Single Amino Acid Substitution of Sendai Virus at the Cleavage Site of the Fusion Protein Confers Trypsin Resistance

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1 J. gen. Virol. (1987), 68, Printed in Great Britain 2939 Key words : Senclai virus~trypsin resistant mutant/fusion protein Single Amino Acid Substitution of Sendai Virus at the Cleavage Site of the Fusion Protein Confers Trypsin Resistance By MASAE ITOH, HIROSHI SHIBUTA 1 AND MORIO HOMMA* Department of Microbiology, Kobe University School of Medicine, Kusunoki-cho, Chuo-ku, Kobe 650 and 1 Department of Viral Infection, Institute of Medical Science, University of Tokyo, Tokyo 108, Japan (Accepted 22 July 1987) SUMMARY Amino acid sequences of fusion (F) proteins of two trypsin-resistant mutants of Sendai virus, TR-2 and TR-5, were deduced from nucleotide analysis of cdna encoding the F gene and were compared with that of the trypsin-sensitive wild-type Sendai virus. In both mutants, amino acid substitutions were found at residues 116 (Arg ~ Ile), the cleavage site of the F protein, and 109 (Asn --, Asp). Two trypsinsensitive revertants, TSrev-52 and TSrev-58, derived from TR-5 were both activated by trypsin similarly to the wild-type virus and had a single amino acid reversion from Ile to Arg at residue 116, leaving Asp as before at residue 109. These results indicate that the trypsin sensitivity of Sendal virus can be changed by a single amino acid substitution at the cleavage site of the F protein and a mutation from Arg to Ile is responsible for the acquisition of resistance to trypsin. Sendai virus (HVJ), has two envelope glycoproteins (Mountcastle et al., 1971) designated HANA and F (Homma et al., 1975). HANA protein possesses both haemagglutinating and neuraminidase activities through which the virus adsorbs to the host cell receptors (Tozawa et al., 1973; Scheid & Choppin, 1974). F protein is a fusion protein and mediates the second step of infection by fusing the viral envelope and the plasma membrane, releasing the nucleocapsid into host cells (Homma & Ohuchi, 1973; Scheid & Choppin, 1974). F protein is initially synthesized as a precursor glycoprotein of Mr 65000; virus having this form of F protein is inactive and exhibits no haemolytic and cell-fusing activities or infectivity. Conversion of virus from the inactive to the active form can be achieved by a mild trypsin treatment in vitro which is accompanied by cleavage of the precursor F protein into its FI (Mr 51000) and F2 (Mr 15000) subunits (Homma, 1971, 1972 a; Homma & Ohuchi, 1973; Homma & Tamagawa, 1973; Scheid & Choppin, 1974; Ohuchi & Homma, 1976). Since the cleavage of the F glycoprotein occurs post-translationally and is accomplished by some protease(s) in the chorioallantoic fluid of embryonated chicken eggs (Muramatsu & Homma, 1980), in tissue culture cells (Shibuta et al., 1971 ; Silver et al., 1978) or in mouse lung (Tashiro & Homma, 1983 a), we and other authors have suggested that the presence of the activating enzyme(s) for Sendai virus will determine its host range and organ tropism (Homma, 1972b; Ishida & Homma, 1978; Silver et al., 1978). A similar proposal has been made concerning the virulence of Newcastle disease virus (Garten et al., 1980; Nagai et al., 1979). In previous reports we isolated a protease activation mutant designated TR-2, by passaging wild-type Sendai virus in the presence of chymotrypsin. TR-2 can be activated in vitro by chymotrypsin but not by trypsin. TR-2 previously activated by chymotrypsin in vitro underwent only one step of replication in mouse lung where no activating proteases for TR-2 were found and brought about negligible lung lesions (Tashiro & Homma, 1983b, 1985). Scheid & Choppin (1976) isolated some protease activation mutants and the sites of amino acid substitutions in SGM

2 2940 Short communication t28 i l i J i i 64 < 32 m v 16 -,~ ~ 8 4 <4... I ; //0-05 O-'lO Trypsin concentration (gg/ml) Fig. 1. Virus growth of the trypsin-sensitive revertants in the presence of various concentrations of trypsin. LLC-MK2 cells were infected with 0-01 p.f.u./cell of virus in the presence of various concentrations of trypsin at 37 C for 120 h. The progeny viruses released from the cells were measured for haemagglutinating activity as haemagglutinating units/ml (HAU/ml). O, Wild-type; [-q, TR-5; A, TSrev-52; II, TSrev-58. these mutants have been determined very recently (Hsu et al., 1987), although the analyses were limited to only 60 to 70 amino acids surrounding the activation cleavage site. There is evidence that a single amino acid substitution occurring at a residue distant from the cleavage site of the F protein could cause conformational change around the NH2 terminus of the F1 subunit (Portner et al., 1987). Accordingly, it is possible that such a mutation may contribute to the alteration of the protease sensitivity of the F protein. Working independently of the above authors, we have determined the full nucleotide sequences of cdna representing the F genes of the trypsinsensitive wild-type Sendal virus, its trypsin-resistant mutants and trypsin-sensitive revertants. By comparison with the whole amino acid sequences deduced from their nucleotide sequences, we aimed to find the mutation point(s) of TR-2 responsible for the trypsin resistance. Three types of viruses were used: the trypsin-sensitive wild-type Fushimi strain of Sendai virus, trypsin-resistant mutants of the Fushimi strain TR-2 (Tashiro & Homma, 1983b) and TR-5 (a further isolate from plaques of TR-2) and the trypsin-sensitive revertants TSrev-52 and TSrev-58 which were obtained by passaging TR-5 in the presence of 3 I~g/ml trypsin. TR-5 was chosen as the parent virus for isolation of revertants because it had a marker amino acid Met at residue 172 (Fig. 4) and the resulting trypsin-sensitive revertants, TSrev-52 and TSrev-58, could be distinguished from the wild-type virus. As shown in Fig. 1, TSrev-52 and TSrev-58 regained trypsin sensitivity and underwent multiple cycles of replication in the presence of trypsin, similar to the wild-type virus, in contrast to their parent virus, TR-5. When analysed by polyacrylamide gel electrophoresis, the F protein of the revertants was hydrolysed into the FL and F2 subunits after treatment with trypsin (data not shown), indicating that activation of the revertants by trypsin is related to the cleavability of F by trypsin and that some mutation(s) must have taken place in the gene coding for the F protein, converting TR into the trypsin-sensitive revertant. To elucidate the trypsin resistance of TR mutants, we first determined the nucleotide sequences of the F gene of the wild-type virus, the TR mutants and the TS revertants, and then compared the amino acid sequences deduced therefrom. Cloning of cdna was performed according to the method of Okayama & Berg (1982) and the clones containing sequences corresponding to the F gene were identified by hybridization with 35S-labelled cdna of the F protein gene of the Sendai virus Z strain. Sequence analysis of the nucleotides was carried out by the dideoxy chain-termination method of Sanger et al. (I977). Fig. 2 shows the nucleotide sequence of the F protein gene of the wild-type Fushimi strain which is the original strain of Sendal virus, and the amino acid sequence deduced therefrom.

3 FO ~D 4~ "I 53* AGGGATAAAGTCCCTTGTGAGTGCTTGGTTGCAAAACTCTCCCCTTGGGAAAC 113" ATG.ACA.GCA.TAT.ATC.CAG.AGG.TCA.CAG.TGC.ATC.TCA.ACA.TCA.CTA.CTG.GTT.GTT.CTC.ACC Met-Thr-Ala-Tyr-Ile-Gln-Arg-Ser-Gln-Cys-Ile-Ser-Thr-Ser-Leu-Leu-Val-Val-Leu-Thr 173" ACA,TTG.GTC.TCG.TGT.CAG.ATT.CCC.AGG.GAT.AGG.CTC,TCT.AAC.ATA.GGG GTC.ATA-GTC-GAT Thr_Leu-val-Ser-Cys-Gln-Ile-Pro-Arg-Asp-Arg-Leu-Ser-Asn-Ile-Gly-Val-Ile-Va -Asp * GAA.GGG.AAA.TCA.CTG.AAG.ATA.GCT GGA.TCC.CAC,GAA.TCG.AGG.TAC.ATA.GTA.CTG.AGT.CTA Glu_Gly-Lys-Ser-Leu-Lys-lle-Ala-Gly-Ser-His-Glu-Ser-Arg-Tyr-Ile-Val-Leu-Ser-Leu * GTT.CCG.GGG.GTA.GAC.CTT.GAG.AAT.GGG.TGC.GGA.ACA.GCC.CAG.GTT.ATC.CAG.TAC.AAG.AGC Val_Pro-Gly-Val-Asp-Leu-Glu-Asn-Gly-Cys-Gly-Thr-Ala-Gln-Val Ile-Gln-Tyr-Lys-ser * CTA.CTG.AAC AGG.CTG.TTA.ATC.CCA.TTG.AGG.GAT.GCC.TTA.GAT.CTT.CAG.GAG.GCT.CTG-ATA Leu_L~u-Asn-Arg-Leu-Leu-Ile-Pro-Leu-Arg-Asp-Ala-Leu-Asp-Leu-Gln-Glu-Ala-Leu-Ile 81 ~ 413" ACT.GTC.ACC.AAT.GAT.ACG.ACA.CAA.AAT.GCC.GGT.GTT.CCA.CAG-TCG.AGA.TTC.TTC.GGT'GCT Thr_Val-Thr-Asn-Asp-Thr-Thr-Gln-Asn-Ala-Gly-Val-Pro-Gln-Ser-Arg-Phe-Phe-Giy-Ala * GTG.ATT.GGT.ACT.ATC.GCA.CTT.GGA.GTG.GCG.ACA.TCA.GCA.CAG.ATC.ACC.GCA.GGG.ATT-GCA Val_ile-Gly-Thr-ile-Ala-Leu-Giy-Val-Ala-Thr-Ser-Ala-Gln-Ile-Thr-Ala-Gly-Ile-Ala * CTA.GCC.GAA.GCG.AGG,GAG.GCC.AAA.AGA.GAC.ATA.GCG.CTC.ATC.AAA.GAA.TCG.ATG.ACA.AAA L~u_Ala-Glu-Ala-Arg-Glu-Ala-Lys-Arg-Asp-Ile-Ala-Leu-Ile-Lys-Glu-Ser-Met-Thr-LyS * ACA.CAC.AAG.TCT.ATA.GAA.CTG.CTG.CAA.AAC.GCT.GTG.GGG.GAA CAA.ATT.CTT-GCT-CTA-AAG Thr_His-Lys-Ser-IIe-GIu-Leu-Leu-GIn-Asn-AIa-VaI-GIy-GIu-GIn-IIe-Leu-AIa-Leu-LyS * ACA.CTC.CAG.GAT.TTC.GTG.AAT.GAT.GAG.ATC.AAA.CCC.GCA.ATA.AGC.GAA.TTA-GGC,TGT-GAG Thr_Leu-Gln-Asp-Phe-Val-Asn-Asp-Glu-Ile-Lys-Pro-Ala-lle-Ser-Glu-Leu-Gly-Cys-Glu " ACT GCT.GCC.TTA.AGA.CTG.GGT.ATA.AAA,TTG.ACA.CAG.CAT.TAC.TCC-GGG.CTG.TTA.ACT GCG Thr-Ala-Ala-Leu-Arg-Leu-Gly-Ile-Lys-Leu-Thr-Gln-His-Tyr-Eer-Gly-Leu-Leu-Thr-Ala * TTC.GGC.TCG.AAT.TTC.GGA.ACC.ATC.GGA.GAG.AAG.AGC.CTC.ACG.CTG.CAG.GCG-CTG-TCT.TCA Phe-GIM-Ser-Asn-Phe-Gly-Thr-Ile-Gly-Glu-Lys-Ser-Leu-Thr-Leu-Gln-Ala-Leu-Ser-Ser 22~ 833* CTT.TAC.TCT.GCT.AAC.ATT.ACT.GAG.ATT.ATG.ACC.ACA.ATC.AGG-ACA.GGG.CAG-TCT-AAC.ATC Leu.Tyr-Ser-Ala-Asn-Ile-Thr-Glu-Ile-Met-Thr-Thr-Ile-Arg-Thr-Gly-Gln-$er-Asn-I e * TAT.GAT.GTC.ATT.TAT.ACA.GAA.CAG.ATC.AAA.GGA.ACG.GTG.ATA.GAT.GTG.GAT.CTA-GAG-AGA T~r-Asp-Val-Ile-Tyr-Thr-Glu-Gln-Ile-Lys-Gly-Thr-Val-Ile-Asp-val-Asp-Leu-Glu-Arg * TAC.ATG.GTT.ACC.CTG.TCT.GTG.AAG.ATC.CCT.ATT.CTT.TCT.GAA.GTC.CCA.GGT-GTG-CTC.ATA T[r-Met-Val-Thr-Leu-Ser-Val-Lys-Ile-Pro-Ile-Leu-Ser-Glo-Val-Pro-Gly-Val-Leu-lle " CAC.AAG.GCA.TCG.TCT.ATT.TCT.TAC.AAC.ATA.GAC.GGG.GAG.GAA.TGG.TAT.GTG.ACT.GTC.CCC His-Lys-Ala-Ser-Ser-Ile-Ser-Tyr-Asn-Ile-Asp-GIy-G1u-Glu-Trp-Tyr-Val-Thr-Val-Pro " AGC.CAT.ATA.CTC. AGT.CGT.GCT*TCT.TTC.TTA.GGG.GGT.GCA.GAC.ATA.ACC.GAT.TGTdGTT.GAG Ser-His-Ile-Leu-Ser-Arg-&la-Ser-Pha-l~u-Gly-Gly-Ala-Asp-Ile-Thr-Asp-Cys-Val-Glu " TCC.AGA.TTG.ACC.TAT.ATA.TGC.CCC.AGG.GAT.CCC.GCA.CAA.CTG.ATA.CCT.GAC.AGC.CAG.CAA Ser-Arg-Leu-Thr-Tyr-lle-Cys-Pro-Arg-Asp-Pro-Ala-Gln-Leu-Ile-Pro-Asp-Ser-Gln-Gln 1193" AAG.TGT.ATC.CTG.GGG.GAC.ACA.ACA.AGG.TGT.CCT.GTC.ACA.AAA.GTT.GTG.GAC.AGC.CTT ATC L~s-Cys-Ile-Leu-Gly-Asp-Th~-Thr-Arg-Cys-Pro-val-Thr-Lys-Val-Val-Asp-Ser-Leu-Ile " CCC.AAG.TTT.GCT.TTT.GTGoAAT.GGG.GGC.GTT.GTT.GCT.AAC.TGC.ATA.GCA.TCC.ACA.TGT.ACC Pro-Lys-Phe-Ala-Phe-Val-Asn-G~y-Gly-Val-Val-Ala-Asn-Cys-Ile-Ala-Ser-Thr-Cys-Thr 1313, TGC.GGG.ACA.GGC.CGA.AGA.CCA.ATC.AGT.CAG.GAT.CGC.TCT.AAA.GGT.GTA.GTA.TTC.CTA.ACC Cys-Gly-Thr-Gly-Arg-Arg-Pro-Ile-Ser-Gln-Asp-Arg-Ser-Lys~Gly-val-Val~Phe-Leu-Thr " CAT.GAC.AAC.TGT.GGT.CTT.ATA.GGT.GTC.AAT.GGG.GTA.GAA.TTG.TAT.GCT-AAC.CGG.AGA-GGG His-Asp-Asn-Cys-Gly-Leu-Ile-Gly-Val-Asn-Gly-Va1-Glu-Leu-Tyr-Ala-Asn-Arg-Arg-Gly 1433" CAC.GAT.GCC.ACT.TGG.GGG.GTC.CAG.AAC.TTG.ACA.GTC.GGT.CCT.GCA.ATT GCT.ATC.AGA.CCC His-Asp-Ala-Thr-Trp-GIy-val-Gln-Asn-Leu-Thr-Val-Gly-Prc-Ala-Ile-Ala~lle-Arg-Pro 1493" ATT.GAT.ATT.TCT.CTC AAC.CTT.GCT.GAT.GCT.ACG.AAT.TTC.TTG.CAA.GAC.TCT.AAG.GCT.GAG Ile-Asp-Ile-Ser-Leu-Asn-Leu-Ala-Asp-Ala-Thr-Asn-Phe-Leu-Gln-Asp-Ser-Lys-Ala-Glu 1553" CTT.GAG.AAA.GCA.CGG.AAA.ATC.CTC.TCT.GAG.GTA.GGT.AGA.TGG.TAC.AAC.TCA.AGA.GAG.ACT Leu-Glu-Lys-Ala-Arg-Lys-Ile-Leu-Ser-Glu-Val-Gly-Arg-Trp-Tyr-Asn-Ser-Arg-G u-thr 1613" GTG ATT A G.ATC.ATA.GTA GTT.ATG.GT.GTA ATA TTG GTG GTC ATT.ATA GTG AT GTC AT Val-lle-Thr-Ile-Ile-Val-Va1-Met-Val-Val-lle-Leu-Val-Val-Ile-Ile-Val-Ile-Val-lle " GTG.CTT.TKT.AGA.CTC.AAA.AGG.TCA.ATG.CTA.ATG.GGT.AAT.CCA.GAT.GAC.CGT.ATA.CCG,AGG Val-Leu-Tyr-Arg-Leu-Lys-Arg-Ser-Met-Leu-Met-Gly-Asn-Pro-Asp-Asp-Arg-Ile-Pro-Arg 1733" GAC.ACA.TAT.ACA.TTA.GAG.CCG.AAG.ATC.AGA.CAT.ATG.TAC.ACA.AAC.GGT.GGG.TTT.GAT GCG Asp-Thr-Tyr-Thr-Leu-Glu-Pro-Lys-Ile-Arg-His-Met-Tyr-Thr-Asn-Gly-Gly-Phe-Asp-Ala ATG.GCT.GAG.AAA.AGA.TGA.TCACGACCATTATCAGATGTCTTGTAAAGCAGGCATGGTATCCGTTGAGACCTGT Met-Ala-Glu-Lys-Arg-*** ~5 1820" ATATAATAAGAAAA Fig 2. Nucleotide sequence of mrna-sense (+) DNA of the F gene of the wild-type Sendal virus Fushimi strain and its predicted amino acid sequence. In the amino acid sequence, the cleavage/activation site by trypsin is shown by an arrow above the sequence and the putative N-linked carbohydrate attachment sites are underlined.

4 2942 Short communication Fushimi Cleavage site,,t4- t T?,,,,,, t? z r?l. r??.trot? +, Phe A a G u Set Vat Ire Arg (66) (112.) (216) (261) (461) (519) (526),,T?I T? TiT [(//Jl ~'/'A K//, I I VaL Phe Arg Arg ALa VaL GLu (165) [215] (233) (394] (417) (461) (536) Ser Vat (453) {461) Fig. 3. Amino acid changes in the F proteins of various strains of Sendai virus [Fushimi (this report), Z (Shioda et al., 1986), an unnamed strain (Blumberg et al., 1985) and RU (Hsu & Choppin, 1984)], and locations of the putative N-linked carbohydrate attachment sites (71) and cysteine residues (O). Bars represent the F proteins with the N-terminal end at the left side, and the hydrophobic regions are represented by hatched areas. Only amino acids different from those of the Fushimi strain are indicated under the bars. There is a single long open reading frame encoding 565 amino acids. When the predicted amino acid sequence was compared with those of other strains of Sendai virus previously reported (Hsu & Choppin, 1984; Blumberg et al., 1985 ; Shioda et al., 1986), minor changes were detected (Fig. 3). The Fushimi strain contained three functionally important hydrophobic sequences corresponding to the transmembrane (anchor) region near the C terminus, the N terminus region of the Ft subunit and the signal peptide at the N-terminal end of the F2 subunit, respectively. The amino acid sequences of these regions were the same for all strains except one amino acid at residue 519 in the anchor region, where Val was replaced by Ile in the Z strain. The Fushimi strain had three putative N-linked carbohydrate attachment sites as reported by Blumberg et al. (1985) but the Z strain possessed a fourth site. The location of cysteines, which are important for the three-dimensional structure of the protein, was the same in the Fushimi and Z strains. Thus, the fundamental structure of the F protein of the Fushimi strain is well preserved. In the case of TR-2, two nucleotide changes were noted, at positions 378 (A --, G) and 400 (G -+ T), both resulting in amino acid substitutions, at residues 109 (Asn -~ Asp) and 116 (Arg --~ Ile), respectively. With TR-5, there was an additional nucleotide change at position 567 (G --, A) corresponding to an amino acid substitution (Va! --, Met) at residue 172 (Fig. 4). Since TR-5 was resistant to trypsin and in this respect is indistinguishable from TR-2, this change may not be responsible for the trypsin resistance. To decide the amino acid change responsible for the trypsin resistance of the TR mutants, the nucleotide sequences of two trypsin-sensitive revertants were examined. We found a transversion from T to G at position 400 in both revertants, but nucleotides G and A at positions 378 and 567, respectively, were conserved (Fig. 4). The former change may cause the reversion of Ile at residue 116 back to Arg. Among the amino acid changes, the Arg -* Ile mutation is the one most likely to induce the trypsin resistance of the TR mutants because trypsin cleaves the F protein at this point (Gething et al., 1978; Richardson et al+, 1980). However, the possible involvement of the Asn -~ Asp mutation at residue 109 should be considered. This is situated near the cleavage site and the simultaneous change of Ash --, Asp and Arg -+ Ile might be necessary to cause trypsin resistance. The present results with the revertants exclude this possibility since Asp at residue 109 remained unchanged irrespective of their trypsin sensitivity. The results show that a single amino acid change at residue 116 is responsible for the change in the trypsin sensitivity of the F protein; when the amino acid is Arg, the F protein is sensitive to

5 Short communication 2943 Cleavage site 37s,0.01 s67 Wild-type -- CAA AAT GCC GGT GTT CCA CAG TCG AGA ]'TC TTC GGT... GTG Gin Asn Ale GIy Vol Pro Gln Ser Arg Phe Phe Gly... VoI -- 10g I TR-2 - o, GAT H,.o,.*,,*,.e.,~- ATA o~* oh,,t... GTG Asp Ile V01 -- TR-5 TSrev-52 --,, - GAT - * * - - -,4..,, *** o** ATk -, 1, - - **$..... ATG As~ lie Met GAT * -- -* , AGA ee. ea. 6o*... AIG Asp At9 Met o TSrev-58 GAT... AGA... ATS -- As~.... ": : Arg Met -- Fig. 4. Amino acid changes around the cleavage site of the F proteins of TR mutants and the trypsinsensitive revertants. In the sequences of the TR mutants and the revertants, only residues 109, 116 and 172 are indicated. The arrows show the putative cleavage site of the wild-type virus and the revertants when activated by trypsin. The symbols indicate (0) nucleotides the same as those found in the wildtype virus and (=) amino acid residues the same as those found in the wild-type virus. 1 trypsin, whereas if it is Ile, it is resistant. In contrast to the TR mutants, the revertants regained pathogenicity for the lungs of mice (unpublished data), suggesting that a single amino acid mutation is sufficient to alter the pneumopathogenicity of Sendal virus in mice. Our results obtained with TR mutants are compatible with the results reported by Hsu et al. (1987) concerning their protease activation mutant, pa-cl, which lost trypsin sensitivity and simultaneously acquired chymotrypsin sensitivity. It should be noted that both the TR and pa-c 1 mutants, although isolated independently, have the same mutations at residues 116 (Arg --, Ile) and 109 (Asn --* Asp). Since the mutation of Arg to Ile has been shown to be responsible for trypsin resistance, the other mutation may be involved in chymotrypsin sensitivity. A difference exists, however, in the chymotrypsin sensitivity of the wild-type Fushimi strain and the parental strain of pa-cl ; the former could be activated by chymotrypsin (Tashiro & Homma, 1983b, 1985) whereas the latter could not (Scheid & Choppin, 1976; Hsu et al., 1987). Since the complete amino acid sequence of the F protein of the latter virus is not available the above difference cannot be elucidated at present. Whether the increased chymotrypsin sensitivity of the TR mutants relates to the mutation of Ash to Asp at residue 109 has yet to be studied; experiments to resolve this question are now under way in our laboratory. This research was supported in part by a Grant-in-Aid for Scientific Research, a Grant-in-Aid for Special Project Research, and a Grant-in-Aid for Co-operative Research from the Ministry of Education, Science and Culture of J apan, and by a Research Programme for Slow Virus Infection from the Ministry of Health and Welfare of Japan, and by Yakult Co. Ltd. This paper has been submitted by M. Itoh as a thesis for the degree of Doctor of Medicine at Kobe University. REFERENCES BLUMBERG, B. M., GIORGI, C., ROSE, K. & KOLAKOFSKY, D. (1985). Sequence determination of the Sendal virus fusion protein gene. Journal of General Virology 66, GARTEN, W., KOHAMA, T. & KLENK, H.-D. (1980). Proteolytic activation of the haemagglutinin neuraminidase of Newcastle disease virus involves loss of a glycopeptide. Journal of General Virology 51, GETHING, M. J., WHITE, J. M. & WATERFIELD, M. D. (1978). Purification of the fusion protein of Sendal virus: analysis of the NH2-terminal sequence generated during precursor activation. Proceedings of the National Academy of Sciences, U.S.A. 75, HOMMA, M. (197 l). Trypsin action on the growth of Sendal virus in tissue culture cells. I. Restoration of infectivity of L cells by direct action of trypsin on L cell-borne Sendal virus. Journal of Virology 8,

6 2944 Short communication HOMMA, M. (1972a). Trypsin action on the growth of Sendai virus in tissue culture cells. II. Restoration of the hemolytic activity of L cell-borne Sendai virus by trypsin. Journal of Virology 9, HOMMA, M. (1972b). Host-induced modification of hemagglutinating virus of Japan (HVJ, Sendai virus). Virus, Tokyo 25, HOMMA, M. & OHUCHI, M. (1973). Trypsin action on the growth of Sendai virus in tissue culture cells. III. Structural difference of Sendai viruses grown in eggs and tissue culture cells. Journal of Virology 12, HOMMA, M. & TAMAGAWA, S. (1973). Restoration of the fusion activity of L cell-borne Sendai virus by trypsin. Journal of General Virology 19, HOMMA, M., TOZAWA, H., SHIMIZU, K. & ISHIDA, N. (1975). A proposal for designation of Sendai virus proteins. Japanese Journal of Microbiology 19, HSU, M. C. & CHOPPIN, P. W. (1984). Analysis of Sendai virus mrnas with cdna clones of viral genes and sequences of biologically important regions of the fusion protein. Proceedings of the National Academy of Sciences, U.S.A. 81, HSU, M. C., SCHEID, A. & CHOPPIN, P. W. (1987). Protease activation mutants of Sendal virus: sequence analysis of the mrna of the fusion protein (F) gene and direct identification of the cleavage-activation site. Virology 156, ISHIDA, N. & HOMMA, M. (1978). Sendai virus. Advances in Virus Research 23, MOUNTCASTLE, W. E., COMPANS, R. W. & CHOPPIN, P. w. (1971). Proteins and glycoproteins of paramyxoviruses: a comparison of simian virus 5, Newcastle disease virus, and Sendai virus. Journal of Virology 7, MURAMATSU, M. & HOMMA, M. (1980). Trypsin action on the growth of Sendai virus in tissue culture cells. V. An activating enzyme for Sendai virus in the chorioallantoic fluid of the embryonated chichen egg. Microbiology and Immunology 24, NAGAI, Y., SHIMOKATA, K., YOSHIDA, T., HAMAGUCHI, M., IINUMA, M., MAENO, K.~ MATSUMOTO~ T., KLENK, H.-D. & ROTT, R. (1979). The spread of a pathogenic and an apathogenic strain of Newcastle disease virus in the chick embryo as depending on the protease sensitivity of the virus glycoproteins. Journal of General Virology 45, OHUCHI, M. & HOMMA, M. (1976). Trypsin action on the growth of Sendai virus in tissue culture cells. IV. Evidence for activation of Sendai virus by cleavage of a glycoprotein. Journal of Virology 18, OKAYAMA, H. & BERG, P. (1982). High-efficiency cloning of full-length cdna. Molecular and Cellular Biology 2, PORTNER, A., SCROGGS, R. A. & NAEVE, C. W. (1987). The fusion glycoprotein of Sendal virus: sequence analysis of an epitope involved in fusion and virus neutralization. Virology 157, RICHARDSON, C. D., SCHEID, A. & CHOPPIN, P. w. (1980). Specific inhibition of paramyxovirus and myxovirus replication by oligopeptides with amino acid sequences similar to those at the N-termini of the F 1 or HA2 viral polypeptides. Virology 105, SANGER, r., NICKLEN, S. & COULSON, A. R. (1977). DNA sequencing with chain-terminating inhibitors. Proceedings of the National Academy of Sciences, U.S.A. 74, SCHEID, A. & CHOPPIN, P. W. (1974). Identification of biological activities of paramyxovirus glycoproteins. Activation of cell fusion, hemolysis, and infectivity by proteolytic cleavage of an inactive precursor protein of Sendal virus. Virology 57, SCHEID, A. & CHOPPIN, P. W. (1976). Protease activation mutants of Sendal virus. Activation of biological properties by specific proteases. Virology 69, SHIBUTA, H., AKAMI, M. & MATSUMOTO, M. (1971). Plaque formation by Sendai virus of parainfluenza virus group, type 1 on monkey, calf kidney and chick embryo cell monolayers. Japanese Journal of Microbiology 15, SHIODA, T., 1WASAKI, K. & SHIBUTA, H. (1986). Determination of the complete nucleotide sequence of the Sendai virus genome RNA and the predicted amino acid sequences of the F, HN and L proteins. Nucleic Acids Research 14, SILVER, S. M., SCHE1D, A. & CHOPPIN, P. W. (1978). Loss on serial passage of rhesus monkey kidney cells of proteolytic activity required for Sendai virus activation. Infection and Immunity 20, TASHIRO, M. & HOMMA, M. (1983a). Evidence of proteolytic activation of Sendai virus in mouse lung. Archives of Virology 77, TASHIRO, M. & HOMMA, M. (1983b). Pneumotropism of Sendai virus in relation to protease-mediated activation in mouse lungs. Injection and Immunity 39, TASHIRO, M. & HOMMA, M. (1985). Protection of mice from wild-type Sendai virus infection by a trypsin-resistant mutant, TR-2. Journal of Virology 53, TOZAWA, H., WATANABE, M. & ISHIDA, N. (1973). Structural components of Sendai virus. Serological and physicochemical characterization of hemagglutinin subunit associated with neuraminidase activity. Virology 55, (Received 25 March 1987)

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