Division of Infection and Immunity, The Roslin Institute, University of Edinburgh, Easter Bush, Midlothian, United Kingdom.
Division of Infection and Pathway Medicine, University of Edinburgh, Edinburgh, United Kingdom.
mBio. 2019 Oct 8;10(5):e01651-19. doi: 10.1128/mBio.01651-19.
As obligate intracellular pathogens, viruses rely on the host cell machinery to replicate efficiently, with the host metabolism extensively manipulated for this purpose. High-throughput small interfering RNA (siRNA) screens provide a systematic approach for the identification of novel host-virus interactions. Here, we report a large-scale screen for host factors important for human cytomegalovirus (HCMV), consisting of 6,881 siRNAs. We identified 47 proviral factors and 68 antiviral factors involved in a wide range of cellular processes, including the mediator complex, proteasome function, and mRNA splicing. Focused characterization of one of the hits, asparagine synthetase (ASNS), demonstrated a strict requirement for asparagine for HCMV replication which leads to an early block in virus replication before the onset of DNA amplification. This effect is specific to HCMV, as knockdown of ASNS had little effect on herpes simplex virus 1 or influenza A virus replication, suggesting that the restriction is not simply due to a failure in protein production. Remarkably, virus replication could be completely rescued 7 days postinfection with the addition of exogenous asparagine, indicating that while virus replication is restricted at an early stage, it maintains the capacity for full replication days after initial infection. This study represents the most comprehensive siRNA screen for the identification of host factors involved in HCMV replication and identifies the nonessential amino acid asparagine as a critical factor in regulating HCMV virus replication. These results have implications for control of viral latency and the clinical treatment of HCMV in patients. HCMV accounts for more than 60% of complications associated with solid organ transplant patients. Prophylactic or preventative treatment with antivirals, such as ganciclovir, reduces the occurrence of early onset HCMV disease. However, late onset disease remains a significant problem, and prolonged treatment, especially in patients with suppressed immune systems, greatly increases the risk of antiviral resistance. Very few antivirals have been developed for use against HCMV since the licensing of ganciclovir, and of these, the same viral genes are often targeted, reducing the usefulness of these drugs against resistant strains. An alternative approach is to target host genes essential for virus replication. Here we demonstrate that HCMV replication is highly dependent on levels of the amino acid asparagine and that knockdown of a critical enzyme involved in asparagine synthesis results in severe attenuation of virus replication. These results suggest that reducing asparagine levels through dietary restriction or chemotherapeutic treatment could limit HCMV replication in patients.
作为专性细胞内病原体，病毒依赖宿主细胞机制高效复制，宿主代谢也因此被广泛调控。高通量小干扰 RNA（siRNA）筛选为鉴定新的宿主-病毒相互作用提供了一种系统的方法。在这里，我们报告了一项针对人类巨细胞病毒（HCMV）的大规模宿主因子筛选，其中包含 6881 个 siRNA。我们鉴定了 47 个促进病毒复制的因子和 68 个抗病毒因子，这些因子涉及广泛的细胞过程，包括中介复合物、蛋白酶体功能和 mRNA 剪接。对其中一个命中目标——天冬酰胺合成酶（ASNS）的重点研究表明，HCMV 复制严格依赖于天冬酰胺，这导致病毒复制在 DNA 扩增之前出现早期阻断。这种效应是 HCMV 特有的，因为敲低 ASNS 对单纯疱疹病毒 1 或流感 A 病毒的复制几乎没有影响，这表明这种限制不是简单地由于蛋白质产生失败所致。值得注意的是，在感染后 7 天添加外源性天冬酰胺可以完全挽救病毒复制，表明尽管病毒复制在早期受到限制，但在最初感染后数天仍具有完全复制的能力。这项研究代表了最全面的 siRNA 筛选，用于鉴定参与 HCMV 复制的宿主因子，并确定非必需氨基酸天冬酰胺是调节 HCMV 病毒复制的关键因素。这些结果对于控制病毒潜伏和 HCMV 患者的临床治疗具有重要意义。HCMV 占实体器官移植患者相关并发症的 60%以上。预防性或预防治疗使用抗病毒药物，如更昔洛韦，可以降低早期 HCMV 疾病的发生。然而，晚期疾病仍然是一个重大问题，延长治疗时间，特别是在免疫系统受到抑制的患者中，会大大增加抗病毒药物耐药的风险。自更昔洛韦获得许可以来，针对 HCMV 开发的抗病毒药物很少，而且这些药物通常针对相同的病毒基因，这降低了这些药物对耐药株的作用。另一种方法是针对病毒复制所必需的宿主基因。在这里，我们证明 HCMV 复制高度依赖于天冬酰胺水平，而敲低参与天冬酰胺合成的关键酶会导致病毒复制严重衰减。这些结果表明，通过饮食限制或化疗治疗降低天冬酰胺水平可能会限制患者体内的 HCMV 复制。
