Murine Hepatitis Virus Nonstructural Protein 4 Regulates Virus-Induced Membrane Modifications and Replication Complex Function
Murine Hepatitis Virus Nonstructural Protein 4 Regulates Virus-Induced Membrane Modifications and Replication Complex Function
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DOI：
10.1128/jvi.01772-09
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发表时间：
2010-01-01
影响因子：
5.4
通讯作者：
Denison, Mark R.
中科院分区：
文献类型：
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作者：
Gadlage, Mark J.;Sparks, Jennifer S.;Denison, Mark R.
正链RNA病毒诱导细胞质膜的修饰以形成复制复合物。对于冠状病毒，复制酶非结构蛋白 4 (nsp4) 被认为在复制复合物的形成和组织中发挥作用。在细胞中 nsp4 的质粒表达过程中，鼠肝炎病毒 (MHV) nsp4 在残基 Asn176 (N176) 和 N237 处被糖基化。为了测试 MHV nsp4 残基 N176 和 N237 在病毒复制过程中是否被糖基化，并确定 N176 和 N237 对 nsp4 功能和 MHV 复制的影响，nsp4 N176、N237 或两者的丙氨酸取代被工程化到 MHV-A59 基因组中。 N176A、N237A 和 N176A/N237A 突变病毒是可行的，并且 N176 和 N237 在野生型 (wt) 和突变病毒感染期间被糖基化。 nsp4糖基化突变体表现出病毒生长和RNA合成受损，其中N237A和N176A/N237A突变体病毒在病毒生长和RNA合成方面表现出更严重的缺陷。对感染细胞超微结构的电子显微镜分析表明，与感染 wt 病毒的细胞相比，nsp4 突变体具有病毒诱导的双膜囊泡 (DMV) 形态异常。 DMV形态改变的程度与nsp4突变病毒的病毒RNA合成和病毒生长的受损程度直接相关。结果表明，nsp4 在 DMV 的组织和稳定性中起着至关重要的作用。结果还支持这样的结论：DMV 的结构对于冠状病毒的有效 RNA 合成和最佳复制至关重要。
Positive-strand RNA viruses induce modifications of cytoplasmic membranes to form replication complexes. For coronaviruses, replicase nonstructural protein 4 (nsp4) has been proposed to function in the formation and organization of replication complexes. Murine hepatitis virus (MHV) nsp4 is glycosylated at residues Asn176 (N176) and N237 during plasmid expression of nsp4 in cells. To test if MHV nsp4 residues N176 and N237 are glycosylated during virus replication and to determine the effects of N176 and N237 on nsp4 function and MHV replication, alanine substitutions of nsp4 N176, N237, or both were engineered into the MHV-A59 genome. The N176A, N237A, and N176A/N237A mutant viruses were viable, and N176 and N237 were glycosylated during infection of wild-type (wt) and mutant viruses. The nsp4 glycosylation mutants exhibited impaired virus growth and RNA synthesis, with the N237A and N176A/N237A mutant viruses demonstrating more profound defects in virus growth and RNA synthesis. Electron microscopic analysis of ultrastructure from infected cells demonstrated that the nsp4 mutants had aberrant morphology of virus-induced double-membrane vesicles (DMVs) compared to those infected with wt virus. The degree of altered DMV morphology directly correlated with the extent of impairment in viral RNA synthesis and virus growth of the nsp4 mutant viruses. The results indicate that nsp4 plays a critical role in the organization and stability of DMVs. The results also support the conclusion that the structure of DMVs is essential for efficient RNA synthesis and optimal replication of coronaviruses.