Human cytomegalovirus pUL79 is an elongation factor of RNA polymerase II for viral gene transcription.
Human cytomegalovirus pUL79 is an elongation factor of RNA polymerase II for viral gene transcription.
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人巨细胞病毒PUL79是病毒基因转录的RNA聚合酶II的伸长因子。
DOI：
10.1371/journal.ppat.1004350
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发表时间：
2014-08
期刊：
影响因子：
6.7
通讯作者：
Yu D
中科院分区：
文献类型：
--
作者：
Perng YC;Campbell JA;Lenschow DJ;Yu D
在这项研究中，我们已经确定了一个独特的机制，其中人巨细胞病毒（HCMV）蛋白pUL 79作为延伸因子，以指导细胞RNA聚合酶II的病毒转录在感染后期。我们和其他人以前报道，pUL 79及其同源物是病毒DNA合成后病毒转录物积累所必需的。我们假设pUL 79代表了在HCMV感染后期调节病毒转录的独特机制。为了验证这一假设，我们通过质谱分析了病毒感染过程中与pUL 79相关的蛋白质组。我们确定了细胞转录因子，包括多个RNA聚合酶II（RNAP II）亚基，和新的病毒反式激活因子，包括pUL 87和pUL 95，作为pUL 79的蛋白结合伴侣。免疫共沉淀（co-IP），随后进行免疫印迹分析，证实了pUL 79-RNAP II相互作用，这种相互作用是独立的任何其他病毒蛋白。使用重组HCMV病毒，其中pUL 79蛋白由蛋白质去稳定化结构域ddFKBP有条件地调节，我们表明这种相互作用不会改变RNAP II的总水平或其向病毒晚期启动子的募集。此外，pUL 79没有改变RNAP II C-末端结构域的磷酸化特征，这对转录调控至关重要。相反，核连续测定表明，在不存在pUL 79的情况下，RNAP II不能伸长并停滞在病毒DNA上。pUL 79依赖性RNAP II延伸是在病毒感染后期从所有三种动力学类型的病毒基因（即，立即早期、早期和晚期）转录所必需的。相反，在HCMV感染过程中宿主基因的转录是独立的pUL 79。总之，我们已经确定了一种新的病毒机制，pUL 79和潜在的其他病毒因子，调节速率的RNAP II转录机制对病毒转录在HCMV感染的晚期阶段。在这项研究中，我们报告了一种新的机制，用于人巨细胞病毒（HCMV）调节RNA聚合酶II（RNAP II）的延伸率，以促进病毒在感染后期的转录。最近，我们和其他人已经确定了几个病毒因子，调节基因表达在晚期感染。这些因子在β-和γ-疱疹病毒中功能保守，表明这两个亚科的病毒共享独特的转录调控。然而，这一机制仍然难以捉摸。在这里，我们表明其中一种因子HCMV pUL 79与RNAP II以及其他参与晚期基因表达的病毒因子相互作用。我们已经开始阐明pUL 79-RNAP II相互作用的性质，发现pUL 79不改变RNAP II的蛋白水平或其向病毒启动子的募集。然而，在感染后期，pUL 79帮助RNAP II有效地沿着病毒DNA模板沿着延伸以转录HCMV基因。宿主基因不受这种pUL 79介导的机制调节。因此，我们的研究发现了一种以前未表征的机制，其中RNAP II活性受病毒因子pUL 79以及潜在的其他病毒因子的调节，用于协调病毒转录。
In this study, we have identified a unique mechanism in which human cytomegalovirus (HCMV) protein pUL79 acts as an elongation factor to direct cellular RNA polymerase II for viral transcription during late times of infection. We and others previously reported that pUL79 and its homologues are required for viral transcript accumulation after viral DNA synthesis. We hypothesized that pUL79 represented a unique mechanism to regulate viral transcription at late times during HCMV infection. To test this hypothesis, we analyzed the proteome associated with pUL79 during virus infection by mass spectrometry. We identified both cellular transcriptional factors, including multiple RNA polymerase II (RNAP II) subunits, and novel viral transactivators, including pUL87 and pUL95, as protein binding partners of pUL79. Co-immunoprecipitation (co-IP) followed by immunoblot analysis confirmed the pUL79-RNAP II interaction, and this interaction was independent of any other viral proteins. Using a recombinant HCMV virus where pUL79 protein is conditionally regulated by a protein destabilization domain ddFKBP, we showed that this interaction did not alter the total levels of RNAP II or its recruitment to viral late promoters. Furthermore, pUL79 did not alter the phosphorylation profiles of the RNAP II C-terminal domain, which was critical for transcriptional regulation. Rather, a nuclear run-on assay indicated that, in the absence of pUL79, RNAP II failed to elongate and stalled on the viral DNA. pUL79-dependent RNAP II elongation was required for transcription from all three kinetic classes of viral genes (i.e. immediate-early, early, and late) at late times during virus infection. In contrast, host gene transcription during HCMV infection was independent of pUL79. In summary, we have identified a novel viral mechanism by which pUL79, and potentially other viral factors, regulates the rate of RNAP II transcription machinery on viral transcription during late stages of HCMV infection. In this study, we report a novel mechanism used by human cytomegalovirus (HCMV) to regulate the elongation rate of RNA polymerase II (RNAP II) to facilitate viral transcription during late stages of infection. Recently, we and others have identified several viral factors that regulate gene expression during late infection. These factors are functionally conserved among beta- and gamma- herpesviruses, suggesting a unique transcriptional regulation shared by viruses of these two subfamilies. However, the mechanism remains elusive. Here we show that HCMV pUL79, one of these factors, interacts with RNAP II as well as other viral factors involved in late gene expression. We have started to elucidate the nature of the pUL79-RNAP II interaction, finding that pUL79 does not alter the protein levels of RNAP II or its recruitment to viral promoters. However, during late times of infection, pUL79 helps RNAP II efficiently elongate along the viral DNA template to transcribe HCMV genes. Host genes are not regulated by this pUL79-mediated mechanism. Therefore, our study discovers a previously uncharacterized mechanism where RNAP II activity is modulated by viral factor pUL79, and potentially other viral factors as well, for coordinated viral transcription.
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