Characterization of the amino acid residues of Sendai virus C protein that are critically involved in its interferon antagonism and RNA synthesis down-regulation
Characterization of the amino acid residues of Sendai virus C protein that are critically involved in its interferon antagonism and RNA synthesis down-regulation
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DOI：
10.1128/jvi.78.14.7443-7454.2004
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发表时间：
2004-07-01
影响因子：
5.4
通讯作者：
Nagai, Y
中科院分区：
文献类型：
--
作者：
Kato, A;Cortese-Grogan, C;Nagai, Y
仙台病毒（SeV）在P基因的交替阅读框中编码两种辅助蛋白，V和C，其通过转录（V）或转录（C）访问。C蛋白表达为使用不同起始密码子的四个C-共末端蛋白C '、C、Y1和Y2的嵌套集合。使用组成型表达各种C蛋白的HeLa细胞系，我们先前发现四种C蛋白中最小的（175个残基Y2）完全能够抵消干扰素（IFN）的抗病毒作用并抑制病毒RNA合成，并且106个残基的C末端的一半足以实现这两种抑制功能（A. Kato等人，J. Virol. 75：3802 - 3810，2001，和A. Kato等人，J. Virol. 76：7114 - 7124，2002）。在这里，我们进一步产生了表达突变C（Cm）蛋白的HeLa细胞系，其中带电氨基酸取代了位置77和80处的丙氨酸残基; 114和115; 139和142; 151、153和154; 156;或173、175和176。我们发现只有151、153和154位的突变消除了IFN拮抗作用。在我们的测定条件下，所有的Cm蛋白都失去了与STAT1结合的能力，而不管它们抑制IFN信号传导的能力如何。另一方面，改变STAT1和STAT2的酪氨酸磷酸化和去磷酸化的Cm蛋白始终保留IFN拮抗作用。因此，磷酸化或去磷酸化的异常似乎是SeV C对IFN拮抗作用的原因。关于病毒RNA合成抑制，除了在位置114和115处具有替换的突变体之外的所有突变体都大大降低了抑制活性，表明C蛋白的抗RNA合成由散布在其C-末端的氨基酸控制。因此，IFN拮抗和RNA合成抑制之间的氨基酸序列要求有很大差异。此外，我们证实，另一种SeV辅助蛋白，V，不拮抗IFN。
Sendai virus (SeV) encodes two accessory proteins, V and C, in the alternative reading frames in the P gene that are accessed transcriptionally (V) or transtationally (C). The C protein is expressed as a nested set of four C-coterminal proteins, C', C, Y1, and Y2, that use different initiation codons. Using HeLa cell lines constitutively expressing the various C proteins, we previously found that the smallest (the 175-residue Y2) of the four C proteins was fully capable of counteracting the antiviral action of interferons (IFNs) and inhibiting viral RNA synthesis and that the C-terminal half of 106 residues was sufficient for both of these inhibitory functions (A. Kato et al., J. Virol. 75:3802-3810, 2001, and A. Kato et al., J. Virol. 76:7114-7124, 2002). Here, we further generated HeLa cell lines expressing the mutated C (Cm) proteins with charged amino acids substituted for alanine residues at either positions 77 and 80; 114 and 115; 139 and 142; 151, 153, and 154; 156; or 173, 175, and 176. We found that only the mutations at positions 151, 153, and 154 abolished IFN antagonism. All the Cm proteins lost the ability to bind with STAT1 under our assay conditions, regardless of their ability to inhibit IFN signaling. On the other hand, the Cm proteins that altered the tyrosine phosphorylation and dephosphorylation of STAT1 and STAT2 always retained IFN antagonism. Thus, the abnormality of phosphorylation or dephosphorylation appeared to be a cause of the IFN antagonism by SeV C. Regarding viral RNA synthesis inhibition, all mutants but the mutant with replacements at positions 114 and 115 greatly reduced the inhibitory activity, indicating that anti-RNA synthesis by the C protein is governed by amino acids scattered across its C-terminal halt Thus, amino acid sequence requirements differ greatly between IFN antagonism and RNA synthesis inhibition. In addition, we confirmed that another SeV accessory protein, V, does not antagonize IFN.