Division of Molecular Virology, Department of Microbiology and Immunology, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan Department of Infectious Disease Control, International Research Center for Infectious Diseases, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan.
Division of Molecular Virology, Department of Microbiology and Immunology, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan Department of Infectious Disease Control, International Research Center for Infectious Diseases, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan
J Virol. 2014 Jul;88(14):7776-85. doi: 10.1128/JVI.00603-14. Epub 2014 Apr 23.
We recently reported that herpes simplex virus 1 (HSV-1) protein kinase Us3 phosphorylated viral dUTPase (vdUTPase) at serine 187 (Ser-187) to upregulate its enzymatic activity, which promoted HSV-1 replication in human neuroblastoma SK-N-SH cells but not in human carcinoma HEp-2 cells. In the present study, we showed that endogenous cellular dUTPase activity in SK-N-SH cells was significantly lower than that in HEp-2 cells and that overexpression of cellular dUTPase in SK-N-SH cells increased the replication of an HSV-1 mutant with an alanine substitution for Ser-187 (S187A) in vdUTPase to the wild-type level. In addition, we showed that knockdown of cellular dUTPase in HEp-2 cells significantly reduced replication of the mutant vdUTPase (S187A) virus but not that of wild-type HSV-1. Furthermore, the replacement of Ser-187 in vdUTPase with aspartic acid, which mimics constitutive phosphorylation, and overexpression of cellular dUTPase restored viral replication to the wild-type level in cellular dUTPase knockdown HEp-2 cells. These results indicated that sufficient dUTPase activity was required for efficient HSV-1 replication and supported the hypothesis that Us3 phosphorylation of vdUTPase Ser-187 upregulated vdUTPase activity in host cells with low cellular dUTPase activity to produce efficient viral replication.virus. Importance: It has long been assumed that dUTPase activity is important for replication of viruses encoding a dUTPase and that the viral dUTPase (vdUTPase) activity was needed if host cell dUTPase activity was not sufficient for efficient viral replication. In the present study, we showed that the S187A mutation in HSV-1 vdUTPase, which impaired its enzymatic activity, reduced viral replication in SK-N-SH cells, which have low endogenous cellular dUTPase activity, and that overexpression of cellular dUTPase restored viral replication to the wild-type level. We also showed that knockdown of cellular dUTPase in HEp-2 cells, which have higher dUTPase activity than do SK-N-SH cells, reduced replication of HSV-1 with the vdUTPase mutation but had no effect on wild-type virus replication. This is the first report, to our knowledge, directly showing that dUTPase activity is critical for efficient viral replication and that vdUTPase compensates for low host cell dUTPase activity to produce efficient viral replication.
我们最近报道，单纯疱疹病毒 1（HSV-1）蛋白激酶 Us3 可将病毒 dUTP 酶（vdUTPase）磷酸化丝氨酸 187（Ser-187），从而上调其酶活性，这促进了 HSV-1 在人神经母细胞瘤 SK-N-SH 细胞中的复制，但在人癌细胞 HEp-2 细胞中却没有。在本研究中，我们发现 SK-N-SH 细胞中的内源性细胞 dUTPase 活性明显低于 HEp-2 细胞，并且在 SK-N-SH 细胞中过表达细胞 dUTPase 可将 vdUTPase 中 Ser-187 取代为丙氨酸的 HSV-1 突变体的复制增加到野生型水平。此外，我们发现细胞 dUTPase 在 HEp-2 细胞中的敲低可显著降低突变体 vdUTPase（S187A）病毒的复制，但对野生型 HSV-1 的复制没有影响。此外，vdUTPase 中的 Ser-187 被天冬氨酸取代，模拟组成性磷酸化，并且在细胞 dUTPase 敲低的 HEp-2 细胞中过表达细胞 dUTPase 可将病毒复制恢复到野生型水平。这些结果表明，有效的 HSV-1 复制需要足够的 dUTPase 活性，并支持 Us3 磷酸化 vdUTPase Ser-187 可上调宿主细胞中低细胞 dUTPase 活性的 vdUTPase 活性以产生有效病毒复制的假说。病毒。重要性：长期以来，人们一直认为 dUTPase 活性对于编码 dUTPase 的病毒的复制很重要，如果宿主细胞 dUTPase 活性不足以进行有效的病毒复制，则需要病毒的 dUTPase（vdUTPase）活性。在本研究中，我们发现 HSV-1 vdUTPase 中的 S187A 突变损害了其酶活性，从而降低了 SK-N-SH 细胞中的病毒复制，而 SK-N-SH 细胞中的内源性细胞 dUTPase 活性较低，而过表达细胞 dUTPase 可将病毒复制恢复至野生型水平。我们还发现，在 HEp-2 细胞（其 dUTPase 活性高于 SK-N-SH 细胞）中敲低细胞 dUTPase 会降低具有 vdUTPase 突变的 HSV-1 的复制，但对野生型病毒的复制没有影响。这是我们所知的第一个直接表明 dUTPase 活性对于有效的病毒复制至关重要并且 vdUTPase 可补偿宿主细胞低 dUTPase 活性以产生有效病毒复制的报告。
