Viral fusion proteins that have been studied in more detail, e.g. the HA protein of influenza A virus or the gp120/gp41 of HIV, are known to undergo a conformational change prior to the fusion reaction (Skehel and Wiley, 2000; Weissenhorn et al., 1999).
This intermolecular rearrangement that makes the protein fusion-active is induced in influenza virus by the low pH encountered within endosomes upon internalisation of the virus. HIV does not require endocytotic uptake. Following attachment to CD4 receptors the interaction with members of the family of chemokine receptors triggers the conformational change that makes gp41 fusion-active (Furuta et al., 1998) . Sequential interaction with cell surface molecules in the initiation of infection has also been reported for members of the herpesvirus family, herpes simplex-1 (HSV-1) and pseudorabies.
With these viruses, attachment is mediated by binding of the viral surface glycoprotein gC to cell surface heparan sulphate proteoglycans (WuDunn and Spear, 1989). Virus entry, i.e. fusion of the viral membrane with the plasma membrane, requires the interaction of the viral glycoprotein gD with a member of the nectin family or an
alternative cell surface receptor (Krummenacher et al., 1998). A conformational change is also expected to render the F protein of RSV fusion active. In theory, interaction with GAG may provide such a stimulus. However, it is also possible that as in the case of herpesviruses, interaction with GAG only mediates attachment and that subsequent interaction with a protein receptor is required to induce the fusion of the viral membrane with the plasma membrane.
80
7 Summary
Characterization of the heparin-binding activity of the bovine respiratory syncytial virus fusion protein
Diana Panayotova
Cell surface glycosaminoglycans (GAGs) are a major factor for respiratory syncytial virus (RSV) attachment to cultured cells leading to infection. The viral glycoprotein G binds to GAGs and was thought to be the only viral attachment protein.
Recently, mutant virus lacking the G protein was shown to be infectious in cell culture suggesting that the F protein, which has long been known for its fusion activity has also receptor binding activities. A linear heparin-binding domain has been identified in the G protein. This domain is characterized by clusters of basic amino acids that are supposed to be important for the interaction with the negatively charged groups of GAGs. The respective domains in the F protein responsible for the interaction with GAGs are not known so far.
The present study was based on the working hypothesis that basic amino acids concentrated in a short domain located in the BRSV fusion protein F2 subunit are involved in heparin-binding. The experimental work was designed to provide a better understanding of the interaction between BRSV F protein and heparin-like structures.
Recombinant BRSV with mutations in the putative-heparin binding domain of the F2 subunit were analyzed. The significance of these mutations with respect to virus infectivity and heparin binding were investigated.
The following results were obtained:
First, 13 recombinant viruses containing point mutations in the F2 subunit of the F protein were generated and characterized with respect to replication and syncytia formation.
Second, an essential role of lysine K75 in F protein function was demonstrated.
Analysis of transfected cells revealed that this mutation do not affect either proteolytic activation, or cell surface transport of the F protein, but had an effect on fusion activity.
Like K75N, no virus could be recovered from mutant K77N. The reason for this is unclear.
Third, inhibition of BRSV infection indicated that the basic amino acids K63, K66, K80, and R85 have modulating effects on virus infectivity.
Fourth, it was demonstrated that even higher concentrations of soluble heparin did not completely abolish virus infection suggesting the existence of additional cellular receptors which are involved in viral attachment.
82
8 Zusammenfassung
Charakterisierung der Heparin-Bindungsaktivität des Fusionsproteins des bovinen respiratorischen Synzytialvirus
Diana Panayotova
Glukosaminoglykane (GAG) auf der Zelloberfläche sind ein wichtiger Faktor für die Bindung des respiratorischen Synzytialvirus (RSV) an Zellen bei der Einleitung einer Infektion. Das virale Glykoprotein G bindet an GAG und galt früher als alleiniges Anheftungsprotein. Kürzlich wurde gezeigt, dass Virusmutanten, denen das G-Protein fehlt, Zellkulturen infizieren können. Daraus folgt, dass das F-Protein neben seiner bekannten Fusionsaktivität auch über Rezeptor-bindende Aktivität verfügt. Auf dem G-Protein ist eine lineare Heparin-bindende Domäne identifiziert worden. Dieser Proteinabschnitt ist gekennzeichnet durch eine Anhäufung basischer Aminosäuren, von denen man annimmt, dass sie für die Wechselwirkung mit den negativ geladenen Gruppen von GAG wichtig sind. Eine entsprechende für die Wechselwirkung mit GAG verantwortliche Domäne war auf dem F-Protein bislang nicht bekannt.
Die vorliegende Studie basiert auf der Arbeitshypothese, dass basische Aminosäuren, die in einem kurzen Abschnitt der F2-Untereinheit gehäuft auftreten, an der Heparin-Bindung beteiligt sind. Die experimentelle Arbeit war darauf ausgerichtet, ein besseres Verständnis der Wechselwirkung zwischen dem BRSV-F-Protein und Heparin-ähnlichen Strukturen zu vermitteln. Rekombinantes BRSV mit Mutationen in der mutmaßlichen Heparin-Bindungsdomäne der F2-Untereinheit wurde analysiert. Die Bedeutung dieser Mutationen hinsichtlich Infektiosität und Heparin-Bindung wurde untersucht.
Folgende Ergebnisse wurden erhalten:
1. Dreizehn rekombinante Viren mit Punktmutationen in der F2-Untereinheit des F-Proteins wurden erzeugt und hinsichtlich Replikation und Synzytium-Bildung charakterisiert.
2. Für die Lysine K75 und K77 wurde eine essentielle Rolle für die Funktion des F-Proteins nachgewiesen. Die Analyse transfizierter Zellen zeigte, dass diese Mutationen weder die proteolytische Aktivierung noch den Oberflächentransport des F-Proteins beeinflussen, wohl aber die Fusionsaktivität (K75). Rekombinantes Virus mit diesen Mutationen konnte nicht isoliert werden.
3. Die Hemmung der BRSV-Infektion zeigte, dass die basischen Aminosäuren K63, K66, K80 und R85 einen modulierenden Effekt auf die Virusinfektiosität haben.
4. Es wurde gezeigt, dass selbst hohe Konzentrationen löslichen Heparins die Virusinfektion nicht völlig verhinderten. Dieses Ergebnis spricht für die Existenz eines zusätzlichen zellulären Rezeptors, der an der Virusbindung beteiligt ist.
9 References
• Baker, J. C., J. A. Ellis, and E. G. Clark. 1997. Bovine respiratory syncytial virus. Vet. Clin. North Am. Food Anim. Pract. 13:425-454.
• Barik, S. 1992. Transcription of human respiratory syncytial virus genome RNA in vitro: requirement of cellular factor(s). J. Virol. 66:6813-6818.
• Bermingham, A. and P. L. Collins. 1999. The M2-2 protein of human respiratory syncytial virus is a regulatory factor involved in the balance between RNA replication and transcription. Proc. Natl. Acad. Sci. U. S. A 96:11259-11264.
• Berthiaume, L., J. Joncas, and V. Pavilanis. 1974. Comparative structure, morphogenesis and biological characteristics of the respiratory syncytial (RS) virus and the pneumonia virus of mice (PVM). Arch. Gesamte Virusforsch. 45:39-51.
• Bossert, B. and K. K. Conzelmann. 2002. Respiratory syncytial virus (RSV) nonstructural (NS) proteins as host range determinants: a chimeric bovine RSV with NS genes from human RSV is attenuated in interferon-competent bovine cells. J. Virol. 76:4287-4293.
• Bourgeois, C., J. B. Bour, K. Lidholt, C. Gauthray, and P. Pothier. 1998.
Heparin-like structures on respiratory syncytial virus are involved in its infectivity in vitro. J. Virol. 72:7221-7227.
• Bowland, S. L. and P. E. Shewen. 2000. Bovine respiratory disease:
commercial vaccines currently available in Canada. Can. Vet. J. 41:33-48.
• Bryson, D. G., J. B. McFerran, H. J. Ball, and S. D. Neill. 1978.
Observations on outbreaks of respiratory disease in housed calves--(1) Epidemiological, clinical and microbiological findings. Vet. Rec. 103:485-489.
• Buchholz, U. J., S. Finke, and K. K. Conzelmann. 1999. Generation of bovine respiratory syncytial virus (BRSV) from cDNA: BRSV NS2 is not
essential for virus replication in tissue culture, and the human RSV leader region acts as a functional BRSV genome promoter. J. Virol. 73:251-259.
• Bukreyev, A., S. S. Whitehead, B. R. Murphy, and P. L. Collins. 1997.
Recombinant respiratory syncytial virus from which the entire SH gene has been deleted grows efficiently in cell culture and exhibits site-specific attenuation in the respiratory tract of the mouse. J. Virol. 71:8973-8982.
• Cardin, A. D. and H. J. Weintraub. 1989. Molecular modeling of protein-glycosaminoglycan interactions. Arteriosclerosis 9:21-32.
• Collins, P. L., M. G. Hill, E. Camargo, H. Grosfeld, R. M. Chanock, and B.
R. Murphy. 1995. Production of infectious human respiratory syncytial virus from cloned cDNA confirms an essential role for the transcription elongation factor from the 5' proximal open reading frame of the M2 mRNA in gene expression and provides a capability for vaccine development.
Proc. Natl. Acad. Sci. U. S. A 92:11563-11567.
• Collins, P. L., M. G. Hill, J. Cristina, and H. Grosfeld. 1996. Transcription elongation factor of respiratory syncytial virus, a nonsegmented negative-strand RNA virus. Proc. Natl. Acad. Sci. U. S. A 93:81-85.
• Collins, P. L., M. G. Hill, and P. R. Johnson. 1990. The two open reading frames of the 22K mRNA of human respiratory syncytial virus: sequence comparison of antigenic subgroups A and B and expression in vitro. J.
Gen. Virol. 71 ( Pt 12):3015-3020.
• Collins, P. L., Y. T. Huang, and G. W. Wertz. 1984. Nucleotide sequence of the gene encoding the fusion (F) glycoprotein of human respiratory syncytial virus. Proc. Natl. Acad. Sci. U. S. A 81:7683-7687.
• Collins, P. L.,K. McIntosh, and R. M. Chanock. 2001. Respiratory syncytial virus, p. 1443-1485. In D. M. Knipe, P. M. Howley, and D. E. Griffin (ed.), Fields virology, 4th ed., vol.2. Lippincott Williams & Wilkins, Philadelphia, Pa.
• Collins, P. L. and G. W. Wertz. 1983. cDNA cloning and transcriptional mapping of nine polyadenylylated RNAs encoded by the genome of human respiratory syncytial virus. Proc. Natl. Acad. Sci. U. S. A 80:3208-3212.
• Dutch, R. E., T. S. Jardetzky, and R. A. Lamb. 2000. Virus membrane fusion proteins: biological machines that undergo a metamorphosis.
Biosci. Rep. 20:597-612.
• Fearns, R. and P. L. Collins. 1999. Role of the M2-1 transcription antitermination protein of respiratory syncytial virus in sequential transcription. J. Virol. 73:5852-5864.
• Feldman, S. A., S. Audet, and J. A. Beeler. 2000. The fusion glycoprotein of human respiratory syncytial virus facilitates virus attachment and infectivity via an interaction with cellular heparan sulfate. J. Virol. 74:6442-6447.
• Feldman, S. A., R. M. Hendry, and J. A. Beeler. 1999. Identification of a linear heparin binding domain for human respiratory syncytial virus attachment glycoprotein G. J. Virol. 73:6610-6617.
• Furuta, R. A., C. T. Wild, Y. Weng, and C. D. Weiss. 1998. Capture of an early fusion-active conformation of HIV-1 gp41. Nat. Struct. Biol. 5:276-279
• Furze, J., G. Wertz, R. Lerch, and G. Taylor. 1994. Antigenic heterogeneity of the attachment protein of bovine respiratory syncytial virus. J. Gen. Virol. 75 ( Pt 2):363-370.
• Gerschwin J., E. Schelegle, R. Gunther, et al., 2000. A bovine model of vaccine enhanced respiratory syncytial virus pathophysiology. Vaccine 16:
1225-1236.
• Ghildyal, R., C. Baulch-Brown, J. Mills, and J. Meanger. 2003. The matrix protein of Human respiratory syncytial virus localises to the nucleus of infected cells and inhibits transcription. Arch. Virol. 148:1419-1429.
• Gonzalez-Reyes, L., M. B. Ruiz-Arguello, B. Garcia-Barreno, L. Calder, J.
A. Lopez, J. P. Albar, J. J. Skehel, D. C. Wiley, and J. A. Melero. 2001.
Cleavage of the human respiratory syncytial virus fusion protein at two distinct sites is required for activation of membrane fusion. Proc. Natl.
Acad. Sci. U. S. A 98:9859-9864.
• Grosfeld, H., M. G. Hill, and P. L. Collins. 1995. RNA replication by respiratory syncytial virus (RSV) is directed by the N, P, and L proteins;
transcription also occurs under these conditions but requires RSV superinfection for efficient synthesis of full-length mRNA. J. Virol. 69:5677-5686.
• Hallak, L. K., P. L. Collins, W. Knudson, and M. E. Peeples. 2000a.
Iduronic acid-containing glycosaminoglycans on target cells are required for efficient respiratory syncytial virus infection. Virology 271:264-275.
• Hallak, L. K., D. Spillmann, P. L. Collins, and M. E. Peeples. 2000b.
Glycosaminoglycan sulfation requirements for respiratory syncytial virus infection. J. Virol. 74:10508-10513.
• Hardy, R. W., S. B. Harmon, and G. W. Wertz. 1999. Diverse gene junctions of respiratory syncytial virus modulate the efficiency of transcription termination and respond differently to M2-mediated antitermination. J. Virol. 73:170-176.
• Heminway, B. R., Y. Yu, Y. Tanaka, K. G. Perrine, E. Gustafson, J. M.
Bernstein, and M. S. Galinski. 1994. Analysis of respiratory syncytial virus F, G, and SH proteins in cell fusion. Virology 200:801-805.
• Hileman, R. E., J. R. Fromm, J. M. Weiler, and R. J. Linhardt. 1998.
Glycosaminoglycan-protein interactions: definition of consensus sites in glycosaminoglycan binding proteins. Bioessays 20:156-167.
• Karger, A., U. Schmidt, and U. J. Buchholz. 2001. Recombinant bovine respiratory syncytial virus with deletions of the G or SH genes: G and F proteins bind heparin. J. Gen. Virol. 82:631-640.
• Karron, R. A., D. A. Buonagurio, A. F. Georgiu, S. S. Whitehead, J. E.
Adamus, M. L. Clements-Mann, D. O. Harris, V. B. Randolph, S. A. Udem, B. R. Murphy, and M. S. Sidhu. 1997. Respiratory syncytial virus (RSV) SH and G proteins are not essential for viral replication in vitro: clinical evaluation and molecular characterization of a cold-passaged, attenuated RSV subgroup B mutant. Proc. Natl. Acad. Sci. U. S. A 94:13961-13966.
• Kimman, T. G. and F. Westenbrink. 1990. Immunity to human and bovine respiratory syncytial virus. Arch. Virol. 112:1-25.
• Kimman, T. G., G. M. Zimmer, F. Westenbrink, J. Mars, and E. van Leeuwen. 1988. Epidemiological study of bovine respiratory syncytial virus infections in calves: influence of maternal antibodies on the outcome of disease. Vet. Rec. 123:104-109.
• Krummenacher, C., A. V. Nicola, J. C. Whitbeck, H. Lou, W. Hou, J. D.
Lambris, R. J. Geraghty, P. G. Spear, G. H. Cohen, and R. J. Eisenberg.
1998. Herpes simplex virus glycoprotein D can bind to poliovirus receptor-related protein 1 or herpesvirus entry mediator, two structurally unreceptor-related mediators of virus entry. J. Virol. 72:7064-7074.
• Krusat, T. and H. J. Streckert. 1997. Heparin-dependent attachment of respiratory syncytial virus (RSV) to host cells. Arch. Virol. 142:1247-1254.
• Kuo, L., R. Fearns, and P. L. Collins. 1997. Analysis of the gene start and gene end signals of human respiratory syncytial virus: quasi-templated initiation at position 1 of the encoded mRNA. J. Virol. 71:4944-4953.
• Kuo, L., H. Grosfeld, J. Cristina, M. G. Hill, and P. L. Collins. 1996. Effects of mutations in the gene-start and gene-end sequence motifs on transcription of monocistronic and dicistronic minigenomes of respiratory syncytial virus. J. Virol. 70:6892-6901.
• Kyhse-Andersen, J. 1984. Electroblotting of multiple gels: a simple apparatus without buffer tank for rapid transfer of proteins from
polyacrylamide to nitrocellulose. J. Biochem. Biophys. Methods 10:203-209.
• Le Bivic, A., F. X. Real, and E. Rodriguez-Boulan. 1989. Vectorial targeting of apical and basolateral plasma membrane proteins in a human adenocarcinoma epithelial cell line. Proc. Natl. Acad. Sci. U. S. A 86:9313-9317.
• Lerch, R. A., K. Anderson, and G. W. Wertz. 1990. Nucleotide sequence analysis and expression from recombinant vectors demonstrate that the attachment protein G of bovine respiratory syncytial virus is distinct from that of human respiratory syncytial virus. J. Virol. 64:5559-5569.
• Levine, S., R. Klaiber-Franco, and P. R. Paradiso. 1987. Demonstration that glycoprotein G is the attachment protein of respiratory syncytial virus.
J. Gen. Virol. 68 ( Pt 9):2521-2524.
• Lindahl, U., K. Lidholt, D. Spillmann, and L, Kjellen. 1994. More to
“heparin” than anticoagulation. Thromb. Res. 75:1-32
• Mallipeddi, S. K. and S. K. Samal. 1993. Sequence variability of the glycoprotein gene of bovine respiratory syncytial virus. J. Gen. Virol. 74 ( Pt 9):2001-2004.
• Moss, B., O. Elroy-Stein, T. Mizukami, W. A. Alexander, and T. R. Fuerst.
1990. Product review. New mammalian expression vectors. Nature 348:91-92.
• Murphy, F. A., P. J. Gibbs, M.C. Horzinek, and M. J. Studdert. 1999.
Paramyxoviridae. p. 412-428. Veterinary Virology. 3rd ed., Academic Press. A division of Harcour Brace & Company.
• Olmsted, R. A. and P. L. Collins. 1989. The 1A protein of respiratory syncytial virus is an integral membrane protein present as multiple, structurally distinct species. J. Virol. 63:2019-2029.
• Paccaud, M. F. and C. Jacquier. 1970. A respiratory syncytial virus of bovine origin. Arch. Gesamte Virusforsch. 30:327-342.
• Samal, S. K. and M. Zamora. 1991. Nucleotide sequence analysis of a matrix and small hydrophobic protein dicistronic mRNA of bovine respiratory syncytial virus demonstrates extensive sequence divergence of the small hydrophobic protein from that of human respiratory syncytial virus. J. Gen. Virol. 72 ( Pt 7):1715-1720.
• Satake, M., J. E. Coligan, N. Elango, E. Norrby, and S. Venkatesan. 1985.
Respiratory syncytial virus envelope glycoprotein (G) has a novel structure. Nucleic Acids Res. 13:7795-7812.
• Schlender, J., B. Bossert, U. Buchholz, and K. K. Conzelmann. 2000.
Bovine respiratory syncytial virus nonstructural proteins NS1 and NS2 cooperatively antagonize alpha/beta interferon-induced antiviral response.
J. Virol. 74:8234-8242.
• Schlender, J., G. Zimmer, G. Herrler, and K. K. Conzelmann. 2003.
Respiratory syncytial virus (RSV) fusion protein subunit F2, not attachment protein G, determines the specificity of RSV infection. J. Virol. 77:4609-4616.
• Schnell, M. J., T. Mebatsion, and K. K. Conzelmann. 1994. Infectious rabies viruses from cloned cDNA. EMBO J. 13:4195-4203.
• Shields, B., J. Mills, R. Ghildyal, P. Gooley, and J. Meanger. 2003. Multiple heparin binding domains of respiratory syncytial virus G mediate binding to mammalian cells. Arch. Virol. 148:1987-2003.
• Skehel, J. J. and D. C. Wiley. 2000. Receptor binding and membrane fusion in virus entry: the influenza hemagglutinin. Annu. Rev. Biochem.
69:531-569.
• Spann, K. M., K. C. Tran, B. Chi, R. L. Rabin, and P. L. Collins. 2004.
Suppression of the induction of alpha, beta, and lambda interferons by the
NS1 and NS2 proteins of human respiratory syncytial virus in human epithelial cells and macrophages [corrected]. J. Virol. 78:4363-4369.
• Sutter, G., M. Ohlmann, and V. Erfle. 1995. Non-replicating vaccinia vector efficiently expresses bacteriophage T7 RNA polymerase. FEBS Lett.
371:9-12.
• Taylor, G., L. H. Thomas, J. M. Furze, R. S. Cook, S. G. Wyld, R. Lerch, R. Hardy, and G. W. Wertz. 1997. Recombinant vaccinia viruses expressing the F, G or N, but not the M2, protein of bovine respiratory syncytial virus (BRSV) induce resistance to BRSV challenge in the calf and protect against the development of pneumonic lesions. J. Gen. Virol.
78 ( Pt 12):3195-3206.
• Techaarpornkul, S., N. Barretto, and M. E. Peeples. 2001. Functional analysis of recombinant respiratory syncytial virus deletion mutants lacking the small hydrophobic and/or attachment glycoprotein gene. J. Virol.
75:6825-6834.
• Techaarpornkul, S., P. L. Collins, and M. E. Peeples. 2002. Respiratory syncytial virus with the fusion protein as its only viral glycoprotein is less dependent on cellular glycosaminoglycans for attachment than complete virus. Virology 294:296-304.
• Teng, M. N. and P. L. Collins. 1998. Identification of the respiratory syncytial virus proteins required for formation and passage of helper-dependent infectious particles. J. Virol. 72:5707-5716.
• Teng, M. N. and P. L. Collins. 2002. The central conserved cystine noose of the attachment G protein of human respiratory syncytial virus is not required for efficient viral infection in vitro or in vivo. J. Virol. 76:6164-6171.
• Teng, M. N., S. S. Whitehead, A. Bermingham, M. St Claire, W. R. Elkins, B. R. Murphy, and P. L. Collins. 2000. Recombinant respiratory syncytial virus that does not express the NS1 or M2-2 protein is highly attenuated and immunogenic in chimpanzees. J. Virol. 74:9317-9321.
• Thomas, L. H., R. S. Cook, S. G. Wyld, J. M. Furze, and G. Taylor. 1998.
Passive protection of gnotobiotic calves using monoclonal antibodies directed at different epitopes on the fusion protein of bovine respiratory syncytial virus. J. Infect. Dis. 177:874-880.
• Valarcher, J. F., F. Schelcher, and H. Bourhy. 2000. Evolution of bovine respiratory syncytial virus. J. Virol. 74:10714-10728.
• Van der Poel, W. H., A. Brand, J. A. Kramps, and J. T. Van Oirschot.
1994. Respiratory syncytial virus infections in human beings and in cattle.
J. Infect. 29:215-228.
• Weissenhorn, W., A. Dessen, L. J. Calder, S. C. Harrison, J. J. Skehel, and D. C. Wiley. 1999. Structural basis for membrane fusion by enveloped viruses. Mol. Membr. Biol. 16:3-9.
• Wertz, G. W., P. L. Collins, Y. Huang, C. Gruber, S. Levine, and L. A. Ball.
1985. Nucleotide sequence of the G protein gene of human respiratory syncytial virus reveals an unusual type of viral membrane protein. Proc.
Natl. Acad. Sci. U. S. A 82:4075-4079.
• West K., L. Petrie, and C. Konoby. 2000. The efficacy of modified-live bovine respiratory syncytial virus vaccines in experimentally infected calves. Vaccine 18:907-919
• West K., L. Petrie, and D. M. Haines. 1999. The effect of formalin-inactivated vaccine of respiratory disease associated with live bovine respiratory syncytial virus infection in calves. Vaccine 17:809-820.
• Whitehead, S. S., A. Bukreyev, M. N. Teng, C. Y. Firestone, M. St Claire, W. R. Elkins, P. L. Collins, and B. R. Murphy. 1999. Recombinant respiratory syncytial virus bearing a deletion of either the NS2 or SH gene is attenuated in chimpanzees. J. Virol. 73:3438-3442.
• WuDunn, D. and P. G. Spear. 1989. Initial interaction of herpes simplex virus with cells is binding to heparan sulfate. J. Virol. 63:52-58.
• Yu, Q., R. W. Hardy, and G. W. Wertz. 1995. Functional cDNA clones of the human respiratory syncytial (RS) virus N, P, and L proteins support replication of RS virus genomic RNA analogs and define minimal trans-acting requirements for RNA replication. J. Virol. 69:2412-2419.
• Zamora, M. and S. K. Samal. 1992. Gene junction sequences of bovine respiratory syncytial virus. Virus Res. 24:115-121.
• Zimmer, G., L. Budz, and G. Herrler. 2001. Proteolytic activation of respiratory syncytial virus fusion protein. Cleavage at two furin consensus sequences. J. Biol. Chem. 276:31642-31650.
• Zimmer, G., K. K. Conzelmann, and G. Herrler. 2002. Cleavage at the furin consensus sequence RAR/KR(109) and presence of the intervening peptide of the respiratory syncytial virus fusion protein are dispensable for virus replication in cell culture. J. Virol. 76:9218-9224.
• Zimmer, G., M. Rohn, G. P. McGregor, M. Schemann, K. K. Conzelmann, and G. Herrler. 2003. Virokinin, a bioactive peptide of the tachykinin family, is released from the fusion protein of bovine respiratory syncytial virus. J.
Biol. Chem. 278:46854-46861
10 Sequences
Amino acid sequence of the complete F protein (amino acid 1-574)
MATTAMRMII SIIFISTYVT HITLCQNITE EFYQSTCSAV SRGYLSALRT GWYTSVVTIE LSKIQKNVCK STDSKVKLIK QELERYNNAV VELQSLMQNE PASFSRAKRG IPELIHYTRN STKKFYGLMG KKRKRRFLGF LLGIGSAVAS GVAVSKVLHL EGEVNKIKNA LLSTNKAVVS LSNGVSVLTS KVLDLKNYID KELLPQVNNH DCRISNIETV IEFQQKNNRL LEIAREFSVN AGITTPLSTY MLTNSELLSL INDMPITNDQ KKLMSSNVQI VRQQSYSIMS VVKEEVIAYV VQLPIYGVID TPCWKLHTSP LCTTDNKEGS NICLTRTDRG WYCDNAGSVS FFPQTETCKV QSNRVFCDTM NSLTLPTDVN LCNTDIFNTK YDCKIMTSKT DISSSVITSI GAIVSCYGKT KCTASNKNRG IIKTFSNGCD YVSNKGVDTV SVGNTLYYVN KLEGKALYIK GEPIINYYDP LVFPSDEFDA SIAQVNAKIN QSLAFIRRSD ELLHSVDVGK STTNVVITTI IIVIVVVILM LIAVGLLFYC KTKSTPIMLG KDQLSGINNL SFSK
Nucleotide sequence of the complete F gene (bp 5570-7294)
a tggcgacaac 5581 agccatgagg atgatcatca gcattatctt catctctacc tatgtgacac atatcacttt 5641 atgccaaaac ataacagaag aattttatca atcaacatgc agtgcagtta gtagaggtta 5701 ccttagtgca ttaagaactg gatggtatac aagtgtggta acaatagagt tgagcaaaat 5761 acaaaaaaat gtgtgtaaaa gtactgattc aaaagtgaaa ttaataaagc aagaactaga 5821 aagatacaac aatgcagtag tggaattgca gtcacttatg caaaatgaac cggcctcctt 5881 cagtagagca aaaagaggga taccagagtt gatacattat acaagaaact ctacaaaaaa 5941 gttttatggg ctaatgggca agaagagaaa aaggagattt ttaggattct tgctaggtat 6001 tggatctgct gttgcaagtg gtgtagcagt gtccaaagta ctacacctgg agggagaggt 6061 gaataaaatt aaaaatgcac tgctatccac aaataaagca gtagttagtc tatccaatgg 6121 agttagtgtc cttactagca aagtacttga tctaaagaac tatatagaca aagagcttct 6181 acctcaagtt aacaatcatg attgtaggat atccaacata gaaactgtga tagaattcca 6241 acaaaaaaac aatagattgt tagaaattgc tagggaattt agtgtaaatg ctggtattac 6301 cacacctctc agtacataca tgttgaccaa tagtgaatta ctatcactaa ttaatgatat 6361 gcctataacg aatgaccaaa aaaagctaat gtcaagtaat gttcaaatag tcaggcaaca
6421 gagttattcc attatgtcag tggtcaaaga agaagtcata gcttatgttg tacaattgcc 6481 tatttatgga gttatagaca ccccctgttg gaaactacac acctctccgt tatgcaccac 6541 tgataataaa gaagggtcaa acatctgctt aactaggaca gatcgtgggt ggtattgtga 6601 caatgcaggc tctgtgtctt ttttcccaca gacagagaca tgtaaggtac aatcaaatag 6661 agtgttctgt gacacaatga acagtttaac tctgcctact gacgttaact tatgcaacac 6721 tgacatattc aatacaaagt atgactgtaa aataatgaca tctaaaactg acataagtag 6781 ctctgtgata acttcaattg gagctattgt atcatgctat gggaagacaa aatgtacagc 6841 ttctaataaa aatcgtggaa tcataaagac tttttccaat gggtgtgatt atgtatcaaa 6901 caaaggagta gatactgtat ctgttggtaa cacactatat tatgtaaata agctagaggg 6961 gaaagcactc tatataaagg gtgaaccaat tattaattac tatgatccac tagtgtttcc 7021 ttctgatgag tttgatgcat caattgccca agtaaacgca aaaataaacc aaagcctggc 7081 cttcatacgt cgatctgatg agttacttca cagtgtagat gtaggaaaat ccaccacaaa 7141 tgtagtaatt actactatta tcatagtgat agttgtagtg atattaatgt taatagctgt 7201 aggattactg ttttactgta agaccaagag tactcctatc atgttaggga aggatcagct 7261 cagtggtatc aacaatcttt cctttagtaa atga
96
I would like to thank to my supervisor PD Dr. Gert Zimmer and to Prof. Dr.
Georg Herrler, for the initiation of this study and for their continuous guidance, help and confidence.
Furthermore, I would like to thank the members of my advisory committee,
Furthermore, I would like to thank the members of my advisory committee,