Human cytomegalovirus protein pUL117 targets the mini-chromosome maintenance complex and suppresses cellular DNA synthesis.
Human cytomegalovirus protein pUL117 targets the mini-chromosome maintenance complex and suppresses cellular DNA synthesis.
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DOI：
10.1371/journal.ppat.1000814
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发表时间：
2010-03-19
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：
Qian Z;Leung-Pineda V;Xuan B;Piwnica-Worms H;Yu D
宿主DNA合成的调节是许多病毒建立生产性感染和导致病毒性疾病的必要条件。人类巨细胞病毒（HCMV）是一种大型DNA病毒，可阻断宿主DNA合成并调节细胞周期进程。我们报告说，pUL117，一种我们最近发现的病毒蛋白，是HCMV阻断宿主DNA合成所必需的。在人包皮成纤维细胞感染24小时后，通过帧移突变或基于蛋白质不稳定的方法破坏pUL117的突变病毒有时无法阻断宿主DNA合成。此外，pUL117缺陷病毒刺激静止成纤维细胞进入s期，证明HCMV促进宿主DNA合成的内在能力，而这种能力被pUL117抑制。我们检查了已知参与HCMV感染中宿主DNA合成抑制的关键蛋白，发现根据它们的表达模式，许多蛋白不太可能参与pUL117的抑制活性，包括双子星蛋白、细胞周期蛋白A和病毒蛋白IE2。然而，在缺乏pUL117的情况下，HCMV延缓以MCM2和MCM4为代表的迷你染色体维持（MCM）复合体蛋白积累并阻止其装载到染色质上的能力受到损害。当单独表达时，pUL117减缓细胞增殖，延迟DNA合成，抑制MCM积累。siRNA敲低MCM蛋白恢复了pul117缺陷病毒阻断细胞DNA合成的能力。因此，靶向MCM复合体是pUL117在HCMV感染期间帮助阻断细胞DNA合成的一种机制。我们的发现证实了一种新兴的观点，即MCM的解除管制是许多病毒阻止宿主DNA合成的保守策略，并有助于阐明大型DNA病毒调节细胞过程以促进感染和发病的复杂策略。抑制宿主DNA合成是许多病毒建立生产性感染和致病的关键。人类巨细胞病毒（HCMV）是导致新生儿出生缺陷的首要病毒，并导致免疫力低下的个体罹患危及生命的疾病。HCMV阻断宿主DNA合成，并创造一个细胞环境来复制自己的基因组。我们在这里报告了pUL117，我们最近发现的一种新的病毒蛋白，是HCMV阻断宿主DNA合成所必需的。从机制上讲，pUL117是减少迷你染色体维持（MCM）复合体积累的必要和充分的，MCM复合体是一种解绕起源并启动细胞DNA复制的复制解旋酶。在HCMV感染期间，pUL117也可能直接作用于阻止MCM装载到染色质上。重要的是，敲除MCM蛋白恢复了pul117缺陷病毒阻断细胞DNA合成的能力。因此，靶向MCM功能是pUL117在HCMV感染期间帮助阻断细胞DNA合成的机制。据报道，其他病毒编码的几种蛋白质通过不同的机制破坏MCM功能，并在宿主细胞中过度表达时抑制宿主DNA合成。因此，MCM已成为病毒阻止宿主DNA合成的保守靶点。我们的研究结果说明了HCMV在感染过程中使用一种新的策略来操纵这个关键的细胞因子。该研究有助于阐明大型DNA病毒调节细胞过程以促进感染和发病机制的复杂策略，并可能揭示真核生物DNA复制的调控。
Modulation of host DNA synthesis is essential for many viruses to establish productive infections and contributes to viral diseases. Human cytomegalovirus (HCMV), a large DNA virus, blocks host DNA synthesis and deregulates cell cycle progression. We report that pUL117, a viral protein that we recently identified, is required for HCMV to block host DNA synthesis. Mutant viruses in which pUL117 was disrupted, either by frame-shift mutation or by a protein destabilization-based approach, failed to block host DNA synthesis at times after 24 hours post infection in human foreskin fibroblasts. Furthermore, pUL117-deficient virus stimulated quiescent fibroblasts to enter S-phase, demonstrating the intrinsic ability of HCMV to promote host DNA synthesis, which was suppressed by pUL117. We examined key proteins known to be involved in inhibition of host DNA synthesis in HCMV infection, and found that many were unlikely involved in the inhibitory activity of pUL117, including geminin, cyclin A, and viral protein IE2, based on their expression patterns. However, the ability of HCMV to delay the accumulation of the mini-chromosome maintenance (MCM) complex proteins, represented by MCM2 and MCM4, and prevent their loading onto chromatin, was compromised in the absence of pUL117. When expressed alone, pUL117 slowed cell proliferation, delayed DNA synthesis, and inhibited MCM accumulation. Knockdown of MCM proteins by siRNA restored the ability of pUL117-deficient virus to block cellular DNA synthesis. Thus, targeting MCM complex is one mechanism pUL117 employs to help block cellular DNA synthesis during HCMV infection. Our finding substantiates an emerging picture that deregulation of MCM is a conserved strategy for many viruses to prevent host DNA synthesis and helps to elucidate the complex strategy used by a large DNA virus to modulate cellular processes to promote infection and pathogenesis. Inhibition of host DNA synthesis is pivotal for many viruses to establish productive infection and cause disease. Human cytomegalovirus (HCMV) is the top viral cause of birth defects in newborns and leads to life-threatening diseases in individuals with compromised immunity. HCMV blocks host DNA synthesis and creates a cellular environment to replicate its own genome. We report here that pUL117, a novel viral protein that we recently identified, is required for HCMV to block host DNA synthesis. Mechanistically, pUL117 is necessary and sufficient to reduce the accumulation of the mini-chromosome maintenance (MCM) complex, a replicative helicase that unwinds the origin and initiates cellular DNA replication. During HCMV infection pUL117 may also have a direct role in preventing MCM loading onto chromatin. Importantly, knockdown of MCM proteins restored the ability of pUL117-deficient virus to block cellular DNA synthesis. Thus, targeting MCM function is a mechanism for pUL117 to help block cellular DNA synthesis during HCMV infection. Several proteins encoded by other viruses have also been reported to subvert MCM function by distinct mechanisms and inhibit host DNA synthesis when over-expressed in host cells. Therefore, MCM has emerged as a conserved target for viruses to prevent host DNA synthesis. Our results illustrate a novel strategy that HCMV uses to manipulate this critical cellular factor during infection. This study helps to elucidate the sophisticated strategies used by a large DNA virus to modulate cellular processes to promote infection and pathogenesis and may also shed light on the regulation of eukaryotic DNA replication.
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影响因子：
2.3
作者：
Hume AJ;Kalejta RF
通讯作者：
Kalejta RF
DOI：
10.1073/pnas.0504583102
发表时间：
2005-09-20
影响因子：
11.1
作者：
Berthet, C;Raj, K;Beard, P
通讯作者：
Beard, P
影响因子：
3.7
作者：
Bresnahan, WA;Boldogh, I;Albrecht, T
通讯作者：
Albrecht, T
影响因子：
4.8
作者：
Edwards, MC;Tutter, AV;Walter, JC
通讯作者：
Walter, JC
影响因子：
5.4
作者：
Hermanns, J;Schulze, A;zurHausen, H
通讯作者：
zurHausen, H