Differential Requirement of Human Cytomegalovirus UL112-113 Protein Isoforms for Viral Replication
Differential Requirement of Human Cytomegalovirus UL112-113 Protein Isoforms for Viral Replication
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DOI：
10.1128/jvi.00254-17
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发表时间：
2017-09-01
影响因子：
5.4
通讯作者：
Brune, Wolfram
中科院分区：
文献类型：
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作者：
Schommartz, Tim;Tang, Jiajia;Brune, Wolfram
UL112-113基因是人巨细胞病毒(HCMV)为数不多的选择性剪接基因。它编码四种磷酸蛋白：p34、p43、p50和p84，所有这些蛋白都以早期动力学方式表达，并在宿主细胞核内的病毒DNA复制部位积聚。虽然已知这些蛋白质在病毒复制周期中扮演重要的、可能是必不可少的角色，但对单个UL112-113蛋白质产物的贡献知之甚少。在这里，我们使用剪接点突变、内含子缺失和替换以及无义突变来阻止每个UL112-113蛋白亚型的单独表达，并研究每个亚型对病毒复制的重要性。我们发现，缺乏p34或p50表达的巨细胞病毒突变株在人成纤维细胞和内皮细胞中复制到高滴度，表明这些蛋白对病毒复制不是必需的，而在p84编码序列内带有停止突变的突变株病毒严重损害了生长。P43表达失活后不能检测到病毒复制，这表明该UL112-113蛋白是病毒复制所必需的。我们还分析了UL112-113蛋白招募其他病毒蛋白到核内复制前间隔的能力。虽然UL112-113的表达足以招募UL44编码的病毒DNA聚合酶处理因子，但它不足以招募病毒UL84和UL117蛋白。值得注意的是，p43和p84亚型都是pUL44有效募集所必需的，这与它们在病毒生命周期中的关键作用是一致的。其中一个基因座UL112-113通过选择性剪接的方式编码四种具有共同N末端的蛋白质。在这项研究中，我们分别灭活了四个UL112-113蛋白的每一个的表达，并确定了它们对HCMV复制的要求。我们发现，UL112-113基因产物中的两个对于病毒在人成纤维细胞和内皮细胞中的复制是必不可少的。相反，当另外两个基因产物中的一个失活时，病毒复制严重减少或消失，这表明它们对病毒复制周期至关重要。我们进一步证明，后两种基因产物参与了pUL44的募集，pUL44是病毒DNA聚合酶的重要辅助因子，被认为是病毒DNA复制的起点。
The UL112-113 gene is one of the few alternatively spliced genes of human cytomegalovirus (HCMV). It codes for four phosphoproteins, p34, p43, p50, and p84, all of which are expressed with early kinetics and accumulate at sites of viral DNA replication within the host cell nucleus. Although these proteins are known to play important, possibly essential, roles in the viral replication cycle, little is known about the contribution of individual UL112-113 protein products. Here we used splice site mutagenesis, intron deletion and substitution, and nonsense mutagenesis to prevent the individual expression of each UL112-113 protein isoform and to investigate the importance of each isoform for viral replication. We show that HCMV mutants lacking p34 or p50 expression replicated to high titers in human fibroblasts and endothelial cells, indicating that these proteins are nonessential for viral replication, while mutant viruses carrying a stop mutation within the p84 coding sequence were severely growth impaired. Viral replication could not be detected upon the inactivation of p43 expression, indicating that this UL112-113 protein is essential for viral replication. We also analyzed the ability of UL112-113 proteins to recruit other viral proteins to intranuclear prereplication compartments. While UL112-113 expression was sufficient to recruit the UL44-encoded viral DNA polymerase processivity factor, it was not sufficient for the recruitment of the viral UL84 and UL117 proteins. Remarkably, both the p43 and p84 isoforms were required for the efficient recruitment of pUL44, which is consistent with their critical role in the viral life cycle.IMPORTANCE Human cytomegalovirus requires gene products from 11 genetic loci for the lytic replication of its genome. One of these loci, UL112-113, encodes four proteins with common N termini by alternative splicing. In this study, we inactivated the expression of each of the four UL112-113 proteins individually and determined their requirement for HCMV replication. We found that two of the UL112-113 gene products were dispensable for viral replication in human fibroblasts and endothelial cells. In contrast, viral replication was severely reduced or absent when one of the other two gene products was inactivated, indicating that they are of crucial importance for the viral replication cycle. We further showed that the latter two gene products are involved in the recruitment of pUL44, an essential cofactor of the viral DNA polymerase, to specific sites within the cell nucleus that are thought to serve as starting points for viral DNA replication.