Department of Microbial Sciences, University of Surrey, Guildford, United Kingdom.
Department of Pathology, University of Cambridge, Cambridge, United Kingdom.
mBio. 2021 May 11;12(3):e02183-20. doi: 10.1128/mBio.02183-20.
Enveloped viruses exploit cellular trafficking pathways for their morphogenesis, providing potential scope for the development of new antiviral therapies. We have previously shown that herpes simplex virus 1 (HSV1) utilizes recycling endocytic membranes as the source of its envelope, in a process involving four Rab GTPases. To identify novel factors involved in HSV1 envelopment, we have screened a small interfering RNA (siRNA) library targeting over 80 human trafficking proteins, including coat proteins, adaptor proteins, fusion factors, fission factors, and Rab effectors. The depletion of 11 factors reduced virus yields by 20- to 100-fold, including three early secretory pathway proteins, four late secretory pathway proteins, and four endocytic pathway proteins, three of which are membrane fission factors. Five of the 11 targets were chosen for further analysis in virus infection, where it was found that the absence of only 1, the fission factor CHMP4C, but not the CHMP4A or CHMP4B paralogues, reduced virus production at the final stage of morphogenesis. Ultrastructural and confocal microscopy of CHMP4C-depleted, HSV1-infected cells showed an accumulation of endocytic membranes; extensive tubulation of recycling, transferrin receptor-positive endosomes indicative of aberrant fission; and a failure in virus envelopment. No effect on the late endocytic pathway was detected, while exogenous CHMP4C was shown to localize to recycling endosomes. Taken together, these data reveal a novel role for the CHMP4C fission factor in the integrity of the recycling endosomal network, which has been unveiled through the dependence of HSV1 on these membranes for the acquisition of their envelopes. Cellular transport pathways play a fundamental role in secretion and membrane biogenesis. Enveloped viruses exploit these pathways to direct their membrane proteins to sites of envelopment and, as such, are powerful tools for unraveling subtle activities of trafficking factors, potentially pinpointing therapeutic targets. Using the sensitive biological readout of virus production, over 80 trafficking factors involved in diverse and poorly defined cellular processes have been screened for involvement in the complex process of HSV1 envelopment. Out of 11 potential targets, CHMP4C, a key component in the cell cycle abscission checkpoint, stood out as being required for the process of virus wrapping in endocytic tubules, where it localized. In the absence of CHMP4C, recycling endocytic membranes failed to undergo scission in infected cells, causing transient tubulation and accumulation of membranes and unwrapped virus. These data reveal a new role for this important cellular factor in the biogenesis of recycling endocytic membranes.
包膜病毒利用细胞运输途径进行形态发生，为开发新的抗病毒疗法提供了潜在的可能性。我们之前已经表明，单纯疱疹病毒 1（HSV1）利用再循环内吞膜作为其包膜的来源，这一过程涉及到四个 Rab GTPases。为了鉴定参与 HSV1 包膜的新因子，我们筛选了一个针对 80 多种人类运输蛋白的小干扰 RNA（siRNA）文库，这些蛋白包括外壳蛋白、衔接蛋白、融合因子、分裂因子和 Rab 效应因子。11 种因子中的 11 种因子的耗竭使病毒产量减少了 20 到 100 倍，其中包括三种早期分泌途径蛋白、四种晚期分泌途径蛋白和四种内吞途径蛋白，其中三种是膜分裂因子。在病毒感染中选择了 11 个目标中的 5 个进行进一步分析，结果发现，只有一种分裂因子 CHMP4C 的缺失，而不是 CHMP4A 或 CHMP4B 同源物的缺失，会降低形态发生的最后阶段的病毒产量。CHMP4C 耗尽的 HSV1 感染细胞的超微结构和共焦显微镜观察显示，内吞膜的积累；循环、转铁蛋白受体阳性内体的广泛管状化，表明异常分裂；以及病毒包膜的失败。没有检测到对晚期内吞途径的影响，而外源性 CHMP4C 被证明定位于再循环内体。总之，这些数据揭示了 CHMP4C 分裂因子在再循环内体网络完整性中的新作用，这是通过 HSV1 对这些膜的依赖来获得其包膜而揭示的。细胞运输途径在分泌和膜生物发生中起着至关重要的作用。包膜病毒利用这些途径将其膜蛋白引导到包膜部位，因此，它们是揭示运输因子微妙活性的有力工具，有可能确定治疗靶点。利用病毒产量的敏感生物学读数，已经筛选了 80 多种参与不同且定义不明确的细胞过程的运输因子，以确定它们是否参与 HSV1 包膜的复杂过程。在 11 个潜在目标中，CHMP4C 脱颖而出，成为病毒包裹在内吞小管中的必需因子，它在这些小管中定位。在没有 CHMP4C 的情况下，再循环内吞膜在感染细胞中无法发生分裂，导致膜的短暂管状化和积累以及未包裹的病毒。这些数据揭示了这个重要的细胞因子在再循环内吞膜生物发生中的新作用。
