Castrucci M R, Hughes M, Calzoletti L, Donatelli I, Wells K, Takada A, Kawaoka Y
Department of Virology, Istituto Superiore di Sanita, Rome, Italy.
Virology. 1997 Nov 10;238(1):128-34. doi: 10.1006/viro.1997.8809.
The M2 protein of influenza A virus functions as an ion channel. It contains three cysteine residues: cysteines 17 and 19, which form disulfide bonds in the ectodomain, and cysteine 50 which is acylated. To understand the role of these cysteine residues in virus replication, we used reverse genetics to create influenza viruses in which the individual cysteines were mutated and a virus in which all three cysteines were changed to serine. The M2 cysteine mutants that lacked either of the cysteine residues in the ectodomain and the mutant that lacked all three residues had appreciably lower amounts of M2 oligomers than did the wild-type virus when examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. None of the mutants, however, were defective in replication, either in vitro or in ferrets and mice. These findings demonstrate that noncovalent interactions are sufficient for the M2 protein to form functional oligomers for virus replication and that its cysteine residues are dispensable for influenza virus replication in vitro and in vivo.
甲型流感病毒的M2蛋白作为一种离子通道发挥作用。它含有三个半胱氨酸残基：位于胞外域中形成二硫键的半胱氨酸17和19，以及被酰化的半胱氨酸50。为了解这些半胱氨酸残基在病毒复制中的作用，我们利用反向遗传学创建了单个半胱氨酸发生突变的流感病毒，以及一个将所有三个半胱氨酸都替换为丝氨酸的病毒。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳检测发现，胞外域缺少任何一个半胱氨酸残基的M2半胱氨酸突变体以及缺少所有三个残基的突变体，其M2寡聚体的量都明显低于野生型病毒。然而，无论是在体外，还是在雪貂和小鼠体内，这些突变体在复制方面均无缺陷。这些发现表明，非共价相互作用足以使M2蛋白形成用于病毒复制的功能性寡聚体，并且其半胱氨酸残基对于流感病毒在体外和体内的复制而言并非必需。
