Murine gammaherpesvirus 68 open reading frame 31 is required for viral replication
Murine gammaherpesvirus 68 open reading frame 31 is required for viral replication
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DOI：
10.1128/jvi.78.12.6610-6620.2004
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发表时间：
2004-06-01
影响因子：
5.4
通讯作者：
Sun, R
中科院分区：
文献类型：
--
作者：
Jia, QM;Wu, TT;Sun, R
小鼠γ疱疹病毒68 （MHV-68）与人类γ疱疹病毒、卡波西肉瘤相关疱疹病毒（KSHV/HHV-8）和爱泼斯坦-巴尔病毒（EBV）有遗传关系。它被认为是伽玛疱疹病毒感染和发病机制的一个模型。开放阅读框31 （ORF31）在Beta-和Gammaherpesvirinae亚家族中是保守的，并且没有已知的哺乳动物同源蛋白。MHV-68 ORF31及其病毒同源物的功能尚未确定。我们在这里描述了该蛋白的主要特征及其对裂解复制的要求。在感染后24 h检测到MRV-68 ORF31的天然水平达到峰值，并且flag标记和绿色荧光蛋白融合的ORF31以弥漫性模式定位于细胞质和细胞核中。然后使用两种独立的实验方法来证明ORF31是裂解复制所必需的。首先，针对ORF31表达产生的小干扰RNA阻断了转染细胞中蛋白的表达和病毒的产生。然后，产生了两个独立的细菌人工染色体来源的ORF31-null MHV-68突变体（31STOP），并发现成纤维细胞中的病毒产生有缺陷。这种缺陷可以通过MHV-68 ORF31转运，更重要的是通过其KSHV同源物转运。在成纤维细胞中通过同源重组获得了31STOP修复病毒。最后，我们发现ORF31的缺陷阻断了后期裂解蛋白的表达。我们的研究结果表明MHV-68 ORF31是病毒裂解复制所必需的，其功能在其KSHV同源物中是保守的。
Murine gammaherpesvirus 68 (MHV-68) is genetically related to the human gammaherpesviruses, Kaposi's sarcoma-associated herpesvirus (KSHV/HHV-8) and Epstein-Barr virus (EBV). It has been proposed as a model for gammaherpesvirus infection and pathogenesis. Open reading frame 31 (ORF31) is conserved among the Beta- and Gammaherpesvirinae subfamily, and there is no known mammalian homologue of this protein. The function of MHV-68 ORF31 and its viral homologues has not yet been determined. We described here a primary characterization of this protein and its requirement for lytic replication. The native MRV-68 ORF31 was detected at peak levels by 24 h postinfection, and the FLAG-tagged and green fluorescent protein fusion ORF31 were localized in the cytoplasm and nucleus in a diffuse pattern. Two independent experimental approaches were then utilized to demonstrate that ORF31 was required for lytic replication. First, small interfering RNA generated against ORF31 expression blocked protein expression and virus production in transfected cells. Then, two-independent bacterial artificial chromosome-derived ORF31-null MHV-68 mutants (31STOP) were generated and found to be defective in virus production in fibroblast cells. This defect can be rescued in trans by MHV-68 ORF31 and importantly by its KSHV homologue. A repair virus of 31STOP was also generated by homologous recombination in fibroblast cells. Finally, we showed that the defect in ORF31 blocked late lytic protein expression. Our results demonstrate that MHV-68 ORF31 is required for viral lytic replication, and its function is conserved in its KSHV homologue.