Newcastle disease virus exhibits a wide range of pathogenicity and virulence which, as with all paramyxoviruses, is directly related to the cleavability of a precursor (F0) of the fusion glycoprotein by cellular proteases. Sequence analyses of the cleavage site of several virulent and avirulent isolates of the Newcastle disease virus serotype reveal a correlation between virulence or pathogenicity and a high content of basic amino acid residues at the cleavage site. A similar correlation has been seen for other paramyxoviruses

Bratt, Michael A.

Glickman, Rhona L.

Iorio, Ronald M.

Sheehan, John P.

Syddall, Richard J.

English

eScholarship@UMMS

University of Massachusetts Medical School eScholarship@UMMS Open Access Articles Open Access Publications by UMMS Authors 1988-01-01 Quantitative basic residue requirements in the cleavage-activation site of the fusion glycoprotein as a determinant of virulence for Newcastle disease virus Rhona L. Glickman University of Massachusetts Medical School Et al. Let us know how access to this document benefits you. Follow this and additional works at: https://escholarship.umassmed.edu/oapubs  Part of the Life Sciences Commons, and the Medicine and Health Sciences Commons Repository Citation Glickman RL, Syddall RJ, Iorio RM, Sheehan JP, Bratt MA. (1988). Quantitative basic residue requirements in the cleavage-activation site of the fusion glycoprotein as a determinant of virulence for Newcastle disease virus. Open Access Articles. Retrieved from https://escholarship.umassmed.edu/oapubs/1590 This material is brought to you by eScholarship@UMMS. It has been accepted for inclusion in Open Access Articles by an authorized administrator of eScholarship@UMMS. For more information, please contact Lisa.Palmer@umassmed.edu. JOURNAL OF VIROLOGY, Jan. 1988, p. 354-3560022-538X/88/010354-03$02.00/0Copyright ©D 1988, American Society for MicrobiologyQuantitative Basic Residue Requirements in the Cleavage-ActivationSite of the Fusion Glycoprotein as a Determinant of Virulence forNewcastle Disease VirusRHONA L. GLICKMAN, RICHARD J. SYDDALL, RONALD M. IORIO, JOHN P. SHEEHAN,AND MICHAEL A. BRATT*Department of Molecular Genetics and Microbiology, University of Massachusetts Medical School, 55 Lake AvenueNorth, Worcester, Massachusetts 01605Received 29 May 1987/Accepted 8 October 1987Newcastle disease virus exhibits a wide range of pathogenicity and virulence which, as with all paramyxo-viruses, is directly related to the cleavability of a precursor (FO) of the fusion glycoprotein by cellular proteases.Sequence analyses of the cleavage site of several virulent and avirulent isolates of the Newcastle disease virusserotype reveal a correlation between virulence or pathogenicity and a high content of basic amino acid residuesat the cleavage site. A similar correlation has been seen for other paramyxoviruses.Paramyxoviruses are a group of negative-stranded RNA-containing enveloped viruses (8), of which Newcastle dis-ease virus (NDV) is unique in that the NDV serotype iscomposed of hundreds of isolates encompassing a widerange of virulence and pathogenicity (18). Paramyxovirionspossess two types of glycoprotein spikes protruding from thecellularly derived envelope. The hemagglutinin-neuramini-dase (HN) and fusion (F) glycoproteins are responsible for,respectively, attachment of the virus to cell surface recep-tors and fusion of the viral and cellular membranes (15). Thelatter is produced as a precursor molecule (FO), which mustbe proteolytically cleaved, producing two disulfide-linkedpolypeptides, F1 and F2 (11, 15). The cleavage activation ofFo is catalyzed by a host-cell protease(s) with trypsinlikeactivity (5). The new amino terminus of F1 is extremelyhydrophobic and functions as the insertion peptide, promot-ing fusion of the viral and cellular envelopes (14).There is a strict correlation between the cleavability of Foin tissue culture cells and the virulence and host cell range ofNDV isolates (10, 11). The FO of virulent isolates is cleaved,and thus, the virus will form plaques in many cell types,including cultured chicken embryo cells. The Fo of avirulentisolates, on the other hand, is cleaved in relatively few celltypes, such as cells of the chorioallantoic membrane ofembryonated hen eggs. These viruses will not form plaquesin cultured chicken embryo cells, unless an exogenousprotease, such as trypsin, is present in the agar overlay ofthe plaque assay.Using primer extension and dideoxy sequencing, we havecompared the deduced amino acid sequences around theproposed cleavage site of the Fo glycoprotein of a number ofvirulent and avirulent isolates of NDV. During the prepara-tion of this manuscript, a report (17) appeared concerningthe role of basic amino acids in the region preceding theF2/F1 cleavage site in five different isolates of NDV, as wellas two isolates used in this study. Their findings, and thedata presented here, suggest a requirement for several basicamino acids at the cleavage site of Fo for proteolytic cleav-age and activation in many cell types.Virulent isolates of NDV used in this study include AV* Corresponding author.(Australia-Victoria, 1932), F (NJ-Roakin, 1946), HP (Israel-HP, 1953), IM (Italy-Milano, 1945), IS (Iowa-Salsbury,1949), L (L-Kansas, 1948), and RO (California RO, 1944).Avirulent isolates include B (B1-Hitchner [Blacksburg]), EF(England F [ARS], 1949), N (NJ-LaSota, 1946), U (Ulster),and W (Wisconsin-Appleton, 1950). Virus was grown in theallantoic sac of 10-day-old embryonated hen eggs at 37°C (3).Virions were purified and concentrated (6, 19) and used as asource of genomic RNA (16).A primer (GACTACTTTGCTCACCC) encompassing nu-cleotides 316 to 332 of the F glycoprotein sequence of theAV isolate (9) was purchased from the DNA SynthesisFacility at the University of Massachusetts Medical School.This region was chosen for the primer because it lies about70 nucleotides upstream from the proposed cleavage site andonly 18 nucleotides upstream from an Arg-Arg sequencewhich has been proposed as a possible second cleavage site(9). The primer was purified by electrophoresis in a 20%polyacrylamide-7 M urea gel, followed by chromatographywith a SEP-PAK C18 cartridge (Waters Associates, Milford,Mass.).Primer extension was performed as described by Air (1)with the following modifications: reactions contained 2 ,ug ofvirion RNA and 100 ng of primer annealed for 6 min at 70°C,7.5 U of reverse transcriptase (Life Sciences, St. Petersburg,Fla.) was added, the reaction was incubated for 60 min at42°C, and the 30-min chase was omitted. Reaction productswere analyzed on 8% polyacrylamide-7 M urea sequencinggels. For better resolution of some regions, 40% formamidewas added to the gel mixture.The nucleotide sequence in the region of the proteolyticcleavage site of Fo was determined for six virulent and fiveavirulent isolates and compared with the correspondingsequence in NDV-AV (9) (Table 1). Also included is thepublished sequence of the Fo of the virulent Beaudette C(BE) isolate (4). A blank space indicates nucleotide homol-ogy with NDV-AV. The deduced amino acid sequences areshown in a similar manner in Table 2. The five amino acidspreceding the new F1 amino terminus produced by cleavageare underlined. In all of the virulent isolates, this pentapep-tide always consists of four basic amino acids with anintervening glutamine residue (Arg-Arg-Gln- Arg-Arg). In354Vol. 62, No. 1 at UNIV OF MASS MED SCH on August 5, 2008 jvi.asm.orgDownloaded from NOTES 355TABLE 1. Comparison of nucleotide sequences among virulent and avirulent NDV isolatesNucleotide sequence differences'375 400 425ACT ACA TCC GGA GGA AGG AGA CAG AAA CGCITTT ATA GGT GCT ATT ATC GGC AGT GTA GCT CTTGGGT G GG C AC G TT G C GT GT G C C T G CT G C C T G GT G C C T G GT G C C T G GT GGT GGT GGT GT GGGGGGGGGGGA GGGGGa Affow points to cleavage site. Blank spaces indicate homology with NDV-AV.b Nucleotide sequence as determined by McGinnes and Morrison (9).' Nucleotide sequence as determined by Chambers et al. (4).contrast, in the five avirulent isolates examined here, aneutral amino acid, glycine, is found in place of the basicarginine residues at positions 1 and 4 of the pentapeptide(Gly-Arg-Gln-Gly-Arg). This suggests that a high content ofbasic residues in the cleavage site may be at least one of therequirements for NDV to form plaques in most cell types,including chicken embryo fibroblasts. It is also noteworthythat the hydrophobic nature of the new amino terminusformed by the proteolytic cleavage is extremely highlyconserved in all virulent NDV isolates, in each case the newamino-terminal residue being a phenylalanine. All avirulentisolates have a leucine residue in this position which, al-though different from the virulent isolates, is a relativelyconservative change, maintaining the hydrophobic nature ofthe terminus of the insertion peptide.TABLE 2. Comparison of deduced amino acid sequences amongvirulent and avirulent NDV isolatesisolates Amino acid sequence differencesaVirulent112 116 125AVb T T S G G R R Q KR IGAIIGSVALGRO R L GHP RIM RIS VF GL GBEc GAvirulentN G G L GB G G L GW G G L GU GK G LEF G G La Arrow points to cleavage site. Blank spaces indicate homology withNDV-AV.b See Table 1, footnote b.c See Table 1, footnote c.The Arg-Arg sequence just downstream from the primerappears not to function as a general cleavage site, as wassuggested by McGinnes and Morrison (9), because it ispresent in avirulent isolates B, N, and W (data not shown),as well as virulent isolates AV (9), BE (4), and F (data notshown). Thus, it cannot account for the difference in sus-ceptibility to cleavage of the F glycoproteins of virulent andavirulent isolates.The amino acid sequences of the fusion glycoproteins ofseveral paramyxoviruses have now been determined. Theseinclude Sendai virus (2), measles virus (13), parainfluenzavirus type 3 (16), respiratory syncytial virus (7), simian virus5 (12), and two virulent isolates of NDV (4, 9). Looking forregions of homology, many of these researchers have madecomparisons of the fusion glycoprotein sequences of dif-ferent paramyxoviruses, including the putative proteolyticcleavage activation site. The location of the new aminoterminus formed upon proteolytic digestion was proposed onthe basis of a high degree of conservation of the region in anumber of paramyxoviruses and the specific inhibition ofparamyxovirus replication by oligopeptides with amino acidsequences similar to that terminus (14). Subsequently,Spriggs and co-workers (16) identified the amino terminus ofF1 of parainfluenza virus type 3 by direct amino acidsequencing. Table 3 shows the amino acid sequences forseveral paramyxoviruses for five residues on either side ofthe cleavage site. The amino terminus of F1 is indeedextremely highly conserved, terminating in phenylalaninefor all of these viruses except the avirulent NDV isolates.Comparison of the F2 carboxy-terminal pentapeptide se-quences reveals what appears to be a relationship betweenthe structure of this region and the ability of the virus togrow in many cell types. All members of the group have anarginine as the ultimate residue at the F2 carboxy terminus.The other residues in this pentapeptide vary in the differentviruses. Those that have four (measles and virulent NDVisolates) or five (respiratory syncytial virus and simian virus5) basic residues in this region are cleaved in many celltypes. For parainfluenza virus type 3, with only three basicresidues in this stretch, the protein is only partially cleavedin several cell types (16). The F glycoproteins of Sendai virusNDVisolatesVirulentAVbROHPIMIsFLBECAvirulentNBwUEFCCCCCCCCCCT G GT G GT G GTVOL. 62, 1988 at UNIV OF MASS MED SCH on August 5, 2008 jvi.asm.orgDownloaded from 356 NOTESTABLE 3. Comparison of paramyxovirus fusion protein cleavage site sequencesAmino acid No. of basic Amino acid F0 cleavedParamyxovirus and reference sequence at residues at sequence at in many cellcarboxyterminus of carboxyterminus aminoterminus of typesF2 of F, FtSimian virus 5 (12) R R R R R 5 F A G V V YesRespiratory synctial virus (7) K R K R R 5 F L G F L YesMeasles virus (13) R R H K R 4 F A G V V YesVirulent NDV isolates R R Q K R 4 F I G A I YesParainfluenza virus type 3 (16) P R T K R 3 F F G G V PartialAvirulent NDV isolates G R Q G R 2 L I G A I NoSendai virus (2) V P Q S R 1 F F G A V Noand the avirulent NDV isolates which, respectively, haveonly one and two basic residues in this region are not cleavedin most cell types, including cultured chicken embryo cellswithout exogenous protease. Thus, the NDV serotype in-cludes isolates which represent both extremes of the pa-ramyxovirus group, those with high basic residue content inthe F2 carboxy terminus and a wide range of cell specificitiesand those with low basic residue content and an inability toform plaques in cultured chicken embryo cells and manyother cell types. The results presented here and thosementioned earlier (17) demonstrate the importance of pairsof basic amino acid residues at the cleavage site for proteo-lytic activation of the fusion protein.We thank Trudy Morrison for helpful discussions and NancyGraham for help in preparation of the manuscript.This work was supported by Public Health Service grants Al-20762-03 and AI-12467-11 from the National Institute of Allergy andInfectious Diseases.ADDENDUMA mutant of the AV isolate (C. H. Madansky and M. A.Bratt, J. Virol. 26:724-729, 1978) has three basic residues atthe cleavage site. Virions have a partially cleaved Fo (C. H.Madansky and M. A. Bratt, J. Virol. 40:691-702, 1981) butstill require trypsin to form plaques in chicken embryo cells.LITERATURE CITED1. Air, G. M. 1979. Nucleotide sequence coding for the "signalpeptide" and N terminus of the hemagglutinin from an Asian(H2N2) strain of influenza virus. Virology 97:468-472.2. Blumberg, B. M., C. Giorgi, K. Rose, and D. Kolakofsky. 1985.Sequence determination of the Sendai virus fusion protein gene.J. Gen. Virol. 66:317-331.3. Bratt, M. A., and W. R. Gallaher. 1969. Preliminary analysis ofthe requirements for fusion from within and fusion from withoutby Newcastle disease virus. Proc. Natl. Acad. Sci. USA 64:536-543.4. Chambers, P., N. S. Millar, and P. T. Emmerson. 1986. Nucle-otide sequence of the gene encoding the fusion glycoprotein ofNewcastle disease virus. J. Gen. Virol. 67:2685-2694.5. Choppin, P. W., and R. W. Compans. 1975. Reproduction ofparamyxoviruses, p. 95-178. In H. Fraenkel-Conrat and R. R.Wagner (ed.), Comprehensive virology, vol. 4. Plenum Publish-ing Corp., New York.6. Clavell, L. A., and M. A. Bratt. 1972. Hemolytic interaction ofNewcastle disease virus and chicken erythrocytes. I1. Deter-mining factors. Appl. Microbiol. 23:461-470.7. Collins, P. L., Y. T. Huang, and G. W. Wertz. 1984. Nucleotidesequence of the gene encoding the fusion (F) glycoprotein ofhuman respiratory syncytial virus. Proc. Natl. Acad. Sci. USA81:7683-7687.8. Kingsbury, D. W., M. A. Bratt, P. W. Choppin, R. P. Hanson,Y. Hosaka, V. terMeulen, E. Norrby, W. Plowright, R. Rott, andW. H. Wunner. 1978. Paramyxoviridae. Intervirology 10:137-152.9. McGinnes, L. W., and T. G. Morrison. 1986. Nucleotide se-quence of the gene encoding the Newcastle disease virus fusionprotein and comparisons of paramyxovirus fusion protein se-quences. Virus Res. 5:343-356.10. Nagai, Y., and H.-D. Klenk. 1977. Activation of precursors toboth glycoproteins of Newcastle disease virus by proteolyticcleavage. Virology 77:125-134.11. Nagai, Y., H.-D Klenk, and R. Rott. 1976. Proteolytic cleavageof the viral glycoproteins and its significance for the virulence ofNewcastle disease virus. Virology 72:484-508.12. Paterson, R., T. Harris, and R. Lamb. 1984. Fusion protein ofthe paramyxovirus simian virus 5: nucleotide sequence ofmRNA predicts a highly hydrophobic glycoprotein. Proc. Natl.Acad. Sci. USA 81:6706-6710.13. Richardson, C., D. Hull, P. Greer, K. Hasel, A. Berkovick, G.Englund, W. Bellini, B. Rima, and R. Lazzarini. 1986. Thenucleotide sequence of the mRNA encoding the fusion proteinof measles virus (Edmonston strain): a comparison of fusionproteins from several different paramyxoviruses. Virology 155:508-523.14. Richardson, C. D., A. Scheid, and P. W. Choppin. 1980. Specificinhibition of paramyxovirus and myxovirus replication by oli-gopeptides with amino acid sequences similar to those at theN-termini of the F1 or HA2 viral polypeptides. Virology 105:205-222.15. Scheid, A., and P. W. Choppin. 1974. Identification of biologicalactivities of paramyxovirus glycoproteins. Activation of cellfusion, hemolysis, and infectivity by proteolytic cleavage of aninactive precursor protein of Sendai virus. Virology 57:475-490.16. Spriggs, M. K., R. A. Olmsted, S. Venkatesan, J. E. Coligan, andP. L. Collins. 1986. Fusion glycoprotein of human parainfluenzavirus type 3: nucleotide sequence of the gene, direct identifica-tion of the cleavage-activation site, and comparison with otherparamyxoviruses. Virology 152:241-251.17. Toyoda, T., T. Sakaguchi, K. Imai, N. M. Inocencio, B. Gotoh,M. Hamaguchi, and Y. Nagai. 1987. Structural comparison ofthe cleavage-activation site of the fusion glycoprotein betweenvirulent and avirulent strains of Newcastle disease virus. Virol-ogy 158:242-247.18. Waterson, A. P., T. H. Pennington, and W. H. Allan. 1967.Virulence in Newcastle disease virus. A preliminary study. Br.Med. Bull. 23:138-143.19. Weiss, S. R., and M. A. Bratt. 1974. Polyadenylate sequences onNewcastle disease virus mRNA synthesized in vivo and in vitro.J. Virol. 13:1220-1230.J. VIROL. at UNIV OF MASS MED SCH on August 5, 2008 jvi.asm.orgDownloaded from 

Quantitative basic residue requirements in the cleavage-activation site of the fusion glycoprotein as a determinant of virulence for Newcastle disease virus

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Quantitative basic residue requirements in the cleavage-activation site of the fusion glycoprotein as a determinant of virulence for Newcastle disease virus

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