MUTATIONS IN ACCESSORY DNA REPLICATING FUNCTIONS ALTER THE RELATIVE MUTATION FREQUENCY OF HERPES-SIMPLEX VIRUS TYPE-1 STRAINS IN CULTURED MURINE CELLS
MUTATIONS IN ACCESSORY DNA REPLICATING FUNCTIONS ALTER THE RELATIVE MUTATION FREQUENCY OF HERPES-SIMPLEX VIRUS TYPE-1 STRAINS IN CULTURED MURINE CELLS
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DOI：
10.1128/jvi.68.7.4514-4524.1994
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发表时间：
1994-07-01
影响因子：
5.4
通讯作者：
THOMPSON, RL
中科院分区：
文献类型：
--
作者：
PYLES, RB;THOMPSON, RL
研究了单纯疱疹病毒1型（HSV-1）编码的尿嘧啶DNA糖基化酶（UNG）、胸苷激酶（TK）和dUTR对培养鼠细胞中病毒相对突变频率（RMF）的贡献。通过用大肠杆菌β-半乳糖苷酶（β-gal）基因破坏菌株17 syn（+）中的酶编码区，产生了一组单一或双重缺乏UNG、TK或dUTR活性的HSV-1突变体。为了建立菌株17 syn（+）的基线RMF，将β-gal基因插入UL 3基因座。在所有病毒中，β-gal插入片段作为RMP的表型标记。一个突变的斑块，通过缺乏β-半乳糖活性来鉴定，在选定的病例中，β-半乳糖序列的原位杂交阳性。在NIH 3 T3细胞中的复制动力学表明，所有的突变体有效地复制，产生具有相等滴度的储备。检查了两种独立生成的UL 3-β-gal病毒，并在NIH 3 T3和LM TK-细胞中确立了与0.5%相似的基线RMF。dUTR活性的丧失导致病毒的RMF增加5倍，表明HSV-1 dUTR具有抗突变功能。观察到的tk（-）病毒的RMF降低了40倍（RMF为0.02%），表明病毒TK具有致突变活性。在有限传代中，蒂托非依赖性UNG（-）病毒的RMF与基线RMF无显著差异;然而，在连续传代后，数据表明UNG活性可作为抗突变剂。这些结果对单纯疱疹病毒的自然史和抗病毒疗法的开发具有影响。
The contribution of the herpes simplex virus type 1 (HSV-1)-encoded uracil DNA glycosylase (UNG), thymidine kinase (TK), and dUTPase to the relative mutant frequency (RMF) of the virus in cultured murine cells was examined. A panel of HSV-1 mutants that lacked singly or doubly the UNG, TK, or dUTPase activity were generated by disruption of the enzyme coding regions with the Escherichia coli beta-galactosidase (beta-gal) gene in strain 17syn(+). To establish a baseline RMF of strain 17syn(+), the beta-gal gene was inserted into the UL3 locus. In all of the viruses, the beta-gal insert served as a phenotypic marker of RMP. A mutant plaque,vas identified by the lack of beta-gal activity and, in selected cases, positive in situ hybridization for beta-gal sequences. Replication kinetics in NIH 3T3 cells demonstrated that all of the mutants replicated efficiently, generating stocks with equivalent titers. Two independently generated UL3-beta-gal viruses were examined and established a baseline RMF of similar to 0.5% in both NIH 3T3 and LM TK- cells. Loss of dUTPase activity resulted in viruses with fivefold-increased RMFs, indicating that the HSV-1 dUTPase has an antimutator function. The RMF observed for the tk(-) viruses was reduced as much as 40-fold (RMF of 0.02%), suggesting that the viral TK is a mutator activity. The RMF of tito independent UNG(-) viruses showed no significant difference from the baseline RMF in limited passage; however, following successive passage, the data suggested that UNG activity serves as an antimutator. These results have implications for the natural history of HSV and the development of antiviral therapies.