Virus-like vesicles of Kaposi’s Sarcoma-Associated Herpesvirus activate lytic replication through triggering differentiation signaling (microRNA)

Virus-like vesicles (VLVs) are membrane derived cellular vesicles that resemble native envelope viruses in organization and conformation, but lack viral capsid and/or genome. During productive virus infection, both infectious virions and non-infectious VLVs are produced and released into the extracellular space. VLVs have been shown to play a role in intercellular communication and in facilitating virus infection. The study of VLVs in the context of gammaherpesvirus infection has been largely restricted due to the technical difficulty of separating VLVs and virions. Here we report a strategy for using a KSHV mutant deficient in capsid assembly to isolate VLVs during infection. Using Mass Spectrometry analysis, we identified that VLVs contain viral glycoproteins required for cellular entry, and tegument proteins involved in regulating lytic replication. Functional analysis showed that VLVs could activate the RTA promoter, the lytic switch for KSHV, and further induce KSHV lytic gene expression from latency. We used RNA sequencing to do a genome-wide analysis of cellular responses triggered by VLVs, and revealed that PRDM1, a master regulator in cell differentiation, was up-regulated. Our data shows that VLVs play an important role in promoting KSHV lytic replication by inducing PRDM1 expression which activates the RTA promoter. Our study significantly extends our current understanding of VLVs. Overall design: Analyze microRNA in KSHV VLVs

病毒样囊泡（Virus-like vesicles, VLVs）是一类源自细胞膜的细胞囊泡，其结构与构象与天然包膜病毒相似，但缺失病毒衣壳与/或基因组。在病毒增殖性感染过程中，感染性病毒粒子与非感染性VLVs均可被合成并释放至细胞外空间。已有研究证实，VLVs可参与细胞间通讯并促进病毒感染进程。由于分离VLVs与病毒粒子存在技术难题，目前针对γ疱疹病毒感染背景下VLVs的研究仍受到极大限制。本研究开发了一种利用衣壳组装缺陷型卡波西肉瘤相关疱疹病毒（Kaposi's sarcoma-associated herpesvirus, KSHV）突变株，在感染过程中分离VLVs的实验策略。通过质谱（Mass Spectrometry）分析，我们发现VLVs携带细胞入侵所需的病毒糖蛋白，以及参与调控病毒裂解复制的被膜蛋白。功能实验结果显示，VLVs可激活RTA启动子——KSHV的裂解激活开关，并进一步诱导潜伏状态下的KSHV裂解基因表达。我们通过RNA测序（RNA Sequencing）对VLVs诱导的细胞应答进行了全基因组分析，发现细胞分化主调控因子PRDM1的表达显著上调。本研究数据表明，VLVs通过诱导PRDM1表达以激活RTA启动子，进而促进KSHV的裂解复制，发挥关键调控功能。本研究极大拓展了当前对VLVs的认知边界。实验整体设计：分析KSHV VLVs中的微小RNA（microRNA）