Jiang Changying, Hwang Ying T, Randell John C W, Coen Donald M, Hwang Charles B C
Department of Microbiology and Immunology, State University of New York, Upstate Medical University, 750 E. Adams St., Syracuse, NY 13210, USA.
J Virol. 2007 Apr;81(7):3495-502. doi: 10.1128/JVI.02359-06. Epub 2007 Jan 17.
The processivity subunit of the herpes simplex virus DNA polymerase, UL42, is essential for viral replication and possesses both Pol- and DNA-binding activities. Previous studies demonstrated that the substitution of alanine for each of four arginine residues, which reside on the positively charged surface of UL42, resulted in decreased DNA binding affinity and a decreased ability to synthesize long-chain DNA by the polymerase. In this study, the effects of each substitution on the production of viral progeny, viral DNA replication, and DNA replication fidelity were examined. Each substitution mutant was able to complement the replication of a UL42 null mutant in transient complementation assays and to support the replication of plasmid DNA containing herpes simplex virus type 1 (HSV-1) origin sequences in transient DNA replication assays. Mutant viruses containing each substitution and a lacZ insertion in a nonessential region of the genome were constructed and characterized. In single-cycle growth assays, the mutants produced significantly less progeny virus than the control virus containing wild-type UL42. Real-time PCR assays revealed that these UL42 mutants synthesized less viral DNA during the early phase of infection. Interestingly, during the late phase of infection, the mutant viruses synthesized larger amounts of viral DNA than the control virus. The frequencies of mutations of the virus-borne lacZ gene increased significantly in the substitution mutants compared to those observed for the control virus. These results demonstrate that the reduced DNA binding of UL42 is associated with significant effects on virus yields, viral DNA replication, and replication fidelity. Thus, a processivity factor can influence replication fidelity in mammalian cells.
单纯疱疹病毒DNA聚合酶的持续合成亚基UL42对病毒复制至关重要，且具有与Pol结合及与DNA结合的活性。先前的研究表明，将位于UL42带正电荷表面的四个精氨酸残基逐个替换为丙氨酸，会导致DNA结合亲和力降低，以及聚合酶合成长链DNA的能力下降。在本研究中，检测了每种替换对病毒子代产生、病毒DNA复制及DNA复制保真度的影响。在瞬时互补试验中，每个替换突变体都能够互补UL42缺失突变体的复制，并在瞬时DNA复制试验中支持含有1型单纯疱疹病毒（HSV-1）起源序列的质粒DNA的复制。构建并鉴定了在基因组非必需区域含有每种替换和一个lacZ插入的突变病毒。在单周期生长试验中，这些突变体产生的子代病毒明显少于含有野生型UL42的对照病毒。实时PCR试验显示，这些UL42突变体在感染早期合成的病毒DNA较少。有趣的是，在感染后期，突变病毒合成的病毒DNA量比对照病毒多。与对照病毒相比，替换突变体中病毒携带的lacZ基因的突变频率显著增加。这些结果表明，UL42与DNA结合能力的降低对病毒产量、病毒DNA复制及复制保真度有显著影响。因此，一个持续合成因子可以影响哺乳动物细胞中的复制保真度。
