J., X. Wang, K. Hulse, L. Meyer, Y. Wu, S. Nye, S.W. Guo, and S. Ghosh. 2003. Nucleic Acids Res., 2003, 31:e14.  b) C. E. Guerra, BioTechniques, 2006, 41 (1), 53–56.
11. Laikhter A. et al. US patent 7,956,169.

-.-

• First the primary antibody is added.  Primary antibody production is usually generated in a host species such as a rabbit, horse, or even exotic animals like llamas.  The antigen is injected into the animal and the animal’s serum is harvested for its antibody.  After the blocking step a small amount of primary antibody is mixed in with the membrane under mild shaking.  The primary antibody is allowed to bind for 1-8 hours.  Different temperatures are employed to affect binding both specific and non-specific.  
• After the primary antibody is added, allowed to incubate, and washed from the membrane the second antibody is added.  This second antibody is against a portion of a host species specifically the animal used to generate the primary antibody.  The secondary antibody is linked to a reporter enzyme such as alkaline phosphatase or HRP.  HRP is the most common reporter used.  A sheet of photographic film is placed against the membrane and then exposed to light.  This reaction forms an image of the antibodies bound to the blot.  Another form of detection in western blots are to use a static fluorophore linked antibody or radioactive labeled antibodies. 

• Chemiluminescent detection:  This method utilizes an enzyme such as HRP which converts a substrate and causes it to luminesce.  A CCD camera is used to take an image of the western blot or photographic film.  The image is then analyzed by densitometry and quantifies the result in terms of OD (optical density).   This method is one of the first methods to be used for western blot detection.
• Fluorphore detection:  These immunoassays require fewer steps because there is no need for a substrate to develop the assay, however special equipment must be used to detect a fluorescent signal.  Recently digital imaging has shifted towards infrared regions of detection.  Near IR and quantum dots has increased the use of fluorescent probes due to their enhanced sensitivity in western blot analysis.    
• Radioactive detection:  Radioactive label can also be used in analysis.  The membrane is placed against an X-ray film and then allowed to develop.  The film beings to show the bands of the protein of interest as dark regions.  This type of detection is rarely used due health risks.  

The Ebola Virus Genome and Proteome

The Ebola virus is a single-stranded, negative-sense mini-genome RNA virus. Zaire Ebola virus is responsible for the recent outbreak in West-Africa. Ebola viruses belong to the filoviridae family, and together with Paramyxoviridae, Rhabdoviridae, and Borna disease virus, Filoviridae viruses belong to the taxonomic order mononegavirales.

Mononegavirales is the term used for "nonsegmented negative-strand RNA viruses" (NNSV). These are enveloped viruses that have mini-genomes consisting of a single RNA molecule of negative or anti-mRNA sense.

Nucleic acids isolated from negative strand RNA viruses or virus-infected cells cannot infect or initiate an infection cycle when introduced into the host cell. This criterion was used to distinguish “positive’ from “negative”-strand RNA viruses. The viral genome needs to be first transcribed to produce mRNAs. Therefore, the purified virion RNA is not infectious. The virus needs to bring its own RNA polymerase into the cell in order to produce mRNA. To allow the virus to be infective a viral polymerase must be part of the viral particle  or virion.

The use of non-infectious synthetic viral RNA allows for the design of PCR primers or probes as well as peptides and recombinant proteins for molecular diagnostics. Similarly, these molecules may lend themselves for the design and production of vaccines against the virus. 

Features of the unsegmented genome of negative-stranded RNA viruses are:

• Negative sense RNA in the virion
• Virion-associated RNA polymerase mediates transcription and replication
• Genome transcribed into 6-10 separate mRNAs from a single promoter
• Replication occurs by synthesis of a complete positive-sense RNA antigenome
• Nucleoprotein is the functional template for synthesis of replicative and mRNA
• Independently assembled nucleocapsids are enveloped at the cell surface at sites containing virus proteins
• Are mainly cytoplasmic
• Can occur in invertebrates, vertebrates and plants
  

Features of the family Filoviridae are:

• Filamentous forms with branching; sometimes U-shaped, 6-shaped or circular 
• Uniform diameter of 80 nm and varying lengths up to 14,000 nm. Infectious particle length is 790 nm for Marburg virus and 970 nm for Ebola virus
• Surface spikes of 10 nm length
• Helical nucleocapsid; 50 nm diameter, with an axial space of 20 nm diameter and helical periodicity of about 5 nm
• Filamentous with a linear ~13-19 kb mini-genome with a negative-sense single-stranded RNA of molecular weight (Mr) =  4.2 x 10⁶ 
• At least five (5) proteins; a large (polymerase) protein, a surface glycoprotein, two (2) nucleocapsid-associated proteins, and at least one other protein of unknown function
• Biology enigmatic; only two antigenically unrelated viruses known; blood borne infection of humans and monkeys

Filoviruses are responsible for newly emerging infections. Filoviruses are considered as Biosafety Level 4 agents, in comparison HIV is only considered as Biosafety Level 2+. Filoviruses can infect mice, hamsters, guinea pigs and monkeys. However, it is not known at presence where the virus originates in the wild. 

Most human epidemics appear to be blood-born spread, in hospitals often transmitted via contaminated needles, and transmitted via close contact with infected persons or their body fluids. Primary infections with Marburg and Ebola are usually 25 to 90% fatal.  Death is thought to occur because of visceral organ necrosis, for example of the liver, due to viral infection of tissue parenchymal cells. 

Viral RNA is not infectious by itself. Therefore, the use of cloned or synthetic viral RNA can be very useful for the development and production of diagnostic tests or the development of vaccines against filoviruses, for example, the Ebola virus. 

Research with the aim to develop a vaccine for Ebola has already been started for several years now. In 1998, the first immunization for Ebola virus infections that was successful was reported. 

{Xu L, Sanchez A, Yang Z, Zaki SR, Nabel EG, Nichol ST, Nabel GJ.; Immunization for Ebola virus infection. Nat Med. 1998 Jan;4(1):37-42.}

The result – a DNA vaccine encoding the glycoprotein (sGP or GP) of the Ebola virus evoked a T-cell based immune response in guinea pigs and protected the animals against infection. Further studies indicated that a DNA vaccine can is useful for vaccination. The use of DNA immunization together with adenovirus vectors encoding viral proteins in nonhuman primates resulted in the protection of crab-eating or cynomolgus macaques (Macaca fascicularis) from the lethal pathogen, the wild-type Zaire virus. 

 {Sullivan NJ, Sanchez A, Rollin PE, Yang ZY, Nabel GJ.; Development of a preventive vaccine for Ebola virus infection in primates. Nature. 2000 Nov 30;408(6812):605-9.}

Results from sequence analysis of Ebola viruses from outbreaks in 1976 and 1995 showed a high degree of genetic conservation for this virus type. An explanation of this could be that Ebola viruses may have coevolved with their natural host reservoirs and do not change a lot in the wild.

Reference

Biology of Negative Strand RNA Viruses: The Power of Reverse Genetics; Y. Kawaoka (Ed.). © Springer-Verlag Berlin Heidelberg 2004.

Ebihara H, Takada A, Kobasa D, Jones S, Neumann G, et al. (2006) Molecular determinants of Ebola virus virulence in mice. PLoS Pathog 2(7): e73. DOI: 10.1371/ journal.ppat.0020073.

MOLECULAR BASIS OF VIRUS EVOLUTION; Edited by ADRIAN J. GIBBS, CHARLES H. CALISHER, and FERNANDO GARCIA-ARENAL, © Cambridge University Press 1995.

http://www.ncbi.nlm.nih.gov/pubmed/?term=ebola+virus+review

The Zaire Ebola Virus Genome and Proteome

Graphical display and FASTA file from Pubmed.

Zaire ebolavirus isolate Ebola virus H.sapiens-tc/COD/1976/Yambuku-Mayinga, complete genome. NCBI Reference Sequence: NC_002549.1. 

Source

http://www.ncbi.nlm.nih.gov/nuccore/10313991?report=graph

LOCUS       NC_002549              18959 bp    cRNA    linear   VRL 27-AUG-2014

DEFINITION  Zaire ebolavirus isolate Ebola virus

            H.sapiens-tc/COD/1976/Yambuku-Mayinga, complete genome.

ACCESSION   NC_002549

VERSION     NC_002549.1  GI:10313991

DBLINK      BioProject: PRJNA14703

KEYWORDS    RefSeq.

SOURCE      Zaire ebolavirus (ZEBOV)

  ORGANISM  Zaire ebolavirus

            Viruses; ssRNA negative-strand viruses; Mononegavirales;

            Filoviridae; Ebolavirus.

REFERENCE   1  (bases 1 to 18959)

  AUTHORS   Volchkov,V.E., Volchkova,V.A., Chepurnov,A.A., Blinov,V.M.,

            Dolnik,O., Netesov,S.V. and Feldmann,H.

  TITLE     Characterization of the L gene and 5' trailer region of Ebola virus

  JOURNAL   J. Gen. Virol. 80 (Pt 2), 355-362 (1999)

   PUBMED   10073695

REFERENCE   2  (bases 1 to 18959)

  AUTHORS   Volchkov,V.E., Volchkova,V.A., Slenczka,W., Klenk,H.D. and

            Feldmann,H.

  TITLE     Release of viral glycoproteins during Ebola virus infection

  JOURNAL   Virology 245 (1), 110-119 (1998)

   PUBMED   9614872

REFERENCE   3  (bases 1 to 18959)

  AUTHORS   Volchkov,V.E., Feldmann,H., Volchkova,V.A. and Klenk,H.D.

  TITLE     Processing of the Ebola virus glycoprotein by the proprotein

            convertase furin

  JOURNAL   Proc. Natl. Acad. Sci. U.S.A. 95 (10), 5762-5767 (1998)

   PUBMED   9576958

REFERENCE   4  (bases 1 to 18959)

  AUTHORS   Volchkov,V.E., Becker,S., Volchkova,V.A., Ternovoj,V.A.,

            Kotov,A.N., Netesov,S.V. and Klenk,H.D.

  TITLE     GP mRNA of Ebola virus is edited by the Ebola virus polymerase and

            by T7 and vaccinia virus polymerases

  JOURNAL   Virology 214 (2), 421-430 (1995)

   PUBMED   8553543

REFERENCE   5  (bases 1 to 18959)

  AUTHORS   Bukreyev,A.A., Volchkov,V.E., Blinov,V.M. and Netesov,S.V.

  TITLE     The VP35 and VP40 proteins of filoviruses. Homology between Marburg

            and Ebola viruses

  JOURNAL   FEBS Lett. 322 (1), 41-46 (1993)

   PUBMED   8482365

REFERENCE   6  (bases 1 to 18959)

  CONSRTM   NCBI Genome Project

  TITLE     Direct Submission

  JOURNAL   Submitted (27-SEP-2000) National Center for Biotechnology

            Information, NIH, Bethesda, MD 20894, USA

REFERENCE   7  (bases 1 to 18959)

  AUTHORS   Volchkov,V.E.

  TITLE     Direct Submission

  JOURNAL   Submitted (02-JUN-2000) Institute of Virology, Philipps-University

            Marburg, Robert-Koch-Str. 17, Marburg 35037, Germany

  REMARK    Sequence update by submitter

REFERENCE   8  (bases 1 to 18959)

  AUTHORS   Volchkov,V.E.

  TITLE     Direct Submission

  JOURNAL   Submitted (20-AUG-1998) Institute of Virology, Philipps-University

            Marburg, Robert-Koch-Str. 17, Marburg 35037, Germany

COMMENT     PROVISIONAL REFSEQ: This record has not yet been subject to final

            NCBI review. The reference sequence is identical to AF086833.

            COMPLETENESS: full length.

FEATURES             Location/Qualifiers

     source          1..18959

                     /organism="Zaire ebolavirus"

                     /mol_type="viral cRNA"

                     /isolate="Ebola virus

                     H.sapiens-tc/COD/1976/Yambuku-Mayinga"

                     /db_xref="taxon:186538"

     5'UTR           1..55

                     /note="putative leader region"

                     /citation=[1]

                     /function="regulation or initiation of RNA replication"

     gene            56..3026

                     /gene="NP"

                     /locus_tag="ZEBOVgp1"

                     /db_xref="GeneID:911830"

     mRNA            56..3026

                     /gene="NP"

                     /locus_tag="ZEBOVgp1"

                     /product="nucleoprotein"

                     /db_xref="GeneID:911830"

     misc_signal     56..67

                     /gene="NP"

                     /locus_tag="ZEBOVgp1"

                     /note="putative; transcription start signal"

                     /citation=[1]

     CDS             470..2689

                     /gene="NP"

                     /locus_tag="ZEBOVgp1"

                     /function="encapsidation of genomic RNA"

                     /codon_start=1

                     /product="nucleoprotein"

                     /protein_id="NP_066243.1"

                     /db_xref="GI:10314000"

                     /db_xref="GeneID:911830"

                     /translation="MDSRPQKIWMAPSLTESDMDYHKILTAGLSVQQGIVRQRVIPVY

                     QVNNLEEICQLIIQAFEAGVDFQESADSFLLMLCLHHAYQGDYKLFLESGAVKYLEGH

                     GFRFEVKKRDGVKRLEELLPAVSSGKNIKRTLAAMPEEETTEANAGQFLSFASLFLPK

                     LVVGEKACLEKVQRQIQVHAEQGLIQYPTAWQSVGHMMVIFRLMRTNFLIKFLLIHQG

                     MHMVAGHDANDAVISNSVAQARFSGLLIVKTVLDHILQKTERGVRLHPLARTAKVKNE

                     VNSFKAALSSLAKHGEYAPFARLLNLSGVNNLEHGLFPQLSAIALGVATAHGSTLAGV

                     NVGEQYQQLREAATEAEKQLQQYAESRELDHLGLDDQEKKILMNFHQKKNEISFQQTN

                     AMVTLRKERLAKLTEAITAASLPKTSGHYDDDDDIPFPGPINDDDNPGHQDDDPTDSQ

                    DTTIPDVVVDPDDGSYGEYQSYSENGMNAPDDLVLFDLDEDDEDTKPVPNRSTKGGQQ

                     KNSQKGQHIEGRQTQSRPIQNVPGPHRTIHHASAPLTDNDRRNEPSGSTSPRMLTPIN

                     EEADPLDDADDETSSLPPLESDDEEQDRDGTSNRTPTVAPPAPVYRDHSEKKELPQDE

                     QQDQDHTQEARNQDSDNTQSEHSFEEMYRHILRSQGPFDAVLYYHMMKDEPVVFSTSD

                     GKEYTYPDSLEEEYPPWLTEKEAMNEENRFVTLDGQQFYWPVMNHKNKFMAILQHHQ"

     misc_feature    524..2671

                     /gene="NP"

                     /locus_tag="ZEBOVgp1"

                     /note="Ebola nucleoprotein; Region: Ebola_NP; pfam05505"

                     /db_xref="CDD:147601"

     polyA_signal    3015..3026

                     /gene="NP"

                     /locus_tag="ZEBOVgp1"

     misc_feature    3027..3031

                     /note="intergenic region"

     gene            3032..4407

                     /gene="VP35"

                     /locus_tag="ZEBOVgp2"

                     /db_xref="GeneID:911827"

     mRNA            3032..4407

                     /gene="VP35"

                     /locus_tag="ZEBOVgp2"

                     /product="VP35"

                     /citation=[5]

                     /db_xref="GeneID:911827"

     misc_signal     3032..3043

                     /gene="VP35"

                     /locus_tag="ZEBOVgp2"

                     /note="putative; transcription start signal"

                     /citation=[5]

     CDS             3129..4151

                     /gene="VP35"

                     /locus_tag="ZEBOVgp2"

                     /function="polymerase complex protein"

                     /citation=[5]

                     /codon_start=1

                     /product="polymerase complex protein"

                     /protein_id="NP_066244.1"

                     /db_xref="GI:10313992"

                     /db_xref="GeneID:911827"

                     /translation="MTTRTKGRGHTAATTQNDRMPGPELSGWISEQLMTGRIPVSDIF

                     CDIENNPGLCYASQMQQTKPNPKTRNSQTQTDPICNHSFEEVVQTLASLATVVQQQTI

                     ASESLEQRITSLENGLKPVYDMAKTISSLNRVCAEMVAKYDLLVMTTGRATATAAATE

                     AYWAEHGQPPPGPSLYEESAIRGKIESRDETVPQSVREAFNNLNSTTSLTEENFGKPD

                     ISAKDLRNIMYDHLPGFGTAFHQLVQVICKLGKDSNSLDIIHAEFQASLAEGDSPQCA

                     LIQITKRVPIFQDAAPPVIHIRSRGDIPRACQKSLRPVPPSPKIDRGWVCVFQLQDGK

                     TLGLKI"

     misc_feature    3186..4148

                     /gene="VP35"

                     /locus_tag="ZEBOVgp2"

                     /note="Filoviridae VP35; Region: Filo_VP35; pfam02097"

                     /db_xref="CDD:145320"

     gene            4390..5894

                     /gene="VP40"

                     /locus_tag="ZEBOVgp3"

                     /db_xref="GeneID:911825"

     mRNA            4390..5894

                     /gene="VP40"

                     /locus_tag="ZEBOVgp3"

                     /product="VP40"

                     /citation=[5]

                     /db_xref="GeneID:911825"

     misc_signal     4390..4401

                     /gene="VP40"

                     /locus_tag="ZEBOVgp3"

                     /note="transcription start signal"

                     /citation=[5]

     polyA_signal    4397..4407

                     /gene="VP35"

                     /locus_tag="ZEBOVgp2"

                     /citation=[5]

     CDS             4479..5459

                     /gene="VP40"

                     /locus_tag="ZEBOVgp3"

                     /citation=[5]

                     /codon_start=1

                     /product="matrix protein"

                     /protein_id="NP_066245.1"

                     /db_xref="GI:10313993"

                     /db_xref="GeneID:911825"

                     /translation="MRRVILPTAPPEYMEAIYPVRSNSTIARGGNSNTGFLTPESVNG

                     DTPSNPLRPIADDTIDHASHTPGSVSSAFILEAMVNVISGPKVLMKQIPIWLPLGVAD

                     QKTYSFDSTTAAIMLASYTITHFGKATNPLVRVNRLGPGIPDHPLRLLRIGNQAFLQE

                     FVLPPVQLPQYFTFDLTALKLITQPLPAATWTDDTPTGSNGALRPGISFHPKLRPILL

                     PNKSGKKGNSADLTSPEKIQAIMTSLQDFKIVPIDPTKNIMGIEVPETLVHKLTGKKV

                     TSKNGQPIIPVLLPKYIGLDPVAPGDLTMVITQDCDTCHSPASLPAVIEK"

     misc_feature    4479..5363

                     /gene="VP40"

                     /locus_tag="ZEBOVgp3"

                     /note="Matrix protein VP40; Region: VP40; pfam07447"

                     /db_xref="CDD:116068"

     polyA_signal    5883..5894

                     /gene="VP40"

                    /locus_tag="ZEBOVgp3"

                     /citation=[5]

     misc_feature    5895..5899

                     /note="intergenic region"

     gene            5900..8305

                     /gene="GP"

                     /locus_tag="ZEBOVgp4"

                     /db_xref="GeneID:911829"

     mRNA            5900..8305

                     /gene="GP"

                     /locus_tag="ZEBOVgp4"

                     /product="sGP"

                     /note="unedited mRNA"

                     /citation=[4]

                     /db_xref="GeneID:911829"

     misc_signal     5900..5911

                     /gene="GP"

                     /locus_tag="ZEBOVgp4"

                     /note="putative; transcription start signal"

                     /citation=[4]

     CDS             join(6039..6923,6923..8068)

                     /gene="GP"

                     /locus_tag="ZEBOVgp4"

                     /function="receptor binding and fusion"

                     /artificial_location="low-quality sequence region"

                     /note="virion spike glycoprotein precursor; an addition A

                     residue is inserted during transcription; encodes two

                     disulfide linked subunits GP1 and GP2"

                     /citation=[2]

                     /citation=[3]

                     /citation=[4]

                     /codon_start=1

                     /product="spike glycoprotein"

                     /protein_id="NP_066246.1"

                     /db_xref="GI:10313995"

                     /db_xref="GeneID:911829"

                     /translation="MGVTGILQLPRDRFKRTSFFLWVIILFQRTFSIPLGVIHNSTLQ

                     VSDVDKLVCRDKLSSTNQLRSVGLNLEGNGVATDVPSATKRWGFRSGVPPKVVNYEAG

                     EWAENCYNLEIKKPDGSECLPAAPDGIRGFPRCRYVHKVSGTGPCAGDFAFHKEGAFF

                     LYDRLASTVIYRGTTFAEGVVAFLILPQAKKDFFSSHPLREPVNATEDPSSGYYSTTI

                     RYQATGFGTNETEYLFEVDNLTYVQLESRFTPQFLLQLNETIYTSGKRSNTTGKLIWK

                     VNPEIDTTIGEWAFWETKKNLTRKIRSEELSFTVVSNGAKNISGQSPARTSSDPGTNT

                     TTEDHKIMASENSSAMVQVHSQGREAAVSHLTTLATISTSPQSLTTKPGPDNSTHNTP

                     VYKLDISEATQVEQHHRRTDNDSTASDTPSATTAAGPPKAENTNTSKSTDFLDPATTT

                     SPQNHSETAGNNNTHHQDTGEESASSGKLGLITNTIAGVAGLITGGRRTRREAIVNAQ

                     PKCNPNLHYWTTQDEGAAIGLAWIPYFGPAAEGIYIEGLMHNQDGLICGLRQLANETT

                     QALQLFLRATTELRTFSILNRKAIDFLLQRWGGTCHILGPDCCIEPHDWTKNITDKID

                     QIIHDFVDKTLPDQGDNDNWWTGWRQWIPAGIGVTGVIIAVIALFCICKFVF"

     misc_feature    7529..7540

                     /gene="GP"

                     /locus_tag="ZEBOVgp4"

                     /note="encodes the glycoprotein cleavage site, precursor

                     GP is cleaved by subtilisin-like cellular protease furin

                     into subunits GP1 and GP2 that are linked by a disulfide

                     bond"

                     /citation=[3]

     misc_feature    7793..7870

                     /gene="GP"

                     /locus_tag="ZEBOVgp4"

                     /note="immunosuppressive motif; other site"

     misc_feature    7988..8053

                     /gene="GP"

                     /locus_tag="ZEBOVgp4"

                     /note="transmembrane anchor; transmembrane region"

     misc_feature    7706..7924

                     /gene="GP"

                     /locus_tag="ZEBOVgp4"

                     /note="heptad repeat 1-heptad repeat 2 region of the

                     transmembrane subunit of Filoviridae viruses, Ebola virus

                     and Marburg virus, and related domains; Region:

                     Ebola-like_HR1-HR2; cd09850"

                     /db_xref="CDD:197367"

     misc_feature    join(6081..6923,6923..7153)

                     /gene="GP"

                     /locus_tag="ZEBOVgp4"

                     /note="Filovirus glycoprotein; Region: Filo_glycop;

                     pfam01611"

                     /db_xref="CDD:110602"

     misc_feature    7706..7732

                     /gene="GP"

                     /locus_tag="ZEBOVgp4"

                     /note="HR1A; other site"

                     /db_xref="CDD:197367"

     misc_feature    7733..7762

                     /gene="GP"

                     /locus_tag="ZEBOVgp4"

                     /note="HR1B; other site"

                     /db_xref="CDD:197367"

     misc_feature    7763..7783

                     /gene="GP"

                     /locus_tag="ZEBOVgp4"

                     /note="HR1C; other site"

                     /db_xref="CDD:197367"

     misc_feature    7784..7831

                     /gene="GP"

                     /locus_tag="ZEBOVgp4"

                     /note="HR1D; other site"

                     /db_xref="CDD:197367"

     misc_feature    7787..7837

                     /gene="GP"

                     /locus_tag="ZEBOVgp4"

                     /note="immunosuppressive region; other site"

                     /db_xref="CDD:197367"

     misc_feature    order(7838..7858,7859..7861)

                     /gene="GP"

                     /locus_tag="ZEBOVgp4"

                     /note="CX(6,7)C motif; other site"

                     /db_xref="CDD:197367"

     misc_feature    7886..7924

                     /gene="GP"

                     /locus_tag="ZEBOVgp4"

                     /note="HR2; other site"

                     /db_xref="CDD:197367"

     misc_feature    order(7784..7786,7793..7795)

                     /gene="GP"

                     /locus_tag="ZEBOVgp4"

                     /note="Cl binding site [ion binding]; other site"

                     /db_xref="CDD:197367"

     misc_feature    order(7706..7714,7718..7723,7727..7732,7736..7744,

                     7748..7756,7760..7765,7769..7777,7781..7807,7811..7819,

                     7823..7828,7844..7849,7856..7858,7865..7876,7880..7882,

                     7889..7894,7901..7903,7910..7915,7922..7924)

                     /gene="GP"

                     /locus_tag="ZEBOVgp4"

                     /note="homotrimer interface [polypeptide binding]; other

                     site"

                     /db_xref="CDD:197367"

     misc_feature    order(7706..7714,7718..7726,7730..7735,7739..7747,

                     7754..7768,7772..7783,7787..7792,7796..7804,7808..7813,

                     7817..7819)

                     /gene="GP"

                     /locus_tag="ZEBOVgp4"

                     /note="HR1-GP1 interface [polypeptide binding]; other

                     site"

                     /db_xref="CDD:197367"

     CDS             6039..7133

                     /gene="GP"

                     /locus_tag="ZEBOVgp4"

                     /note="sGP, small non-structural, secreted glycoprotein;

                     sGP secreted as a anti-parallel oriented homodimer"

                     /citation=[4]

                     /codon_start=1

                     /product="small secreted glycoprotein"

                     /protein_id="NP_066247.1"

                     /db_xref="GI:10313994"

                     /db_xref="GeneID:911829"

                     /translation="MGVTGILQLPRDRFKRTSFFLWVIILFQRTFSIPLGVIHNSTLQ

                     VSDVDKLVCRDKLSSTNQLRSVGLNLEGNGVATDVPSATKRWGFRSGVPPKVVNYEAG

                     EWAENCYNLEIKKPDGSECLPAAPDGIRGFPRCRYVHKVSGTGPCAGDFAFHKEGAFF

                     LYDRLASTVIYRGTTFAEGVVAFLILPQAKKDFFSSHPLREPVNATEDPSSGYYSTTI

                     RYQATGFGTNETEYLFEVDNLTYVQLESRFTPQFLLQLNETIYTSGKRSNTTGKLIWK

                     VNPEIDTTIGEWAFWETKKTSLEKFAVKSCLSQLYQTEPKTSVVRVRRELLPTQGPTQ

                     QLKTTKSWLQKIPLQWFKCTVKEGKLQCRI"

     misc_feature    6081..7130

                     /gene="GP"

                     /locus_tag="ZEBOVgp4"

                     /note="Filovirus glycoprotein; Region: Filo_glycop;

                     pfam01611"

                     /db_xref="CDD:110602"

     CDS             join(6039..6922,6924..6933)

                     /gene="GP"

                     /locus_tag="ZEBOVgp4"

                     /artificial_location="low-quality sequence region"

                     /note="ssGP; second non-structural secreted glycoprotein;

                     secreted in a monomeric form; one A residue is deleted or

                     two additional A residues are inserted at the editing site

                     during transcription of the GP gene"

                     /citation=[4]

                     /codon_start=1

                     /product="second secreted glycoprotein"

                     /protein_id="NP_066248.1"

                     /db_xref="GI:10313996"

                     /db_xref="GeneID:911829"

                     /translation="MGVTGILQLPRDRFKRTSFFLWVIILFQRTFSIPLGVIHNSTLQ

                     VSDVDKLVCRDKLSSTNQLRSVGLNLEGNGVATDVPSATKRWGFRSGVPPKVVNYEAG

                     EWAENCYNLEIKKPDGSECLPAAPDGIRGFPRCRYVHKVSGTGPCAGDFAFHKEGAFF

                     LYDRLASTVIYRGTTFAEGVVAFLILPQAKKDFFSSHPLREPVNATEDPSSGYYSTTI

                     RYQATGFGTNETEYLFEVDNLTYVQLESRFTPQFLLQLNETIYTSGKRSNTTGKLIWK

                     VNPEIDTTIGEWAFWETKKPH"

     misc_feature    join(6081..6922,6924..>6924)

                     /gene="GP"

                     /locus_tag="ZEBOVgp4"

                     /note="Filovirus glycoprotein; Region: Filo_glycop;

                     pfam01611"

                     /db_xref="CDD:110602"

     misc_signal     6918..6924

                     /gene="GP"

                     /locus_tag="ZEBOVgp4"

                     /note="additional A residues are inserted or deleted

                     during transcription of the GP gene by the viral

                     polymerase"

                     /citation=[4]

                     /function="RNA editing"

     gene            8288..9740

                     /gene="VP30"

                     /locus_tag="ZEBOVgp5"

                     /db_xref="GeneID:911826"

     mRNA            8288..9740

                     /gene="VP30"

                     /locus_tag="ZEBOVgp5"

                     /product="VP30"

                     /db_xref="GeneID:911826"

     misc_signal     8288..8299

                     /gene="VP30"

                     /locus_tag="ZEBOVgp5"

                     /note="putative; transcription start signal"

     polyA_signal    8295..8305

                     /gene="GP"

                     /locus_tag="ZEBOVgp4"

                     /citation=[4]

     CDS             8509..9375

                     /gene="VP30"

                     /locus_tag="ZEBOVgp5"

                     /note="polymerase complex protein"

                     /codon_start=1

                     /product="minor nucleoprotein"

                     /protein_id="NP_066249.1"

                     /db_xref="GI:10313997"

                     /db_xref="GeneID:911826"

                     /translation="MEASYERGRPRAARQHSRDGHDHHVRARSSSRENYRGEYRQSRS

                     ASQVRVPTVFHKKRVEPLTVPPAPKDICPTLKKGFLCDSSFCKKDHQLESLTDRELLL

                     LIARKTCGSVEQQLNITAPKDSRLANPTADDFQQEEGPKITLLTLIKTAEHWARQDIR

                     TIEDSKLRALLTLCAVMTRKFSKSQLSLLCETHLRREGLGQDQAEPVLEVYQRLHSDK

                     GGSFEAALWQQWDRQSLIMFITAFLNIALQLPCESSAVVVSGLRTLVPQSDNEEASTN

                     PGTCSWSDEGTP"

     misc_feature    8932..9321

                     /gene="VP30"

                     /locus_tag="ZEBOVgp5"

                     /note="Ebola virus-specific transcription factor VP30;

                     Region: Transcript_VP30; pfam11507"

                     /db_xref="CDD:151944"

     polyA_signal    9730..9740

                     /gene="VP30"

                     /locus_tag="ZEBOVgp5"

                     /note="putative"

     misc_feature    9741..9884

                     /note="intergenic region"

     gene            9885..11518

                     /gene="VP24"

                     /locus_tag="ZEBOVgp6"

                     /note="putative"

                     /db_xref="GeneID:911828"

     mRNA            9885..11496

                     /gene="VP24"

                     /locus_tag="ZEBOVgp6"

                     /product="VP24"

                     /db_xref="GeneID:911828"

     misc_signal     9885..9896

                     /gene="VP24"

                     /locus_tag="ZEBOVgp6"

                     /note="transcription start signal"

     CDS             10345..11100

                     /gene="VP24"

                     /locus_tag="ZEBOVgp6"

                     /codon_start=1

                     /product="membrane-associated protein"

                     /protein_id="NP_066250.1"

                     /db_xref="GI:10313998"

                     /db_xref="GeneID:911828"

                     /translation="MAKATGRYNLISPKKDLEKGVVLSDLCNFLVSQTIQGWKVYWAG

                     IEFDVTHKGMALLHRLKTNDFAPAWSMTRNLFPHLFQNPNSTIESPLWALRVILAAGI

                     QDQLIDQSLIEPLAGALGLISDWLLTTNTNHFNMRTQRVKEQLSLKMLSLIRSNILKF

                     INKLDALHVVNYNGLLSSIEIGTQNHTIIITRTNMGFLVELQEPDKSAMNRMKPGPAK

                     FSLLHESTLKAFTQGSSTRMQSLILEFNSSLAI"

     misc_feature    10369..11040

                     /gene="VP24"

                     /locus_tag="ZEBOVgp6"

                     /note="Filovirus membrane-associated protein VP24; Region:

                     Filo_VP24; pfam06389"

                     /db_xref="CDD:253701"

     polyA_signal    11485..11496

                     /gene="VP24"

                     /locus_tag="ZEBOVgp6"

                     /note="putative"

     misc_feature    11497..11500

                     /gene="VP24"

                     /locus_tag="ZEBOVgp6"

                     /note="intergenic region"

     gene            11501..18282

                     /gene="L"

                     /locus_tag="ZEBOVgp7"

                     /db_xref="GeneID:911824"

     mRNA            11501..18282

                     /gene="L"

                     /locus_tag="ZEBOVgp7"

                     /product="polymerase"

                     /citation=[1]

                     /db_xref="GeneID:911824"

     misc_signal     11501..11512

                     /gene="L"

                     /locus_tag="ZEBOVgp7"

                     /note="transcription start signal"

                     /citation=[1]

     polyA_signal    11508..11518

                     /gene="VP24"

                     /locus_tag="ZEBOVgp6"

     CDS             11581..18219

                     /gene="L"

                     /locus_tag="ZEBOVgp7"

                     /function="synthesis of viral RNAs; transcriptional RNA

                     editing"

                     /note="polymerase"

                     /citation=[1]

                     /codon_start=1

                     /product="RNA-dependent RNA polymerase"

                     /protein_id="NP_066251.1"

                     /db_xref="GI:10313999"

                     /db_xref="GeneID:911824"

                     /translation="MATQHTQYPDARLSSPIVLDQCDLVTRACGLYSSYSLNPQLRNC

                     KLPKHIYRLKYDVTVTKFLSDVPVATLPIDFIVPVLLKALSGNGFCPVEPRCQQFLDE

                     IIKYTMQDALFLKYYLKNVGAQEDCVDEHFQEKILSSIQGNEFLHQMFFWYDLAILTR

                     RGRLNRGNSRSTWFVHDDLIDILGYGDYVFWKIPISMLPLNTQGIPHAAMDWYQASVF

                     KEAVQGHTHIVSVSTADVLIMCKDLITCRFNTTLISKIAEIEDPVCSDYPNFKIVSML

                     YQSGDYLLSILGSDGYKIIKFLEPLCLAKIQLCSKYTERKGRFLTQMHLAVNHTLEEI

                     TEMRALKPSQAQKIREFHRTLIRLEMTPQQLCELFSIQKHWGHPVLHSETAIQKVKKH

                     ATVLKALRPIVIFETYCVFKYSIAKHYFDSQGSWYSVTSDRNLTPGLNSYIKRNQFPP

                     LPMIKELLWEFYHLDHPPLFSTKIISDLSIFIKDRATAVERTCWDAVFEPNVLGYNPP

                     HKFSTKRVPEQFLEQENFSIENVLSYAQKLEYLLPQYRNFSFSLKEKELNVGRTFGKL

                     PYPTRNVQTLCEALLADGLAKAFPSNMMVVTEREQKESLLHQASWHHTSDDFGEHATV

                     RGSSFVTDLEKYNLAFRYEFTAPFIEYCNRCYGVKNVFNWMHYTIPQCYMHVSDYYNP

                     PHNLTLENRDNPPEGPSSYRGHMGGIEGLQQKLWTSISCAQISLVEIKTGFKLRSAVM

                     GDNQCITVLSVFPLETDADEQEQSAEDNAARVAASLAKVTSACGIFLKPDETFVHSGF

                     IYFGKKQYLNGVQLPQSLKTATRMAPLSDAIFDDLQGTLASIGTAFERSISETRHIFP

                     CRITAAFHTFFSVRILQYHHLGFNKGFDLGQLTLGKPLDFGTISLALAVPQVLGGLSF

                     LNPEKCFYRNLGDPVTSGLFQLKTYLRMIEMDDLFLPLIAKNPGNCTAIDFVLNPSGL

                     NVPGSQDLTSFLRQIVRRTITLSAKNKLINTLFHASADFEDEMVCKWLLSSTPVMSRF

                     AADIFSRTPSGKRLQILGYLEGTRTLLASKIINNNTETPVLDRLRKITLQRWSLWFSY

                     LDHCDNILAEALTQITCTVDLAQILREYSWAHILEGRPLIGATLPCMIEQFKVFWLKP

                     YEQCPQCSNAKQPGGKPFVSVAVKKHIVSAWPNASRISWTIGDGIPYIGSRTEDKIGQ

                     PAIKPKCPSAALREAIELASRLTWVTQGSSNSDLLIKPFLEARVNLSVQEILQMTPSH

                     YSGNIVHRYNDQYSPHSFMANRMSNSATRLIVSTNTLGEFSGGGQSARDSNIIFQNVI

                     NYAVALFDIKFRNTEATDIQYNRAHLHLTKCCTREVPAQYLTYTSTLDLDLTRYRENE

                     LIYDSNPLKGGLNCNISFDNPFFQGKRLNIIEDDLIRLPHLSGWELAKTIMQSIISDS

                     NNSSTDPISSGETRSFTTHFLTYPKIGLLYSFGAFVSYYLGNTILRTKKLTLDNFLYY

                     LTTQIHNLPHRSLRILKPTFKHASVMSRLMSIDPHFSIYIGGAAGDRGLSDAARLFLR

                     TSISSFLTFVKEWIINRGTIVPLWIVYPLEGQNPTPVNNFLYQIVELLVHDSSRQQAF

                     KTTISDHVHPHDNLVYTCKSTASNFFHASLAYWRSRHRNSNRKYLARDSSTGSSTNNS

                     DGHIERSQEQTTRDPHDGTERNLVLQMSHEIKRTTIPQENTHQGPSFQSFLSDSACGT

                     ANPKLNFDRSRHNVKFQDHNSASKREGHQIISHRLVLPFFTLSQGTRQLTSSNESQTQ

                     DEISKYLRQLRSVIDTTVYCRFTGIVSSMHYKLDEVLWEIESFKSAVTLAEGEGAGAL

                     LLIQKYQVKTLFFNTLATESSIESEIVSGMTTPRMLLPVMSKFHNDQIEIILNNSASQ

                     ITDITNPTWFKDQRARLPKQVEVITMDAETTENINRSKLYEAVYKLILHHIDPSVLKA

                     VVLKVFLSDTEGMLWLNDNLAPFFATGYLIKPITSSARSSEWYLCLTNFLSTTRKMPH

                     QNHLSCKQVILTALQLQIQRSPYWLSHLTQYADCELHLSYIRLGFPSLEKVLYHRYNL

                     VDSKRGPLVSITQHLAHLRAEIRELTNDYNQQRQSRTQTYHFIRTAKGRITKLVNDYL

                     KFFLIVQALKHNGTWQAEFKKLPELISVCNRFYHIRDCNCEERFLVQTLYLHRMQDSE

                     VKLIERLTGLLSLFPDGLYRFD"

     misc_feature    11608..14853

                     /gene="L"

                     /locus_tag="ZEBOVgp7"

                     /note="Mononegavirales RNA dependent RNA polymerase;

                     Region: Mononeg_RNA_pol; pfam00946"

                     /db_xref="CDD:250248"

     misc_feature    15223..18192

                     /gene="L"

                     /locus_tag="ZEBOVgp7"

                     /note="mRNA capping enzyme, paramyxovirus family; Region:

                     paramyx_RNAcap; TIGR04198"

                     /db_xref="CDD:234496"

     polyA_signal    18272..18282

                     /gene="L"

                     /locus_tag="ZEBOVgp7"

                     /citation=[1]

     3'UTR           18283..18959

                     /note="putative trailer region"

                     /citation=[1]

                     /function="regulation or initiation of RNA replication"

ORIGIN      

        1 cggacacaca aaaagaaaga agaattttta ggatcttttg tgtgcgaata actatgagga

       61 agattaataa ttttcctctc attgaaattt atatcggaat ttaaattgaa attgttactg

      121 taatcacacc tggtttgttt cagagccaca tcacaaagat agagaacaac ctaggtctcc

      181 gaagggagca agggcatcag tgtgctcagt tgaaaatccc ttgtcaacac ctaggtctta

      241 tcacatcaca agttccacct cagactctgc agggtgatcc aacaacctta atagaaacat

      301 tattgttaaa ggacagcatt agttcacagt caaacaagca agattgagaa ttaaccttgg

      361 ttttgaactt gaacacttag gggattgaag attcaacaac cctaaagctt ggggtaaaac

      421 attggaaata gttaaaagac aaattgctcg gaatcacaaa attccgagta tggattctcg

      481 tcctcagaaa atctggatgg cgccgagtct cactgaatct gacatggatt accacaagat

      541 cttgacagca ggtctgtccg ttcaacaggg gattgttcgg caaagagtca tcccagtgta

      601 tcaagtaaac aatcttgaag aaatttgcca acttatcata caggcctttg aagcaggtgt

      661 tgattttcaa gagagtgcgg acagtttcct tctcatgctt tgtcttcatc atgcgtacca

      721 gggagattac aaacttttct tggaaagtgg cgcagtcaag tatttggaag ggcacgggtt

      781 ccgttttgaa gtcaagaagc gtgatggagt gaagcgcctt gaggaattgc tgccagcagt

      841 atctagtgga aaaaacatta agagaacact tgctgccatg ccggaagagg agacaactga

      901 agctaatgcc ggtcagtttc tctcctttgc aagtctattc cttccgaaat tggtagtagg

      961 agaaaaggct tgccttgaga aggttcaaag gcaaattcaa gtacatgcag agcaaggact

     1021 gatacaatat ccaacagctt ggcaatcagt aggacacatg atggtgattt tccgtttgat

     1081 gcgaacaaat tttctgatca aatttctcct aatacaccaa gggatgcaca tggttgccgg

     1141 gcatgatgcc aacgatgctg tgatttcaaa ttcagtggct caagctcgtt tttcaggctt

     1201 attgattgtc aaaacagtac ttgatcatat cctacaaaag acagaacgag gagttcgtct

     1261 ccatcctctt gcaaggaccg ccaaggtaaa aaatgaggtg aactccttta aggctgcact

     1321 cagctccctg gccaagcatg gagagtatgc tcctttcgcc cgacttttga acctttctgg

     1381 agtaaataat cttgagcatg gtcttttccc tcaactatcg gcaattgcac tcggagtcgc

     1441 cacagcacac gggagtaccc tcgcaggagt aaatgttgga gaacagtatc aacaactcag

     1501 agaggctgcc actgaggctg agaagcaact ccaacaatat gcagagtctc gcgaacttga

     1561 ccatcttgga cttgatgatc aggaaaagaa aattcttatg aacttccatc agaaaaagaa

     1621 cgaaatcagc ttccagcaaa caaacgctat ggtaactcta agaaaagagc gcctggccaa

     1681 gctgacagaa gctatcactg ctgcgtcact gcccaaaaca agtggacatt acgatgatga

     1741 tgacgacatt ccctttccag gacccatcaa tgatgacgac aatcctggcc atcaagatga

     1801 tgatccgact gactcacagg atacgaccat tcccgatgtg gtggttgatc ccgatgatgg

     1861 aagctacggc gaataccaga gttactcgga aaacggcatg aatgcaccag atgacttggt

     1921 cctattcgat ctagacgagg acgacgagga cactaagcca gtgcctaata gatcgaccaa

     1981 gggtggacaa cagaagaaca gtcaaaaggg ccagcatata gagggcagac agacacaatc

     2041 caggccaatt caaaatgtcc caggccctca cagaacaatc caccacgcca gtgcgccact

     2101 cacggacaat gacagaagaa atgaaccctc cggctcaacc agccctcgca tgctgacacc

     2161 aattaacgaa gaggcagacc cactggacga tgccgacgac gagacgtcta gccttccgcc

     2221 cttggagtca gatgatgaag agcaggacag ggacggaact tccaaccgca cacccactgt

     2281 cgccccaccg gctcccgtat acagagatca ctctgaaaag aaagaactcc cgcaagacga

     2341 gcaacaagat caggaccaca ctcaagaggc caggaaccag gacagtgaca acacccagtc

     2401 agaacactct tttgaggaga tgtatcgcca cattctaaga tcacaggggc catttgatgc

     2461 tgttttgtat tatcatatga tgaaggatga gcctgtagtt ttcagtacca gtgatggcaa

     2521 agagtacacg tatccagact cccttgaaga ggaatatcca ccatggctca ctgaaaaaga

     2581 ggctatgaat gaagagaata gatttgttac attggatggt caacaatttt attggccggt

     2641 gatgaatcac aagaataaat tcatggcaat cctgcaacat catcagtgaa tgagcatgga

     2701 acaatgggat gattcaaccg acaaatagct aacattaagt agtcaaggaa cgaaaacagg

     2761 aagaattttt gatgtctaag gtgtgaatta ttatcacaat aaaagtgatt cttatttttg

     2821 aatttaaagc tagcttatta ttactagccg tttttcaaag ttcaatttga gtcttaatgc

     2881 aaataggcgt taagccacag ttatagccat aattgtaact caatattcta actagcgatt

     2941 tatctaaatt aaattacatt atgcttttat aacttaccta ctagcctgcc caacatttac

     3001 acgatcgttt tataattaag aaaaaactaa tgatgaagat taaaaccttc atcatcctta

     3061 cgtcaattga attctctagc actcgaagct tattgtcttc aatgtaaaag aaaagctggt

     3121 ctaacaagat gacaactaga acaaagggca ggggccatac tgcggccacg actcaaaacg

     3181 acagaatgcc aggccctgag ctttcgggct ggatctctga gcagctaatg accggaagaa

     3241 ttcctgtaag cgacatcttc tgtgatattg agaacaatcc aggattatgc tacgcatccc

     3301 aaatgcaaca aacgaagcca aacccgaaga cgcgcaacag tcaaacccaa acggacccaa

     3361 tttgcaatca tagttttgag gaggtagtac aaacattggc ttcattggct actgttgtgc

     3421 aacaacaaac catcgcatca gaatcattag aacaacgcat tacgagtctt gagaatggtc

     3481 taaagccagt ttatgatatg gcaaaaacaa tctcctcatt gaacagggtt tgtgctgaga

     3541 tggttgcaaa atatgatctt ctggtgatga caaccggtcg ggcaacagca accgctgcgg

     3601 caactgaggc ttattgggcc gaacatggtc aaccaccacc tggaccatca ctttatgaag

     3661 aaagtgcgat tcggggtaag attgaatcta gagatgagac cgtccctcaa agtgttaggg

     3721 aggcattcaa caatctaaac agtaccactt cactaactga ggaaaatttt gggaaacctg

     3781 acatttcggc aaaggatttg agaaacatta tgtatgatca cttgcctggt tttggaactg

     3841 ctttccacca attagtacaa gtgatttgta aattgggaaa agatagcaac tcattggaca

     3901 tcattcatgc tgagttccag gccagcctgg ctgaaggaga ctctcctcaa tgtgccctaa

     3961 ttcaaattac aaaaagagtt ccaatcttcc aagatgctgc tccacctgtc atccacatcc

     4021 gctctcgagg tgacattccc cgagcttgcc agaaaagctt gcgtccagtc ccaccatcgc

     4081 ccaagattga tcgaggttgg gtatgtgttt ttcagcttca agatggtaaa acacttggac

     4141 tcaaaatttg agccaatctc ccttccctcc gaaagaggcg aataatagca gaggcttcaa

     4201 ctgctgaact atagggtacg ttacattaat gatacacttg tgagtatcag ccctggataa

     4261 tataagtcaa ttaaacgacc aagataaaat tgttcatatc tcgctagcag cttaaaatat

     4321 aaatgtaata ggagctatat ctctgacagt attataatca attgttatta agtaacccaa

     4381 accaaaagtg atgaagatta agaaaaacct acctcggctg agagagtgtt ttttcattaa

     4441 ccttcatctt gtaaacgttg agcaaaattg ttaaaaatat gaggcgggtt atattgccta

     4501 ctgctcctcc tgaatatatg gaggccatat accctgtcag gtcaaattca acaattgcta

     4561 gaggtggcaa cagcaataca ggcttcctga caccggagtc agtcaatggg gacactccat

     4621 cgaatccact caggccaatt gccgatgaca ccatcgacca tgccagccac acaccaggca

     4681 gtgtgtcatc agcattcatc cttgaagcta tggtgaatgt catatcgggc cccaaagtgc

     4741 taatgaagca aattccaatt tggcttcctc taggtgtcgc tgatcaaaag acctacagct

     4801 ttgactcaac tacggccgcc atcatgcttg cttcatacac tatcacccat ttcggcaagg

     4861 caaccaatcc acttgtcaga gtcaatcggc tgggtcctgg aatcccggat catcccctca

     4921 ggctcctgcg aattggaaac caggctttcc tccaggagtt cgttcttccg ccagtccaac

     4981 taccccagta tttcaccttt gatttgacag cactcaaact gatcacccaa ccactgcctg

     5041 ctgcaacatg gaccgatgac actccaacag gatcaaatgg agcgttgcgt ccaggaattt

     5101 catttcatcc aaaacttcgc cccattcttt tacccaacaa aagtgggaag aaggggaaca

     5161 gtgccgatct aacatctccg gagaaaatcc aagcaataat gacttcactc caggacttta

     5221 agatcgttcc aattgatcca accaaaaata tcatgggaat cgaagtgcca gaaactctgg

     5281 tccacaagct gaccggtaag aaggtgactt ctaaaaatgg acaaccaatc atccctgttc

     5341 ttttgccaaa gtacattggg ttggacccgg tggctccagg agacctcacc atggtaatca

     5401 cacaggattg tgacacgtgt cattctcctg caagtcttcc agctgtgatt gagaagtaat

     5461 tgcaataatt gactcagatc cagttttata gaatcttctc agggatagtg ataacatcta

     5521 tttagtaatc cgtccattag aggagacact tttaattgat caatatacta aaggtgcttt

     5581 acaccattgt cttttttctc tcctaaatgt agaacttaac aaaagactca taatatactt

     5641 gtttttaaag gattgattga tgaaagatca taactaataa cattacaaat aatcctacta

     5701 taatcaatac ggtgattcaa atgttaatct ttctcattgc acatactttt tgcccttatc

     5761 ctcaaattgc ctgcatgctt acatctgagg atagccagtg tgacttggat tggaaatgtg

     5821 gagaaaaaat cgggacccat ttctaggttg ttcacaatcc aagtacagac attgcccttc

     5881 taattaagaa aaaatcggcg atgaagatta agccgacagt gagcgtaatc ttcatctctc

     5941 ttagattatt tgttttccag agtaggggtc gtcaggtcct tttcaatcgt gtaaccaaaa

     6001 taaactccac tagaaggata ttgtggggca acaacacaat gggcgttaca ggaatattgc

     6061 agttacctcg tgatcgattc aagaggacat cattctttct ttgggtaatt atccttttcc

     6121 aaagaacatt ttccatccca cttggagtca tccacaatag cacattacag gttagtgatg

     6181 tcgacaaact agtttgtcgt gacaaactgt catccacaaa tcaattgaga tcagttggac

     6241 tgaatctcga agggaatgga gtggcaactg acgtgccatc tgcaactaaa agatggggct

     6301 tcaggtccgg tgtcccacca aaggtggtca attatgaagc tggtgaatgg gctgaaaact

     6361 gctacaatct tgaaatcaaa aaacctgacg ggagtgagtg tctaccagca gcgccagacg

     6421 ggattcgggg cttcccccgg tgccggtatg tgcacaaagt atcaggaacg ggaccgtgtg

     6481 ccggagactt tgccttccat aaagagggtg ctttcttcct gtatgatcga cttgcttcca

     6541 cagttatcta ccgaggaacg actttcgctg aaggtgtcgt tgcatttctg atactgcccc

     6601 aagctaagaa ggacttcttc agctcacacc ccttgagaga gccggtcaat gcaacggagg

     6661 acccgtctag tggctactat tctaccacaa ttagatatca ggctaccggt tttggaacca

     6721 atgagacaga gtacttgttc gaggttgaca atttgaccta cgtccaactt gaatcaagat

     6781 tcacaccaca gtttctgctc cagctgaatg agacaatata tacaagtggg aaaaggagca

     6841 ataccacggg aaaactaatt tggaaggtca accccgaaat tgatacaaca atcggggagt

     6901 gggccttctg ggaaactaaa aaaacctcac tagaaaaatt cgcagtgaag agttgtcttt

     6961 cacagttgta tcaaacggag ccaaaaacat cagtggtcag agtccggcgc gaacttcttc

     7021 cgacccaggg accaacacaa caactgaaga ccacaaaatc atggcttcag aaaattcctc

     7081 tgcaatggtt caagtgcaca gtcaaggaag ggaagctgca gtgtcgcatc taacaaccct

     7141 tgccacaatc tccacgagtc cccaatccct cacaaccaaa ccaggtccgg acaacagcac

     7201 ccataataca cccgtgtata aacttgacat ctctgaggca actcaagttg aacaacatca

     7261 ccgcagaaca gacaacgaca gcacagcctc cgacactccc tctgccacga ccgcagccgg

     7321 acccccaaaa gcagagaaca ccaacacgag caagagcact gacttcctgg accccgccac

     7381 cacaacaagt ccccaaaacc acagcgagac cgctggcaac aacaacactc atcaccaaga

     7441 taccggagaa gagagtgcca gcagcgggaa gctaggctta attaccaata ctattgctgg

     7501 agtcgcagga ctgatcacag gcgggagaag aactcgaaga gaagcaattg tcaatgctca

     7561 acccaaatgc aaccctaatt tacattactg gactactcag gatgaaggtg ctgcaatcgg

     7621 actggcctgg ataccatatt tcgggccagc agccgaggga atttacatag aggggctaat

     7681 gcacaatcaa gatggtttaa tctgtgggtt gagacagctg gccaacgaga cgactcaagc

     7741 tcttcaactg ttcctgagag ccacaactga gctacgcacc ttttcaatcc tcaaccgtaa

     7801 ggcaattgat ttcttgctgc agcgatgggg cggcacatgc cacattctgg gaccggactg

     7861 ctgtatcgaa ccacatgatt ggaccaagaa cataacagac aaaattgatc agattattca

     7921 tgattttgtt gataaaaccc ttccggacca gggggacaat gacaattggt ggacaggatg

     7981 gagacaatgg ataccggcag gtattggagt tacaggcgtt ataattgcag ttatcgcttt

     8041 attctgtata tgcaaatttg tcttttagtt tttcttcaga ttgcttcatg gaaaagctca

     8101 gcctcaaatc aatgaaacca ggatttaatt atatggatta cttgaatcta agattacttg

     8161 acaaatgata atataataca ctggagcttt aaacatagcc aatgtgattc taactccttt

     8221 aaactcacag ttaatcataa acaaggtttg acatcaatct agttatctct ttgagaatga

     8281 taaacttgat gaagattaag aaaaaggtaa tctttcgatt atctttaatc ttcatccttg

     8341 attctacaat catgacagtt gtctttagtg acaagggaaa gaagcctttt tattaagttg

     8401 taataatcag atctgcgaac cggtagagtt tagttgcaac ctaacacaca taaagcattg

     8461 gtcaaaaagt caatagaaat ttaaacagtg agtggagaca acttttaaat ggaagcttca

     8521 tatgagagag gacgcccacg agctgccaga cagcattcaa gggatggaca cgaccaccat

     8581 gttcgagcac gatcatcatc cagagagaat tatcgaggtg agtaccgtca atcaaggagc

     8641 gcctcacaag tgcgcgttcc tactgtattt cataagaaga gagttgaacc attaacagtt

     8701 cctccagcac ctaaagacat atgtccgacc ttgaaaaaag gatttttgtg tgacagtagt

     8761 ttttgcaaaa aagatcacca gttggagagt ttaactgata gggaattact cctactaatc

     8821 gcccgtaaga cttgtggatc agtagaacaa caattaaata taactgcacc caaggactcg

     8881 cgcttagcaa atccaacggc tgatgatttc cagcaagagg aaggtccaaa aattaccttg

     8941 ttgacactga tcaagacggc agaacactgg gcgagacaag acatcagaac catagaggat

     9001 tcaaaattaa gagcattgtt gactctatgt gctgtgatga cgaggaaatt ctcaaaatcc

     9061 cagctgagtc ttttatgtga gacacaccta aggcgcgagg ggcttgggca agatcaggca

     9121 gaacccgttc tcgaagtata tcaacgatta cacagtgata aaggaggcag ttttgaagct

     9181 gcactatggc aacaatggga ccgacaatcc ctaattatgt ttatcactgc attcttgaat

     9241 attgctctcc agttaccgtg tgaaagttct gctgtcgttg tttcagggtt aagaacattg

     9301 gttcctcaat cagataatga ggaagcttca accaacccgg ggacatgctc atggtctgat

     9361 gagggtaccc cttaataagg ctgactaaaa cactatataa ccttctactt gatcacaata

     9421 ctccgtatac ctatcatcat atatttaatc aagacgatat cctttaaaac ttattcagta

     9481 ctataatcac tctcgtttca aattaataag atgtgcatga ttgccctaat atatgaagag

     9541 gtatgataca accctaacag tgatcaaaga aaatcataat ctcgtatcgc tcgtaatata

     9601 acctgccaag catacctctt gcacaaagtg attcttgtac acaaataatg ttttactcta

     9661 caggaggtag caacgatcca tcccatcaaa aaataagtat ttcatgactt actaatgatc

     9721 tcttaaaata ttaagaaaaa ctgacggaac ataaattctt tatgcttcaa gctgtggagg

     9781 aggtgtttgg tattggctat tgttatatta caatcaataa caagcttgta aaaatattgt

     9841 tcttgtttca agaggtagat tgtgaccgga aatgctaaac taatgatgaa gattaatgcg

     9901 gaggtctgat aagaataaac cttattattc agattaggcc ccaagaggca ttcttcatct

     9961 ccttttagca aagtactatt tcagggtagt ccaattagtg gcacgtcttt tagctgtata

    10021 tcagtcgccc ctgagatacg ccacaaaagt gtctctaagc taaattggtc tgtacacatc

    10081 ccatacattg tattaggggc aataatatct aattgaactt agccgtttaa aatttagtgc

    10141 ataaatctgg gctaacacca ccaggtcaac tccattggct gaaaagaagc ttacctacaa

    10201 cgaacatcac tttgagcgcc ctcacaatta aaaaatagga acgtcgttcc aacaatcgag

    10261 cgcaaggttt caaggttgaa ctgagagtgt ctagacaaca aaatattgat actccagaca

    10321 ccaagcaaga cctgagaaaa aaccatggct aaagctacgg gacgatacaa tctaatatcg

    10381 cccaaaaagg acctggagaa aggggttgtc ttaagcgacc tctgtaactt cttagttagc

    10441 caaactattc aggggtggaa ggtttattgg gctggtattg agtttgatgt gactcacaaa

    10501 ggaatggccc tattgcatag actgaaaact aatgactttg cccctgcatg gtcaatgaca

    10561 aggaatctct ttcctcattt atttcaaaat ccgaattcca caattgaatc accgctgtgg

    10621 gcattgagag tcatccttgc agcagggata caggaccagc tgattgacca gtctttgatt

    10681 gaacccttag caggagccct tggtctgatc tctgattggc tgctaacaac caacactaac

    10741 catttcaaca tgcgaacaca acgtgtcaag gaacaattga gcctaaaaat gctgtcgttg

    10801 attcgatcca atattctcaa gtttattaac aaattggatg ctctacatgt cgtgaactac

    10861 aacggattgt tgagcagtat tgaaattgga actcaaaatc atacaatcat cataactcga

    10921 actaacatgg gttttctggt ggagctccaa gaacccgaca aatcggcaat gaaccgcatg

    10981 aagcctgggc cggcgaaatt ttccctcctt catgagtcca cactgaaagc atttacacaa

    11041 ggatcctcga cacgaatgca aagtttgatt cttgaattta atagctctct tgctatctaa

    11101 ctaaggtaga atacttcata ttgagctaac tcatatatgc tgactcaata gttatcttga

    11161 catctctgct ttcataatca gatatataag cataataaat aaatactcat atttcttgat

    11221 aatttgttta accacagata aatcctcact gtaagccagc ttccaagttg acacccttac

    11281 aaaaaccagg actcagaatc cctcaaacaa gagattccaa gacaacatca tagaattgct

    11341 ttattatatg aataagcatt ttatcaccag aaatcctata tactaaatgg ttaattgtaa

    11401 ctgaacccgc aggtcacatg tgttaggttt cacagattct atatattact aactctatac

    11461 tcgtaattaa cattagataa gtagattaag aaaaaagcct gaggaagatt aagaaaaact

    11521 gcttattggg tctttccgtg ttttagatga agcagttgaa attcttcctc ttgatattaa

    11581 atggctacac aacataccca atacccagac gctaggttat catcaccaat tgtattggac

    11641 caatgtgacc tagtcactag agcttgcggg ttatattcat catactccct taatccgcaa

    11701 ctacgcaact gtaaactccc gaaacatatc taccgtttga aatacgatgt aactgttacc

    11761 aagttcttga gtgatgtacc agtggcgaca ttgcccatag atttcatagt cccagttctt

    11821 ctcaaggcac tgtcaggcaa tggattctgt cctgttgagc cgcggtgcca acagttctta

    11881 gatgaaatca ttaagtacac aatgcaagat gctctcttct tgaaatatta tctcaaaaat

    11941 gtgggtgctc aagaagactg tgttgatgaa cactttcaag agaaaatctt atcttcaatt

    12001 cagggcaatg aatttttaca tcaaatgttt ttctggtatg atctggctat tttaactcga

    12061 aggggtagat taaatcgagg aaactctaga tcaacatggt ttgttcatga tgatttaata

    12121 gacatcttag gctatgggga ctatgttttt tggaagatcc caatttcaat gttaccactg

    12181 aacacacaag gaatccccca tgctgctatg gactggtatc aggcatcagt attcaaagaa

    12241 gcggttcaag ggcatacaca cattgtttct gtttctactg ccgacgtctt gataatgtgc

    12301 aaagatttaa ttacatgtcg attcaacaca actctaatct caaaaatagc agagattgag

    12361 gatccagttt gttctgatta tcccaatttt aagattgtgt ctatgcttta ccagagcgga

    12421 gattacttac tctccatatt agggtctgat gggtataaaa ttattaagtt cctcgaacca

    12481 ttgtgcttgg ccaaaattca attatgctca aagtacactg agaggaaggg ccgattctta

    12541 acacaaatgc atttagctgt aaatcacacc ctagaagaaa ttacagaaat gcgtgcacta

    12601 aagccttcac aggctcaaaa gatccgtgaa ttccatagaa cattgataag gctggagatg

    12661 acgccacaac aactttgtga gctattttcc attcaaaaac actgggggca tcctgtgcta

    12721 catagtgaaa cagcaatcca aaaagttaaa aaacatgcta cggtgctaaa agcattacgc

    12781 cctatagtga ttttcgagac atactgtgtt tttaaatata gtattgccaa acattatttt

    12841 gatagtcaag gatcttggta cagtgttact tcagatagga atctaacacc gggtcttaat

    12901 tcttatatca aaagaaatca attccctccg ttgccaatga ttaaagaact actatgggaa

    12961 ttttaccacc ttgaccaccc tccacttttc tcaaccaaaa ttattagtga cttaagtatt

    13021 tttataaaag acagagctac cgcagtagaa aggacatgct gggatgcagt attcgagcct

    13081 aatgttctag gatataatcc acctcacaaa tttagtacta aacgtgtacc ggaacaattt

    13141 ttagagcaag aaaacttttc tattgagaat gttctttcct acgcacaaaa actcgagtat

    13201 ctactaccac aatatcggaa cttttctttc tcattgaaag agaaagagtt gaatgtaggt

    13261 agaaccttcg gaaaattgcc ttatccgact cgcaatgttc aaacactttg tgaagctctg

    13321 ttagctgatg gtcttgctaa agcatttcct agcaatatga tggtagttac ggaacgtgag

    13381 caaaaagaaa gcttattgca tcaagcatca tggcaccaca caagtgatga ttttggtgaa

    13441 catgccacag ttagagggag tagctttgta actgatttag agaaatacaa tcttgcattt

    13501 agatatgagt ttacagcacc ttttatagaa tattgcaacc gttgctatgg tgttaagaat

    13561 gtttttaatt ggatgcatta tacaatccca cagtgttata tgcatgtcag tgattattat

    13621 aatccaccac ataacctcac actggagaat cgagacaacc cccccgaagg gcctagttca

    13681 tacaggggtc atatgggagg gattgaagga ctgcaacaaa aactctggac aagtatttca

    13741 tgtgctcaaa tttctttagt tgaaattaag actggtttta agttacgctc agctgtgatg

    13801 ggtgacaatc agtgcattac tgttttatca gtcttcccct tagagactga cgcagacgag

    13861 caggaacaga gcgccgaaga caatgcagcg agggtggccg ccagcctagc aaaagttaca

    13921 agtgcctgtg gaatcttttt aaaacctgat gaaacatttg tacattcagg ttttatctat

    13981 tttggaaaaa aacaatattt gaatggggtc caattgcctc agtcccttaa aacggctaca

    14041 agaatggcac cattgtctga tgcaattttt gatgatcttc aagggaccct ggctagtata

    14101 ggcactgctt ttgagcgatc catctctgag acacgacata tctttccttg caggataacc

    14161 gcagctttcc atacgttttt ttcggtgaga atcttgcaat atcatcatct cgggttcaat

    14221 aaaggttttg accttggaca gttaacactc ggcaaacctc tggatttcgg aacaatatca

    14281 ttggcactag cggtaccgca ggtgcttgga gggttatcct tcttgaatcc tgagaaatgt

    14341 ttctaccgga atctaggaga tccagttacc tcaggcttat tccagttaaa aacttatctc

    14401 cgaatgattg agatggatga tttattctta cctttaattg cgaagaaccc tgggaactgc

    14461 actgccattg actttgtgct aaatcctagc ggattaaatg tccctgggtc gcaagactta

    14521 acttcatttc tgcgccagat tgtacgcagg accatcaccc taagtgcgaa aaacaaactt

    14581 attaatacct tatttcatgc gtcagctgac ttcgaagacg aaatggtttg taaatggcta

    14641 ttatcatcaa ctcctgttat gagtcgtttt gcggccgata tcttttcacg cacgccgagc

    14701 gggaagcgat tgcaaattct aggatacctg gaaggaacac gcacattatt agcctctaag

    14761 atcatcaaca ataatacaga gacaccggtt ttggacagac tgaggaaaat aacattgcaa

    14821 aggtggagcc tatggtttag ttatcttgat cattgtgata atatcctggc ggaggcttta

    14881 acccaaataa cttgcacagt tgatttagca cagattctga gggaatattc atgggctcat

    14941 attttagagg gaagacctct tattggagcc acactcccat gtatgattga gcaattcaaa

    15001 gtgttttggc tgaaacccta cgaacaatgt ccgcagtgtt caaatgcaaa gcaaccaggt

    15061 gggaaaccat tcgtgtcagt ggcagtcaag aaacatattg ttagtgcatg gccgaacgca

    15121 tcccgaataa gctggactat cggggatgga atcccataca ttggatcaag gacagaagat

    15181 aagataggac aacctgctat taaaccaaaa tgtccttccg cagccttaag agaggccatt

    15241 gaattggcgt cccgtttaac atgggtaact caaggcagtt cgaacagtga cttgctaata

    15301 aaaccatttt tggaagcacg agtaaattta agtgttcaag aaatacttca aatgacccct

    15361 tcacattact caggaaatat tgttcacagg tacaacgatc aatacagtcc tcattctttc

    15421 atggccaatc gtatgagtaa ttcagcaacg cgattgattg tttctacaaa cactttaggt

    15481 gagttttcag gaggtggcca gtctgcacgc gacagcaata ttattttcca gaatgttata

    15541 aattatgcag ttgcactgtt cgatattaaa tttagaaaca ctgaggctac agatatccaa

    15601 tataatcgtg ctcaccttca tctaactaag tgttgcaccc gggaagtacc agctcagtat

    15661 ttaacataca catctacatt ggatttagat ttaacaagat accgagaaaa cgaattgatt

    15721 tatgacagta atcctctaaa aggaggactc aattgcaata tctcattcga taatccattt

    15781 ttccaaggta aacggctgaa cattatagaa gatgatctta ttcgactgcc tcacttatct

    15841 ggatgggagc tagccaagac catcatgcaa tcaattattt cagatagcaa caattcatct

    15901 acagacccaa ttagcagtgg agaaacaaga tcattcacta cccatttctt aacttatccc

    15961 aagataggac ttctgtacag ttttggggcc tttgtaagtt attatcttgg caatacaatt

    16021 cttcggacta agaaattaac acttgacaat tttttatatt acttaactac tcaaattcat

    16081 aatctaccac atcgctcatt gcgaatactt aagccaacat tcaaacatgc aagcgttatg

    16141 tcacggttaa tgagtattga tcctcatttt tctatttaca taggcggtgc tgcaggtgac

    16201 agaggactct cagatgcggc caggttattt ttgagaacgt ccatttcatc ttttcttaca

    16261 tttgtaaaag aatggataat taatcgcgga acaattgtcc ctttatggat agtatatccg

    16321 ctagagggtc aaaacccaac acctgtgaat aattttctct atcagatcgt agaactgctg

    16381 gtgcatgatt catcaagaca acaggctttt aaaactacca taagtgatca tgtacatcct

    16441 cacgacaatc ttgtttacac atgtaagagt acagccagca atttcttcca tgcatcattg

    16501 gcgtactgga ggagcagaca cagaaacagc aaccgaaaat acttggcaag agactcttca

    16561 actggatcaa gcacaaacaa cagtgatggt catattgaga gaagtcaaga acaaaccacc

    16621 agagatccac atgatggcac tgaacggaat ctagtcctac aaatgagcca tgaaataaaa

    16681 agaacgacaa ttccacaaga aaacacgcac cagggtccgt cgttccagtc ctttctaagt

    16741 gactctgctt gtggtacagc aaatccaaaa ctaaatttcg atcgatcgag acacaatgtg

    16801 aaatttcagg atcataactc ggcatccaag agggaaggtc atcaaataat ctcacaccgt

    16861 ctagtcctac ctttctttac attatctcaa gggacacgcc aattaacgtc atccaatgag

    16921 tcacaaaccc aagacgagat atcaaagtac ttacggcaat tgagatccgt cattgatacc

    16981 acagtttatt gtagatttac cggtatagtc tcgtccatgc attacaaact tgatgaggtc

    17041 ctttgggaaa tagagagttt caagtcggct gtgacgctag cagagggaga aggtgctggt

    17101 gccttactat tgattcagaa ataccaagtt aagaccttat ttttcaacac gctagctact

    17161 gagtccagta tagagtcaga aatagtatca ggaatgacta ctcctaggat gcttctacct

    17221 gttatgtcaa aattccataa tgaccaaatt gagattattc ttaacaactc agcaagccaa

    17281 ataacagaca taacaaatcc tacttggttt aaagaccaaa gagcaaggct acctaagcaa

    17341 gtcgaggtta taaccatgga tgcagagaca acagagaata taaacagatc gaaattgtac

    17401 gaagctgtat ataaattgat cttacaccat attgatccta gcgtattgaa agcagtggtc

    17461 cttaaagtct ttctaagtga tactgagggt atgttatggc taaatgataa tttagccccg

    17521 ttttttgcca ctggttattt aattaagcca ataacgtcaa gtgctagatc tagtgagtgg

    17581 tatctttgtc tgacgaactt cttatcaact acacgtaaga tgccacacca aaaccatctc

    17641 agttgtaaac aggtaatact tacggcattg caactgcaaa ttcaacgaag cccatactgg

    17701 ctaagtcatt taactcagta tgctgactgt gagttacatt taagttatat ccgccttggt

    17761 tttccatcat tagagaaagt actataccac aggtataacc tcgtcgattc aaaaagaggt

    17821 ccactagtct ctatcactca gcacttagca catcttagag cagagattcg agaattaact

    17881 aatgattata atcaacagcg acaaagtcgg actcaaacat atcactttat tcgtactgca

    17941 aaaggacgaa tcacaaaact agtcaatgat tatttaaaat tctttcttat tgtgcaagca

    18001 ttaaaacata atgggacatg gcaagctgag tttaagaaat taccagagtt gattagtgtg

    18061 tgcaataggt tctaccatat tagagattgc aattgtgaag aacgtttctt agttcaaacc

    18121 ttatatttac atagaatgca ggattctgaa gttaagctta tcgaaaggct gacagggctt

    18181 ctgagtttat ttccggatgg tctctacagg tttgattgaa ttaccgtgca tagtatcctg

    18241 atacttgcaa aggttggtta ttaacataca gattataaaa aactcataaa ttgctctcat

    18301 acatcatatt gatctaatct caataaacaa ctatttaaat aacgaaagga gtccctatat

    18361 tatatactat atttagcctc tctccctgcg tgataatcaa aaaattcaca atgcagcatg

    18421 tgtgacatat tactgccgca atgaatttaa cgcaacataa taaactctgc actctttata

    18481 attaagcttt aacgaaaggt ctgggctcat attgttattg atataataat gttgtatcaa

    18541 tatcctgtca gatggaatag tgttttggtt gataacacaa cttcttaaaa caaaattgat

    18601 ctttaagatt aagtttttta taattatcat tactttaatt tgtcgtttta aaaacggtga

    18661 tagccttaat ctttgtgtaa aataagagat taggtgtaat aaccttaaca tttttgtcta

    18721 gtaagctact atttcataca gaatgataaa attaaaagaa aaggcaggac tgtaaaatca

    18781 gaaatacctt ctttacaata tagcagacta gataataatc ttcgtgttaa tgataattaa

    18841 gacattgacc acgctcatca gaaggctcgc cagaataaac gttgcaaaaa ggattcctgg

    18901 aaaaatggtc gcacacaaaa atttaaaaat aaatctattt cttctttttt gtgtgtcca

//Ebola Genome

Zaire ebolavirus isolate Ebola virus H.sapiens-tc/COD/1976/Yambuku-Mayinga, complete genome. NCBI Reference Sequence: NC_002549.1. 

http://www.ncbi.nlm.nih.gov/nuccore/10313991?report=graph

LOCUS       NC_002549              18959 bp    cRNA    linear   VRL 27-AUG-2014

DEFINITION  Zaire ebolavirus isolate Ebola virus

            H.sapiens-tc/COD/1976/Yambuku-Mayinga, complete genome.

ACCESSION   NC_002549

VERSION     NC_002549.1  GI:10313991

DBLINK      BioProject: PRJNA14703

KEYWORDS    RefSeq.

SOURCE      Zaire ebolavirus (ZEBOV)

  ORGANISM  Zaire ebolavirus

            Viruses; ssRNA negative-strand viruses; Mononegavirales;

            Filoviridae; Ebolavirus.

REFERENCE   1  (bases 1 to 18959)

  AUTHORS   Volchkov,V.E., Volchkova,V.A., Chepurnov,A.A., Blinov,V.M.,

            Dolnik,O., Netesov,S.V. and Feldmann,H.

  TITLE     Characterization of the L gene and 5' trailer region of Ebola virus

  JOURNAL   J. Gen. Virol. 80 (Pt 2), 355-362 (1999)

   PUBMED   10073695

REFERENCE   2  (bases 1 to 18959)

  AUTHORS   Volchkov,V.E., Volchkova,V.A., Slenczka,W., Klenk,H.D. and

            Feldmann,H.

  TITLE     Release of viral glycoproteins during Ebola virus infection

  JOURNAL   Virology 245 (1), 110-119 (1998)

   PUBMED   9614872

REFERENCE   3  (bases 1 to 18959)

  AUTHORS   Volchkov,V.E., Feldmann,H., Volchkova,V.A. and Klenk,H.D.

  TITLE     Processing of the Ebola virus glycoprotein by the proprotein

            convertase furin

  JOURNAL   Proc. Natl. Acad. Sci. U.S.A. 95 (10), 5762-5767 (1998)

   PUBMED   9576958

REFERENCE   4  (bases 1 to 18959)

  AUTHORS   Volchkov,V.E., Becker,S., Volchkova,V.A., Ternovoj,V.A.,

            Kotov,A.N., Netesov,S.V. and Klenk,H.D.

  TITLE     GP mRNA of Ebola virus is edited by the Ebola virus polymerase and

            by T7 and vaccinia virus polymerases

  JOURNAL   Virology 214 (2), 421-430 (1995)

   PUBMED   8553543

REFERENCE   5  (bases 1 to 18959)

  AUTHORS   Bukreyev,A.A., Volchkov,V.E., Blinov,V.M. and Netesov,S.V.

  TITLE     The VP35 and VP40 proteins of filoviruses. Homology between Marburg

            and Ebola viruses

  JOURNAL   FEBS Lett. 322 (1), 41-46 (1993)

   PUBMED   8482365

REFERENCE   6  (bases 1 to 18959)

  CONSRTM   NCBI Genome Project

  TITLE     Direct Submission

  JOURNAL   Submitted (27-SEP-2000) National Center for Biotechnology

            Information, NIH, Bethesda, MD 20894, USA

REFERENCE   7  (bases 1 to 18959)

  AUTHORS   Volchkov,V.E.

  TITLE     Direct Submission

  JOURNAL   Submitted (02-JUN-2000) Institute of Virology, Philipps-University

            Marburg, Robert-Koch-Str. 17, Marburg 35037, Germany

  REMARK    Sequence update by submitter

REFERENCE   8  (bases 1 to 18959)

  AUTHORS   Volchkov,V.E.

  TITLE     Direct Submission

  JOURNAL   Submitted (20-AUG-1998) Institute of Virology, Philipps-University

            Marburg, Robert-Koch-Str. 17, Marburg 35037, Germany

COMMENT     PROVISIONAL REFSEQ: This record has not yet been subject to final

            NCBI review. The reference sequence is identical to AF086833.

            COMPLETENESS: full length.

FEATURES             Location/Qualifiers

     source          1..18959

                     /organism="Zaire ebolavirus"

                     /mol_type="viral cRNA"

                     /isolate="Ebola virus

                     H.sapiens-tc/COD/1976/Yambuku-Mayinga"

                     /db_xref="taxon:186538"

     5'UTR           1..55

                     /note="putative leader region"

                     /citation=[1]

                     /function="regulation or initiation of RNA replication"

     gene            56..3026

                     /gene="NP"

                     /locus_tag="ZEBOVgp1"

                     /db_xref="GeneID:911830"

     mRNA            56..3026

                     /gene="NP"

                     /locus_tag="ZEBOVgp1"

                     /product="nucleoprotein"

                     /db_xref="GeneID:911830"

     misc_signal     56..67

                     /gene="NP"

                     /locus_tag="ZEBOVgp1"

                     /note="putative; transcription start signal"

                     /citation=[1]

     CDS             470..2689

                     /gene="NP"

                     /locus_tag="ZEBOVgp1"

                     /function="encapsidation of genomic RNA"

                     /codon_start=1

                     /product="nucleoprotein"

                     /protein_id="NP_066243.1"

                     /db_xref="GI:10314000"

                     /db_xref="GeneID:911830"

                     /translation="MDSRPQKIWMAPSLTESDMDYHKILTAGLSVQQGIVRQRVIPVY

                     QVNNLEEICQLIIQAFEAGVDFQESADSFLLMLCLHHAYQGDYKLFLESGAVKYLEGH

                     GFRFEVKKRDGVKRLEELLPAVSSGKNIKRTLAAMPEEETTEANAGQFLSFASLFLPK

                     LVVGEKACLEKVQRQIQVHAEQGLIQYPTAWQSVGHMMVIFRLMRTNFLIKFLLIHQG

                     MHMVAGHDANDAVISNSVAQARFSGLLIVKTVLDHILQKTERGVRLHPLARTAKVKNE

                     VNSFKAALSSLAKHGEYAPFARLLNLSGVNNLEHGLFPQLSAIALGVATAHGSTLAGV

                     NVGEQYQQLREAATEAEKQLQQYAESRELDHLGLDDQEKKILMNFHQKKNEISFQQTN

                     AMVTLRKERLAKLTEAITAASLPKTSGHYDDDDDIPFPGPINDDDNPGHQDDDPTDSQ

                    DTTIPDVVVDPDDGSYGEYQSYSENGMNAPDDLVLFDLDEDDEDTKPVPNRSTKGGQQ

                     KNSQKGQHIEGRQTQSRPIQNVPGPHRTIHHASAPLTDNDRRNEPSGSTSPRMLTPIN

                     EEADPLDDADDETSSLPPLESDDEEQDRDGTSNRTPTVAPPAPVYRDHSEKKELPQDE

                     QQDQDHTQEARNQDSDNTQSEHSFEEMYRHILRSQGPFDAVLYYHMMKDEPVVFSTSD

                     GKEYTYPDSLEEEYPPWLTEKEAMNEENRFVTLDGQQFYWPVMNHKNKFMAILQHHQ"

     misc_feature    524..2671

                     /gene="NP"

                     /locus_tag="ZEBOVgp1"

                     /note="Ebola nucleoprotein; Region: Ebola_NP; pfam05505"

                     /db_xref="CDD:147601"

     polyA_signal    3015..3026

                     /gene="NP"

                     /locus_tag="ZEBOVgp1"

     misc_feature    3027..3031

                     /note="intergenic region"

     gene            3032..4407

                     /gene="VP35"

                     /locus_tag="ZEBOVgp2"

                     /db_xref="GeneID:911827"

     mRNA            3032..4407

                     /gene="VP35"

                     /locus_tag="ZEBOVgp2"

                     /product="VP35"

                     /citation=[5]

                     /db_xref="GeneID:911827"

     misc_signal     3032..3043

                     /gene="VP35"

                     /locus_tag="ZEBOVgp2"

                     /note="putative; transcription start signal"

                     /citation=[5]

     CDS             3129..4151

                     /gene="VP35"

                     /locus_tag="ZEBOVgp2"

                     /function="polymerase complex protein"

                     /citation=[5]

                     /codon_start=1

                     /product="polymerase complex protein"

                     /protein_id="NP_066244.1"

                     /db_xref="GI:10313992"

                     /db_xref="GeneID:911827"

                     /translation="MTTRTKGRGHTAATTQNDRMPGPELSGWISEQLMTGRIPVSDIF

                     CDIENNPGLCYASQMQQTKPNPKTRNSQTQTDPICNHSFEEVVQTLASLATVVQQQTI

                     ASESLEQRITSLENGLKPVYDMAKTISSLNRVCAEMVAKYDLLVMTTGRATATAAATE

                     AYWAEHGQPPPGPSLYEESAIRGKIESRDETVPQSVREAFNNLNSTTSLTEENFGKPD

                     ISAKDLRNIMYDHLPGFGTAFHQLVQVICKLGKDSNSLDIIHAEFQASLAEGDSPQCA

                     LIQITKRVPIFQDAAPPVIHIRSRGDIPRACQKSLRPVPPSPKIDRGWVCVFQLQDGK

                     TLGLKI"

     misc_feature    3186..4148

                     /gene="VP35"

                     /locus_tag="ZEBOVgp2"

                     /note="Filoviridae VP35; Region: Filo_VP35; pfam02097"

                     /db_xref="CDD:145320"

     gene            4390..5894

                     /gene="VP40"

                     /locus_tag="ZEBOVgp3"

                     /db_xref="GeneID:911825"

     mRNA            4390..5894

                     /gene="VP40"

                     /locus_tag="ZEBOVgp3"

                     /product="VP40"

                     /citation=[5]

                     /db_xref="GeneID:911825"

     misc_signal     4390..4401

                     /gene="VP40"

                     /locus_tag="ZEBOVgp3"

                     /note="transcription start signal"

                     /citation=[5]

     polyA_signal    4397..4407

                     /gene="VP35"

                     /locus_tag="ZEBOVgp2"

                     /citation=[5]

     CDS             4479..5459

                     /gene="VP40"

                     /locus_tag="ZEBOVgp3"

                     /citation=[5]

                     /codon_start=1

                     /product="matrix protein"

                     /protein_id="NP_066245.1"

                     /db_xref="GI:10313993"

                     /db_xref="GeneID:911825"

                     /translation="MRRVILPTAPPEYMEAIYPVRSNSTIARGGNSNTGFLTPESVNG

                     DTPSNPLRPIADDTIDHASHTPGSVSSAFILEAMVNVISGPKVLMKQIPIWLPLGVAD

                     QKTYSFDSTTAAIMLASYTITHFGKATNPLVRVNRLGPGIPDHPLRLLRIGNQAFLQE

                     FVLPPVQLPQYFTFDLTALKLITQPLPAATWTDDTPTGSNGALRPGISFHPKLRPILL

                     PNKSGKKGNSADLTSPEKIQAIMTSLQDFKIVPIDPTKNIMGIEVPETLVHKLTGKKV

                     TSKNGQPIIPVLLPKYIGLDPVAPGDLTMVITQDCDTCHSPASLPAVIEK"

     misc_feature    4479..5363

                     /gene="VP40"

                     /locus_tag="ZEBOVgp3"

                     /note="Matrix protein VP40; Region: VP40; pfam07447"

                     /db_xref="CDD:116068"

     polyA_signal    5883..5894

                     /gene="VP40"

                    /locus_tag="ZEBOVgp3"

                     /citation=[5]

     misc_feature    5895..5899

                     /note="intergenic region"

     gene            5900..8305

                     /gene="GP"

                     /locus_tag="ZEBOVgp4"

                     /db_xref="GeneID:911829"

     mRNA            5900..8305

                     /gene="GP"

                     /locus_tag="ZEBOVgp4"

                     /product="sGP"

                     /note="unedited mRNA"

                     /citation=[4]

                     /db_xref="GeneID:911829"

     misc_signal     5900..5911

                     /gene="GP"

                     /locus_tag="ZEBOVgp4"

                     /note="putative; transcription start signal"

                     /citation=[4]

     CDS             join(6039..6923,6923..8068)

                     /gene="GP"

                     /locus_tag="ZEBOVgp4"

                     /function="receptor binding and fusion"

                     /artificial_location="low-quality sequence region"

                     /note="virion spike glycoprotein precursor; an addition A

                     residue is inserted during transcription; encodes two

                     disulfide linked subunits GP1 and GP2"

                     /citation=[2]

                     /citation=[3]

                     /citation=[4]

                     /codon_start=1

                     /product="spike glycoprotein"

                     /protein_id="NP_066246.1"

                     /db_xref="GI:10313995"

                     /db_xref="GeneID:911829"

                     /translation="MGVTGILQLPRDRFKRTSFFLWVIILFQRTFSIPLGVIHNSTLQ

                     VSDVDKLVCRDKLSSTNQLRSVGLNLEGNGVATDVPSATKRWGFRSGVPPKVVNYEAG

                     EWAENCYNLEIKKPDGSECLPAAPDGIRGFPRCRYVHKVSGTGPCAGDFAFHKEGAFF

                     LYDRLASTVIYRGTTFAEGVVAFLILPQAKKDFFSSHPLREPVNATEDPSSGYYSTTI

                     RYQATGFGTNETEYLFEVDNLTYVQLESRFTPQFLLQLNETIYTSGKRSNTTGKLIWK

                     VNPEIDTTIGEWAFWETKKNLTRKIRSEELSFTVVSNGAKNISGQSPARTSSDPGTNT

                     TTEDHKIMASENSSAMVQVHSQGREAAVSHLTTLATISTSPQSLTTKPGPDNSTHNTP

                     VYKLDISEATQVEQHHRRTDNDSTASDTPSATTAAGPPKAENTNTSKSTDFLDPATTT

                     SPQNHSETAGNNNTHHQDTGEESASSGKLGLITNTIAGVAGLITGGRRTRREAIVNAQ

                     PKCNPNLHYWTTQDEGAAIGLAWIPYFGPAAEGIYIEGLMHNQDGLICGLRQLANETT

                     QALQLFLRATTELRTFSILNRKAIDFLLQRWGGTCHILGPDCCIEPHDWTKNITDKID

                     QIIHDFVDKTLPDQGDNDNWWTGWRQWIPAGIGVTGVIIAVIALFCICKFVF"

     misc_feature    7529..7540

                     /gene="GP"

                     /locus_tag="ZEBOVgp4"

                     /note="encodes the glycoprotein cleavage site, precursor

                     GP is cleaved by subtilisin-like cellular protease furin

                     into subunits GP1 and GP2 that are linked by a disulfide

                     bond"

                     /citation=[3]

     misc_feature    7793..7870

                     /gene="GP"

                     /locus_tag="ZEBOVgp4"

                     /note="immunosuppressive motif; other site"

     misc_feature    7988..8053

                     /gene="GP"

                     /locus_tag="ZEBOVgp4"

                     /note="transmembrane anchor; transmembrane region"

     misc_feature    7706..7924

                     /gene="GP"

                     /locus_tag="ZEBOVgp4"

                     /note="heptad repeat 1-heptad repeat 2 region of the

                     transmembrane subunit of Filoviridae viruses, Ebola virus

                     and Marburg virus, and related domains; Region:

                     Ebola-like_HR1-HR2; cd09850"

                     /db_xref="CDD:197367"

     misc_feature    join(6081..6923,6923..7153)

                     /gene="GP"

                     /locus_tag="ZEBOVgp4"

                     /note="Filovirus glycoprotein; Region: Filo_glycop;

                     pfam01611"

                     /db_xref="CDD:110602"

     misc_feature    7706..7732

                     /gene="GP"

                     /locus_tag="ZEBOVgp4"

                     /note="HR1A; other site"

                     /db_xref="CDD:197367"

     misc_feature    7733..7762

                     /gene="GP"

                     /locus_tag="ZEBOVgp4"

                     /note="HR1B; other site"

                     /db_xref="CDD:197367"

     misc_feature    7763..7783

                     /gene="GP"

                     /locus_tag="ZEBOVgp4"

                     /note="HR1C; other site"

                     /db_xref="CDD:197367"

     misc_feature    7784..7831

                     /gene="GP"

                     /locus_tag="ZEBOVgp4"

                     /note="HR1D; other site"

                     /db_xref="CDD:197367"

     misc_feature    7787..7837

                     /gene="GP"

                     /locus_tag="ZEBOVgp4"

                     /note="immunosuppressive region; other site"

                     /db_xref="CDD:197367"

     misc_feature    order(7838..7858,7859..7861)

                     /gene="GP"

                     /locus_tag="ZEBOVgp4"

                     /note="CX(6,7)C motif; other site"

                     /db_xref="CDD:197367"

     misc_feature    7886..7924

                     /gene="GP"

                     /locus_tag="ZEBOVgp4"

                     /note="HR2; other site"

                     /db_xref="CDD:197367"

     misc_feature    order(7784..7786,7793..7795)

                     /gene="GP"

                     /locus_tag="ZEBOVgp4"

                     /note="Cl binding site [ion binding]; other site"

                     /db_xref="CDD:197367"

     misc_feature    order(7706..7714,7718..7723,7727..7732,7736..7744,

                     7748..7756,7760..7765,7769..7777,7781..7807,7811..7819,

                     7823..7828,7844..7849,7856..7858,7865..7876,7880..7882,

                     7889..7894,7901..7903,7910..7915,7922..7924)

                     /gene="GP"

                     /locus_tag="ZEBOVgp4"

                     /note="homotrimer interface [polypeptide binding]; other

                     site"

                     /db_xref="CDD:197367"

     misc_feature    order(7706..7714,7718..7726,7730..7735,7739..7747,

                     7754..7768,7772..7783,7787..7792,7796..7804,7808..7813,

                     7817..7819)

                     /gene="GP"

                     /locus_tag="ZEBOVgp4"

                     /note="HR1-GP1 interface [polypeptide binding]; other

                     site"

                     /db_xref="CDD:197367"

     CDS             6039..7133

                     /gene="GP"

                     /locus_tag="ZEBOVgp4"

                     /note="sGP, small non-structural, secreted glycoprotein;

                     sGP secreted as a anti-parallel oriented homodimer"

                     /citation=[4]

                     /codon_start=1

                     /product="small secreted glycoprotein"

                     /protein_id="NP_066247.1"

                     /db_xref="GI:10313994"

                     /db_xref="GeneID:911829"

                     /translation="MGVTGILQLPRDRFKRTSFFLWVIILFQRTFSIPLGVIHNSTLQ

                     VSDVDKLVCRDKLSSTNQLRSVGLNLEGNGVATDVPSATKRWGFRSGVPPKVVNYEAG

                     EWAENCYNLEIKKPDGSECLPAAPDGIRGFPRCRYVHKVSGTGPCAGDFAFHKEGAFF

                     LYDRLASTVIYRGTTFAEGVVAFLILPQAKKDFFSSHPLREPVNATEDPSSGYYSTTI

                     RYQATGFGTNETEYLFEVDNLTYVQLESRFTPQFLLQLNETIYTSGKRSNTTGKLIWK

                     VNPEIDTTIGEWAFWETKKTSLEKFAVKSCLSQLYQTEPKTSVVRVRRELLPTQGPTQ

                     QLKTTKSWLQKIPLQWFKCTVKEGKLQCRI"

     misc_feature    6081..7130

                     /gene="GP"

                     /locus_tag="ZEBOVgp4"

                     /note="Filovirus glycoprotein; Region: Filo_glycop;

                     pfam01611"

                     /db_xref="CDD:110602"

     CDS             join(6039..6922,6924..6933)

                     /gene="GP"

                     /locus_tag="ZEBOVgp4"

                     /artificial_location="low-quality sequence region"

                     /note="ssGP; second non-structural secreted glycoprotein;

                     secreted in a monomeric form; one A residue is deleted or

                     two additional A residues are inserted at the editing site

                     during transcription of the GP gene"

                     /citation=[4]

                     /codon_start=1

                     /product="second secreted glycoprotein"

                     /protein_id="NP_066248.1"

                     /db_xref="GI:10313996"

                     /db_xref="GeneID:911829"

                     /translation="MGVTGILQLPRDRFKRTSFFLWVIILFQRTFSIPLGVIHNSTLQ

                     VSDVDKLVCRDKLSSTNQLRSVGLNLEGNGVATDVPSATKRWGFRSGVPPKVVNYEAG

                     EWAENCYNLEIKKPDGSECLPAAPDGIRGFPRCRYVHKVSGTGPCAGDFAFHKEGAFF

                     LYDRLASTVIYRGTTFAEGVVAFLILPQAKKDFFSSHPLREPVNATEDPSSGYYSTTI

                     RYQATGFGTNETEYLFEVDNLTYVQLESRFTPQFLLQLNETIYTSGKRSNTTGKLIWK

                     VNPEIDTTIGEWAFWETKKPH"

     misc_feature    join(6081..6922,6924..>6924)

                     /gene="GP"

                     /locus_tag="ZEBOVgp4"

                     /note="Filovirus glycoprotein; Region: Filo_glycop;

                     pfam01611"

                     /db_xref="CDD:110602"

     misc_signal     6918..6924

                     /gene="GP"

                     /locus_tag="ZEBOVgp4"

                     /note="additional A residues are inserted or deleted

                     during transcription of the GP gene by the viral

                     polymerase"

                     /citation=[4]

                     /function="RNA editing"

     gene            8288..9740

                     /gene="VP30"

                     /locus_tag="ZEBOVgp5"

                     /db_xref="GeneID:911826"

     mRNA            8288..9740

                     /gene="VP30"

                     /locus_tag="ZEBOVgp5"

                     /product="VP30"

                     /db_xref="GeneID:911826"

     misc_signal     8288..8299

                     /gene="VP30"

                     /locus_tag="ZEBOVgp5"

                     /note="putative; transcription start signal"

     polyA_signal    8295..8305

                     /gene="GP"

                     /locus_tag="ZEBOVgp4"

                     /citation=[4]

     CDS             8509..9375

                     /gene="VP30"

                     /locus_tag="ZEBOVgp5"

                     /note="polymerase complex protein"

                     /codon_start=1

                     /product="minor nucleoprotein"

                     /protein_id="NP_066249.1"

                     /db_xref="GI:10313997"

                     /db_xref="GeneID:911826"

                     /translation="MEASYERGRPRAARQHSRDGHDHHVRARSSSRENYRGEYRQSRS

                     ASQVRVPTVFHKKRVEPLTVPPAPKDICPTLKKGFLCDSSFCKKDHQLESLTDRELLL

                     LIARKTCGSVEQQLNITAPKDSRLANPTADDFQQEEGPKITLLTLIKTAEHWARQDIR

                     TIEDSKLRALLTLCAVMTRKFSKSQLSLLCETHLRREGLGQDQAEPVLEVYQRLHSDK

                     GGSFEAALWQQWDRQSLIMFITAFLNIALQLPCESSAVVVSGLRTLVPQSDNEEASTN

                     PGTCSWSDEGTP"

     misc_feature    8932..9321

                     /gene="VP30"

                     /locus_tag="ZEBOVgp5"

                     /note="Ebola virus-specific transcription factor VP30;

                     Region: Transcript_VP30; pfam11507"

                     /db_xref="CDD:151944"

     polyA_signal    9730..9740

                     /gene="VP30"

                     /locus_tag="ZEBOVgp5"

                     /note="putative"

     misc_feature    9741..9884

                     /note="intergenic region"

     gene            9885..11518

                     /gene="VP24"

                     /locus_tag="ZEBOVgp6"

                     /note="putative"

                     /db_xref="GeneID:911828"

     mRNA            9885..11496

                     /gene="VP24"

                     /locus_tag="ZEBOVgp6"

                     /product="VP24"

                     /db_xref="GeneID:911828"

     misc_signal     9885..9896

                     /gene="VP24"

                     /locus_tag="ZEBOVgp6"

                     /note="transcription start signal"

     CDS             10345..11100

                     /gene="VP24"

                     /locus_tag="ZEBOVgp6"

                     /codon_start=1

                     /product="membrane-associated protein"

                     /protein_id="NP_066250.1"

                     /db_xref="GI:10313998"

                     /db_xref="GeneID:911828"

                     /translation="MAKATGRYNLISPKKDLEKGVVLSDLCNFLVSQTIQGWKVYWAG

                     IEFDVTHKGMALLHRLKTNDFAPAWSMTRNLFPHLFQNPNSTIESPLWALRVILAAGI

                     QDQLIDQSLIEPLAGALGLISDWLLTTNTNHFNMRTQRVKEQLSLKMLSLIRSNILKF

                     INKLDALHVVNYNGLLSSIEIGTQNHTIIITRTNMGFLVELQEPDKSAMNRMKPGPAK

                     FSLLHESTLKAFTQGSSTRMQSLILEFNSSLAI"

     misc_feature    10369..11040

                     /gene="VP24"

                     /locus_tag="ZEBOVgp6"

                     /note="Filovirus membrane-associated protein VP24; Region:

                     Filo_VP24; pfam06389"

                     /db_xref="CDD:253701"