Identification of Host Factors Involved in Human Cytomegalovirus Replication, Assembly, and Egress Using a Two-Step Small Interfering RNA Screen.
Identification of Host Factors Involved in Human Cytomegalovirus Replication, Assembly, and Egress Using a Two-Step Small Interfering RNA Screen.
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DOI：
10.1128/mbio.00716-18
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发表时间：
2018-06-26
期刊：
影响因子：
6.4
通讯作者：
Grey F
中科院分区：
文献类型：
--
作者：
McCormick D;Lin YT;Grey F
作为专性细胞内寄生虫，病毒完全依赖宿主因子进行复制。复杂病毒颗粒的组装和输出，如人类巨细胞病毒（HCMV），可能需要许多宿主因子。尽管如此，相对较少的已被确定和表征。本研究描述了一种新的高通量，两步小干扰RNA （siRNA）筛选，它独立测量病毒复制和病毒产生。通过结合复制和病毒产生的数据，确定了多个候选基因，其中敲低导致病毒产生的大量损失，对初级复制的影响有限，这表明在病毒组装和输出等后期阶段发挥作用。敲除顶级候选基因ERC1、RAB4B、COPA和COPB2，造成病毒生产的严重损失。尽管据报道COPA和COPB2在相同的复合物中起作用，但敲低这些基因会产生不同的表型。此外，COPA的敲低导致病毒晚期基因的表达增加，尽管病毒DNA复制受到了实质性的抑制。这表明，高效的病毒基因组复制并不需要晚期基因表达。最后，我们发现RAB4B在感染HCMV后重新定位到病毒组装室，并且RAB4B的敲低减少了完整病毒粒子的释放，这表明它在病毒粒子的组装和出口中起作用。本研究展示了一种强大的高通量筛选方法，用于鉴定宿主-病毒相互作用，鉴定了与HCMV组装和输出相关的多个宿主基因，并揭示了涂层组分COPA和COPB2在感染过程中的潜在独立功能。人巨细胞病毒感染是免疫功能低下人群、心脏病患者以及实体器官和骨髓移植接受者的重要病因。HCMV也是传染性先天性出生缺陷的主要原因。临床使用的大多数抗病毒药物使用病毒的靶成分特异性地抑制复制。然而，这种方法的一个主要缺点是耐药性的出现。另一种方法是瞄准病毒成功感染所需的宿主因子。在这项研究中，发现了多种宿主因子，这些因子对新感染性人巨细胞病毒的产生至关重要。确定哪些宿主基因是病毒复制所必需的，扩展了我们对病毒如何复制和细胞如何功能的理解，并为新型抗病毒药物提供了潜在的靶点。
As obligate intracellular parasites, viruses are completely dependent on host factors for replication. Assembly and egress of complex virus particles, such as human cytomegalovirus (HCMV), are likely to require many host factors. Despite this, relatively few have been identified and characterized. This study describes a novel high-throughput, two-step small interfering RNA (siRNA) screen, which independently measures virus replication and virus production. By combining data from replication and virus production, multiple candidate genes were identified in which knockdown resulted in substantial loss of virus production with limited effect on primary replication, suggesting roles in later stages such as virus assembly and egress. Knockdown of the top candidates, ERC1, RAB4B, COPA, and COPB2, caused profound loss of virus production. Despite COPA and COPB2 being reported to function in the same complex, knockdown of these genes produced distinct phenotypes. Furthermore, knockdown of COPA caused increased expression of viral late genes despite substantial inhibition of viral DNA replication. This suggests that efficient viral genome replication is not required for late gene expression. Finally, we show that RAB4B relocates to the viral assembly compartment following infection with HCMV and knockdown of RAB4B reduces the release of intact virion particles, suggesting that it plays a role in virion assembly and egress. This study demonstrates a powerful high-throughput screen for identification of host-virus interactions, identifies multiple host genes associated with HCMV assembly and egress, and uncovers potentially independent functions for coatomer components COPA and COPB2 during infection. Human cytomegalovirus infection is a significant cause of disease in immunocompromised populations, individuals with heart disease, and recipients of solid organ and bone marrow transplants. HCMV is also the leading cause of infectious congenital birth defects. The majority of antivirals in clinical use target components of the virus to specifically inhibit replication. However, a major drawback of this approach is the emergence of resistance. An alternative approach is to target host factors that the virus requires for successful infection. In this study, multiple host factors were identified that were found to be essential for the production of newly infectious human cytomegalovirus. Identifying which host genes are necessary for virus replication extends our understanding of how viruses replicate and how cells function and provides potential targets for novel antivirals.