The product of the herpes simplex virus type 1 UL25 gene is required for encapsidation but not for cleavage of replicated viral DNA
The product of the herpes simplex virus type 1 UL25 gene is required for encapsidation but not for cleavage of replicated viral DNA
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DOI：
10.1128/jvi.72.2.1060-1070.1998
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发表时间：
1998-02-01
影响因子：
5.4
通讯作者：
Homa, FL
中科院分区：
文献类型：
--
作者：
McNab, AR;Desai, P;Homa, FL
单纯疱疹病毒1型（HSV-1）UL 25基因含有一个580个氨基酸的开放阅读框，编码一种必需蛋白。Ali等人，Virology 216：278-283，1996）参与病毒穿透和衣壳组装（C. Addison等人，Virology 138：246-259，1984）。在这项研究中，我们描述了一个UL 25突变体（KUL 25 NS）的分离，这是通过在UL 25开放阅读框中插入一个符合读框的终止密码子构建的，并在互补细胞系上繁殖。虽然该突变体能够合成病毒DNA，但它不会在非互补细胞中形成噬斑或产生感染性病毒。使用对UL 25蛋白特异的抗体证明KUL 25 NS感染的Vero细胞不表达UL 25蛋白，Western免疫印迹显示UL 25蛋白与纯化的野生型HSV A、B和C衣壳相关。透射电镜观察表明，KUL 25 NS感染的Vero细胞核内含有大量的A和B衣壳，但不含C衣壳。通过蔗糖梯度沉降分析对感染细胞的分析证实，在KUL 25 NS感染的Vero细胞中，A与B衣壳的比率升高。限制性内切酶消化后，在从KUL 25 NS感染的Vero细胞分离的DNA中观察到特异性末端片段，表明UL 25基因对于复制的病毒DNA的切割不是必需的。后一结果通过脉冲场凝胶电泳（PFGE）证实，其显示在KUL 25 NS感染的Vero细胞中存在基因组大小的病毒DNA，在PFGE之前的DNase I处理证明在不存在UL 25蛋白的情况下单体HSV DNA不被包装。我们的结果表明，UL 25基因的产物是包装所需的，但不是复制的病毒DNA的切割。
The herpes simplex virus type 1 (HSV-1) UL25 gene contains a 580-amino-acid open reading frame that codes for an essential protein, Previous studies have shown that the UL25 gene product is a virion component (M.A. Ali et al., Virology 216:278-283, 1996) involved in virus penetration and capsid assembly (C. Addison et al., Virology 138:246-259, 1984). In this study we describe the isolation of a UL25 mutant (KUL25NS) that was constructed by insertion of an in-frame stop codon in the UL25 open reading frame and propagated on a complementing cell line. Although the mutant was capable of synthesis of viral DNA, it did not form plaques or produce infectious virus in noncomplementing cells. Antibodies specific for the UL25 protein were used to demonstrate that KUL25NS-infected Vero cells did not express the UL25 protein, Western immunoblotting showed that the UL25 protein was associated with purified, wild-type HSV A, B, and C capsids. Transmission electron microscopy indicated that the nucleus of Vero cells infected with KUL25NS contained large numbers of both A and B capsids but no C capsids. Analysis of infected cells by sucrose gradient sedimentation analysis confirmed that the ratio of A to B capsids was elevated in KUL25NS-infected Vero cells. Following restriction enzyme digestion, specific terminal fragments were observed in DNA isolated from KUL25NS-infected Vero cells, indicating that the UL25 gene was not required for cleavage of replicated viral DNA. The latter result was confirmed by pulsed-field gel electrophoresis (PFGE), which showed the presence of genome-size viral DNA in KUL25NS-infected Vero cells, DNase I treatment prior to PFGE demonstrated that monomeric HSV DNA was not packaged in the absence of the UL25 protein, Our results indicate that the product of the UL25 gene is required for packaging but not cleavage of replicated viral DNA.