Modification of the major tegument protein pp65 of human cytomegalovirus inhibits virus growth and leads to the enhancement of a protein complex with pUL69 and pUL97 in infected cells
Modification of the major tegument protein pp65 of human cytomegalovirus inhibits virus growth and leads to the enhancement of a protein complex with pUL69 and pUL97 in infected cells
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DOI：
10.1099/vir.0.022293-0
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发表时间：
2010-10-01
影响因子：
3.8
通讯作者：
Reyda, Sabine
中科院分区：
文献类型：
--
作者：
Becke, Sabine;Fabre-Mersseman, Veronique;Reyda, Sabine
人巨细胞病毒(HCMV)被膜蛋白pp65在裂解感染的人包皮成纤维细胞(HFF)、病毒粒子和亚病毒致密小体(DB)中含量丰富。尽管如此，我们之前已经证明pp65对于HFF的生长是必不可少的。在提纯基于DB的候选疫苗的过程中，产生了不同的HCMV突变体，表达了融合到pp65的IE1蛋白的一个主要的HLA-A2呈现的多肽。其中一种突变病毒(RV-VM1)令人惊讶地显示出从HFF释放病毒的显著障碍。我们假设，对RV-VM1表型变化的分析将提供对pp65功能的洞察，到目前为止还没有明确的定义。免疫共沉淀实验表明，野生型(Wt)pp65和pp65-VM1是病毒pUL97蛋白的体外底物，与病毒RNA输出蛋白pUL69和感染细胞裂解物中的pUL97形成复合体。在这个复合体中没有发现pUL97受损的证据。然而，RV-VM1在感染细胞中的复制对pUL97抑制剂具有抵抗力，pUL97抑制剂与突变体相似，pp65保留在细胞核中。结果表明，RV-VM1的生命周期在转录早期-晚期、RNA输出或衣壳成熟阶段受阻。WT-pp65可能在感染的这些阶段发挥作用，与pUL69和pUL97形成的复合体可能对该功能起重要作用。
The tegument protein pp65 of human cytomegalovirus (HCMV) is abundant in lytically infected human foreskin fibroblasts (HFF), as well as in virions and subviral dense bodies (DB). Despite this, we showed previously that pp65 is dispensable for growth in HFF. In the process of refining a DB-based vaccine candidate, different HCMV mutants were generated, expressing a dominant HLA-A2-presented peptide of the IE1 protein fused to pp65. One of the mutant viruses (RV-VM1) surprisingly showed marked impairment in virus release from HFF. We hypothesized that analysis of the phenotypic alterations of RV-VM1 would provide insight into the functions of pp65, poorly defined thus far. RV-VM1 infection resulted in nuclear retention of the fusion protein and reorganization of nuclear inclusion bodies Coimmunoprecipitation experiments suggested that wild-type (wt) pp65 and pp65-VM1 were substrates of the viral pUL97 kinase in vitro and formed a complex with the viral RNA-export protein pUL69 and with pUL97 in lysates of infected cells. No evidence for an impairment of pUL97 within this complex was found. However, RV-VM1 replication in infected cells was resistant to a pUL97 inhibitor, and pUL97 inhibitors mimicked the mutant in terms of pp65 being retained in the nucleus The results suggest that the life cycle of RV-VM1 was impeded at the stages of early-late transcription, RNA export or capsid maturation. wt-pp65 may play a role at these stages of infection, and complex formation with pUL69 and pUL97 may be important for that function.