Jia Qingmei, Wu Ting-Ting, Liao Hsiang-I, Chernishof Vasili, Sun Ren
Department of Molecular and Medical Pharmacology, University of California at Los Angeles, Los Angeles, CA 90095, USA.
J Virol. 2004 Jun;78(12):6610-20. doi: 10.1128/JVI.78.12.6610-6620.2004.
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 MHV-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.
小鼠γ-疱疹病毒68（MHV-68）在基因上与人类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）相关。它已被提议作为γ-疱疹病毒感染和发病机制的模型。开放阅读框31（ORF31）在β-疱疹病毒亚科和γ-疱疹病毒亚科中是保守的，并且该蛋白没有已知的哺乳动物同源物。MHV-68 ORF31及其病毒同源物的功能尚未确定。我们在此描述了该蛋白的初步特征及其对裂解复制的需求。感染后24小时可检测到天然MHV-68 ORF31的峰值水平，带有FLAG标签和绿色荧光蛋白融合的ORF31以弥散模式定位于细胞质和细胞核中。然后利用两种独立的实验方法证明ORF31是裂解复制所必需的。首先，针对ORF31表达产生的小干扰RNA阻断了转染细胞中的蛋白表达和病毒产生。然后，产生了两个独立的源自细菌人工染色体的ORF31缺失的MHV-68突变体（31STOP），并发现它们在成纤维细胞中的病毒产生存在缺陷。这种缺陷可以通过MHV-68 ORF31反式挽救，重要的是可以通过其KSHV同源物挽救。还通过在成纤维细胞中的同源重组产生了31STOP的修复病毒。最后，我们表明ORF31缺陷阻断了晚期裂解蛋白表达。我们的结果表明，MHV-68 ORF31是病毒裂解复制所必需的，并且其功能在其KSHV同源物中是保守的。
