Alteration of Sendai virus morphogenesis and nucleocapsid incorporation due to mutation of cysteine residues of the matrix protein
Alteration of Sendai virus morphogenesis and nucleocapsid incorporation due to mutation of cysteine residues of the matrix protein
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DOI：
10.1128/jvi.76.4.1682-1690.2002
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发表时间：
2002-02-01
影响因子：
5.4
通讯作者：
Yoshida, T
中科院分区：
文献类型：
--
作者：
Sakaguchi, T;Uchiyama, T;Yoshida, T
仙台病毒(SeV)的基质(M)蛋白有5个半胱氨酸残基，分别位于83、106、158、251和295位。为了确定半胱氨酸残基在病毒组装中的作用，我们构建了半胱氨酸残基中的一个或全部半胱氨酸残基上具有丝氨酸取代作用的突变体M。一些突变的M蛋白在培养细胞中表达时是不稳定的，这表明半胱氨酸残基可能通过破坏正确的构象来影响蛋白质的稳定性。在从cDNA中提取病毒的尝试中，成功地从cDNA中回收了SeV M-C83S、SeV M-C106S和SeV M-C295s，而具有其他突变的重组SEV则没有成功。SEV M-C83S和SEV M-C106S的病毒颗粒均小于野生型，而SEV M-C295S的病毒颗粒较大且大小不均。此外，SeV M-C106S含有大量无核衣壳的空颗粒。这些结果表明，M蛋白半胱氨酸残基的单点突变影响了病毒的形态和核衣壳的掺入，表明M蛋白直接参与了SeV的组装。半胱氨酸依赖的M蛋白的构象不是由于二硫键的形成，因为半胱氨酸在病毒的整个生命周期中是自由的。
The matrix (M) protein of Sendai virus (SeV) has five cysteine residues, at positions 83, 106, 158, 251, and 295. To determine the roles of the cysteine residues in viral assembly, we generated mutant M cDNA possessing a substitution to serine at one of the cysteine residues or at all of the cysteine residues. Some mutant M proteins were unstable when expressed in cultured cells, suggesting that cysteine residues affect protein stability, probably by disrupting the proper conformation. In an attempt to generate virus from cDNA, SeV M-C83S, SeV M-C106S, and SeV M-C295S were successfully recovered from cDNA, while recombinant SeVs possessing other mutations were not. SeV M-C83S and SeV M-C106S,S had smaller virus particles than did the wild-type SeV, whereas SeV M-C295S had larger and heterogeneously sized particles. Furthermore, SeV M-C106S had a significant amount of empty particles lacking nucleocapsids. These results indicate that a single-point mutation at a cysteine residue of the M protein affects virus morphology and nucleocapsid incorporation, showing direct involvement of the M protein in SeV assembly. Cysteine-dependent conformation of the M protein was not due to disulfide bond formation, since the cysteines were shown to be free throughout the viral life cycle.