A host range mutant of Newcastle disease virus with an altered cleavage site for proteolytic activation of the F protein

231

Virus Research, 15 (1990) 231-242 Elsevier

VIRUS 00557

A host range mutant of Newcastle disease virus with an altered cleavage site for proteolytic activation of the F protein Ellen Pritzer

I, Kazumichi

Kuroda ‘, Wolfgang Garten and Hans-Dieter Klenk ’

I, Yoshiyuki

Nagai

2

’ Institut fG Virologie, Philipps-Universittit,

Marburg, F.R.G. and 2 Research Institute for Disease Mechanism and Control, Nagoya University, Nagoya, Japan (Accepted 14 November 1989)

The primary structure of the F protein of a host range mutant of the Ulster strain of Newcastle Disease virus (NDV) has been determined by nucleotide sequence analysis and compared to that of the wild type and other NDV strains. The cleavage site of the mutant had the sequence Gly-Lys-Gln-Arg-Arg as compared to two isolated basic ammo acids [Gly-Lys(Arg)-Gln-Gly-Arg] with the apathogenic strains and two pairs of basic amino acids [Arg-Arg-Gln-Lys(Arg)-Arg] with the pathogenic strains. The data indicate that the cleavability of the F protein of NDV increases with the number of arginine and lysine residues at the cleavage site and that the susceptibility of the pathogenic strains to ubiquitous host proteases depends on both pairs of basic amino acids. Newcastle disease virus; Host range mutant

The fusion (F) glycoprotein of Newcastle disease virus (NDV) is synthesized as the inactive precursor F, that is proteolytically cleaved into the fragments FI and F2. Cleavage generates a hydrophobic ammo acid sequence at the N-terminus of Ft that is responsible for the fusion between the viral envelope with the target cell membrane and is therefore essential for the initiation of infection (Nagai et al.,

Correspondence

to: H.D. Klenk, Institut fiir Virologie, Philipps-Universitlt,

0168-1702/90/$03.50

Marburg, F.R.G.

0 1990 Ekevier Science Publishers B.V. (Biomedical Division)

238

1976; Scheid and Choppin, 1977; Richardson et al., 1980). The important role of the activation process for the pathogenicity of NDV has well been established. Fb of pathogenic strains is cleaved in a wide variety of cells both in vivo and in vitro, whereas F, of apathogenic strains is processed only in a limited number of cells, such as the endodermal cells of chick embryos. The high cleavability of the F protein of virulent NDV strains permits rapid spread of infectious virus particles ~ou~out the organism, resulting in systemic fatal disease (Nagai et al., 1976, 1979). Sequence analysis of F has revealed variations in the structure of the cleavage site which appear to account for the differences in NDV pathogenicity. With the pathogenic strains, this site consists of two pairs of the basic amino acids lysine and arginine. With the apathogenic strains, in contrast, one of the basic residues in each pair is replaced by an uncharged amino acid (Toyoda et al., 1987). Thus, it appears that the ubiquitous proteases responsible for the activation of the pathogenic strains require paired basic residues as a cleavage signal, whereas the rarely occurring proteases responsible for the activation of the apathogenic viruses recognize single basic residues. In the present study we have carried out a comparative sequence analysis of the F gene of NDV field strains and of a protease activation mutant derived by chemical mutagenesis from the apathogenic strain Ulster. We have previously shown that the F protein of this mutant is cleaved not only in chorio-allantoic membrane cells, but also in MDBK cells, and allows therefore multiple cycle replication in cells not permissive for the wild-type. The mutant was not activated, however, in other cells, such as MDCK, BHK, or chick embryo cells. This indicated that the increase in host range was not high enough to convert the mutant into a pantropic virus, which was therefore indistinguishable in its pathogemcity from the wild-type (Carten et al., 1980). Thus, compared to the vast differences between pathogenic and apathogenie field strains, the mutant exhibits only a subtle shift in host range and should therefore provide additional information on the factors determining the cleavability of F. Nucleotide sequence analysis was done on vRNA and mRNA. Virus was grown in 11-day-old chick embryos and purified by centrifugation on sucrose density gradients (Nagai et al., 1976). Viral RNA was isolated by digestion of purified virus with proteinase K followed by phenol and chloroform extraction. For preparation of mRNA, chick embryo fibroblasts were infected at an MO1 of 10 pfu/cell. After 8 h at 37 o C, cells were harvested, and CsCl pelleted RNA was prepared as described (Okayama and Berg, 1982). Poly(A + ) RNA was isolated by the oligo(dT) cellulose method following the protocol of Maniatis and coworkers (1982). Nucleotide sequences of the F genes were determined from total viral RNA and from poly(A + ) RNA,by the dideoxy chain termination method with the use of reverse transcriptase and synthetic oligonucleotides (Air et al., 1981). Eight oligonucleotide primers complements to the F gene of strain Miyadera and two primers implemental to the mRNA were synthesized. The reaction products of reverse transcription were resolved on 6% polyacrylamide-7 M urea thin slab gels in TBE buffer. Computer-assisted sequence analysis was performed using the Microgenie system (Beckman, Palo Alto, CA).

239

Fig. 1 shows the amino acid sequences derived from the nucleotide sequences of the F genes of Ulster mutant and wild-type, in addition to those of other NDV field strains that have been either also analyzed in the present study or have been derived from the literature. The list includes all F sequences determined to our knowledge to date, except for those of strains Bl (Toyoda et al., 1989) and Sato (Toyoda et al., 1988). As has been pointed out before (Toyoda et al., 1989), comparison of the sequences reveals high homology at the nucleotide (data not shown) and the amino acid level. Only the signal sequence at the N-terminus of the F2 subunit (residues 1-31) and the transmembrane region (residues 500-526) show significant differences. The cysteine positions are well conserved within all strains, including a cluster of six residues between positions 337 and 426. Six potential glycosylation sites were detected. One is located in the F2 subunit, three are in the ectodomain of the FI subunit and one is located in the cytoplasmic tail which may not be glycosylated. With strains Miyadera and Field Pheasant, there are two overlapping attachment sites at positions 191-194, of which most likely only one is glycosylated. The F protein of the NDV mutant differs from that of the wild-type by two point mutations. One of them, involving the exchange of Ala 125 for Val, is most likely functionally irrelevant, because all other strains contain also valine in this position. Crucial for the shift in cleavability, however, appears to be the other mutation which replaces Gly 115 by Arg and converts the sequence at the cleavage site from Gly-Lys-Gin-Gly-Arg to Gly-Lys-Gln-Arg-Arg. Thus, there is a gradient in the number of basic ammo acids at the cleavage site, ranging from two isolated residues with the apathogenic strains over one isolated and two paired residues with the mutant to four paired residues with the pathogenic strains, and this gradient matches the stepwise increase in cleavability. These observations further emphasize the general concept that the number of lysine and a&nine residues is an important dete~nant for this type of cleavage activation. Furthermore, they show specifically that both pairs of basic residues are required to render the F protein of NDV susceptible to ubiquitous cleavage, that the pair at positions 115 and 116 is insufficient for this property, and that each basic residue may contribute in a given cell to the cleavability of this glycoprotein. There is increasing evidence that a general consensus sequence for “high cleavability” is difficult to define. For instance, the ubiquitously cleaved precursor glycoproteins of Semliki Forest virus (Garoff et al., 1980) and Sindbis virus (Rice et al., 1981) resemble in their cleavage site the F protein of the Ulster mutant, whereas the cleavage sites of peptide hormones that are also cleaved in a large variety of different cells contain usually only one Lys-Arg or Arg-Arg pair (Steiner et al., 1980). Thus, it appears that the protease susceptibility of the clustered basic residues can be modulated by other structural features of the protein. With the influenza virus hemagglutinin, a carbohydrate side chain has been identified as one of these factors (Deshpande et al., 1988; Ohuchi et al.; 1989) which in most other instances, however, are not known. Studies on the influenza virus hemagglutinin have also provided evidence that the alteration of the cleavage site responsible for increased cleavability can be accomplished by point mutations resulting in the exchange of acidic or neutral for basic

240

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241

amino acids (Kawaoka and Webster, 1988; Ohuchi et al, 1989). Alternatively, there may be an insertion of basic residues or even an uncharged peptide, and evidence has been obtained that such insertions may originate from recombination with foreign RNA (Khatchikian et al, 1989). As has been pointed out before, only the former mechanism appears to occur with NDV (Toyoda et al., 1989) and this concept is also supported by the present study.

Acknowledgements

We thank Beate Siegmann for technical assistance and Sabine Fischbach for secretarial help. This work was supported by the Deutsche Forschungsgemeinschaft (KI 238/1-l) and by the Fonds der Chem. Industrie.

References Air, G.M. and Hall, R.M. (1981) In: D.P. Nayak (Ed.), Genetic Variation among Influenza Viruses, pp. 29-44, Academic Press, New York. Chambers, Ph., Millar, N.S. and Emmerson, P.T. (1986) J. Gen Virol. 67, 2685-2694. Deshpande, K.L., Fried, V.A., Ando, M. and Webster, R.G. (1987) Proc. Nat1 Acad. Sci. USA 84, 36-40. Espion, D., de Henau, S., Letellier, C., Wemers, C.D., Brasseur, R., Young, J.F., Gross, M., Rosenberg, M., Meulemans, G. and Burny, A. (1987) Arch. Virol. 95, 79-95. Garoff, H., Frischauf, A.M., Simons, K., Lehrach, H. and Delius, H. (1980) Nature (London) 288, 236-241. Garten, W., Berk, W., Nagai, Y., Rott, R. and Klenk, H.-D. (1980) J. Gen. Virol. 50, 135-147. Kawaoka, Y. and Webster, R.G. (1988) Proc. Natl. Acad. Sci. USA 85, 324-328. Khatchikian, D., Orlich, M. and Rott, R. (1989) Nature (London) 340, 156-157. Maniatis, T., Fritsch, E.F. and Sambrook, J. (1982) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. McGinnes, L.W. and Morrison, T.G. (1986) Virus Res. 5, 343-356. Millar, N.S., Chambers, Ph. and Emmerson, P.T. (1988) J. Gen. Virol. 69, 613-620. Nagai, Y., Klenk, H.-D. and Rott, R. (1976) Virology 72, 494-508. Nagai, Y., Shimokata, K., Yoshida, T., Hamaguchi, M., Iinuma, M., Maeno, K., Matsumoto, T., Klenk, H.-D. and Rott, R. (1979) J. Gen. Virol. 45, 263-272. Ohuchi, M., Grlich, M., Ohuchi, R., Simpson, B.E.J., Garten, W., KIenk, H.-D. and Rott, R. (1989) Virology 168, 274-280.

Fig. 1. Comparison of the amino acid sequences of the F protein of Ulster mutant and wild-type and 11 additional NDV strains. Differences from the reference strain Ulster are indicated. Hydrophobic domains are marked by straight lines, N-glycosylation sites by waved lines. The cleavage site is boxed. Sequences shown here for the fist time: ulmu = Ulster mutant, phea = Field Pheasant, warw = Warwick. Reexaminations of published sequences: ulst = Ulster wild type (Millar et al., 1988), laso = La Sota (Toyoda et al., 1989) texa = Texas (Schaper et al., 1988), ita-= Italien (Espion et al., 1987). Sequences obtained from the literature: d26-= (Sato et al., 1987), quee = Queensland (Toyoda et al., 1989), beau = Beaudette (Chambers et al., 1986), aust = Australia (MC Ginnes and Morrison, 1986), hert = Herts (Toyoda et al., 1989), miya = Miyadera (Toyoda et al., 1987).

242 Okayama, H., and Berg, P. (1982) Mol. Cell. Biol. 2, 161-172. Oroszlan, S., Henderson, L.E., Copeland, T.D., Schultz, A.M. and Rabin, E.M. (1980) In: G. Koch and D. Richter (Eds.), Biosynthesis, Modification and Processing of Cellular and Viral Proteins, pp. 236-241. Academic Press, New York. Rice, C.M. and Strauss, J.H. (1981) Proc. Natl. Acad. Sci. USA 178, 2062-2066. Richardson, C.D., Scheid, A. and Choppin, P.W. (1980) Virology 105,205-222. Sato, H., Oh-him, M., Ishida, N., Imamura, Y.. Hattori, S. and Kawakita, M. (1987) Virus Res. 7, 241-255. Schaper, U.M., Fuller, F.J., Ward, M.D.W., Mehrotra. Y., Stone, H.O., Stripp, B.R. and De Buyscher, E.V. (1988) Virology 165, 291-295. Scheid, A. and Choppin, P.W. (1977) Virology 80, 54-66. Steiner, D.F., Quinn, P.S., Chan, S.J., Marsk, J. and Tager, D.F. (1980) Ann. N.Y. Acad. Sci. 343, 1-16. Toyoda, T., Sakaguchi, T., Hirota, H., Gotoh, B., Kuma, K., Miyata, M. and Nagai, Y. (1989) Virology 169,273-282. Toyoda, T., Gotoh, B., Sakaguchi, T., Kida, H. and Nagai, Y. (1988) J. Virol. 62,4427-4430. Toyoda, T., Sakaguchi, T., Imai, U., Inocencio, N.M., Gotoh, B., Hamaguchi, M. and Nagai, Y. (1987) Virology 158, 242-247. (Received 8 September 1989; revision accepted 14 November 1989)