Cavaletto Noemi, Luganini Anna, Gribaudo Giorgio
Department of Life Sciences and Systems Biology, University of Turin, Turin, Italy.
Department of Life Sciences and Systems Biology, University of Turin, Turin, Italy
J Virol. 2015 Nov;89(21):11092-106. doi: 10.1128/JVI.01141-15. Epub 2015 Aug 26.
The human cytomegalovirus (HCMV) US12 gene family includes a group of 10 contiguous genes (US12 to US21) encoding predicted seven-transmembrane-domain (7TMD) proteins that are nonessential for replication within cultured fibroblasts. Nevertheless, inactivation of some US12 family members affects virus replication in other cell types; e.g., deletion of US16 or US18 abrogates virus growth in endothelial and epithelial cells or in human gingival tissue, respectively, suggesting a role for some US12 proteins in HCMV cell tropism. Here, we provide evidence that another member, US20, impacts the ability of a clinical strain of HCMV to replicate in endothelial cells. Through the use of recombinant HCMV encoding tagged versions of the US20 protein, we investigated the expression pattern, localization, and topology of the US20-encoded protein (pUS20). We show that pUS20 is expressed as a partially glycosylated 7TMD protein which accumulates late in infection in endoplasmic reticulum-derived peripheral structures localized outside the cytoplasmic virus assembly compartment (cVAC). US20-deficient mutants generated in the TR clinical strain of HCMV exhibited major growth defects in different types of endothelial cells, whereas they replicated normally in fibroblasts and epithelial cells. While the attachment and entry phases in endothelial cells were not significantly affected by the absence of US20 protein, US20-null viruses failed to replicate viral DNA and express representative E and L mRNAs and proteins. Taken together, these results indicate that US20 sustains the HCMV replication cycle at a stage subsequent to entry but prior to E gene expression and viral DNA synthesis in endothelial cells.
Human cytomegalovirus (HCMV) is a major pathogen in newborns and immunocompromised individuals. A hallmark of HCMV pathogenesis is its ability to productively replicate in an exceptionally broad range of target cells, including endothelial cells, which represent a key target for viral dissemination and replication in the host, and to contribute to both viral persistence and associated inflammation and vascular diseases. Replication in endothelial cells depends on the activities of a set of viral proteins that regulate different stages of the HCMV replication cycle in an endothelial cell type-specific manner and thereby act as determinants of viral tropism. Here, we report the requirement of a HCMV protein as a postentry tropism factor in endothelial cells. The identification and characterization of HCMV endotheliotropism-regulating proteins will advance our understanding of the molecular mechanisms of HCMV-related pathogenesis and help lead to the design of new antiviral strategies able to exploit these functions.
人类巨细胞病毒（HCMV）的US12基因家族包含一组10个相邻基因（US12至US21），这些基因编码预测的七跨膜结构域（7TMD）蛋白，它们对于在培养的成纤维细胞中复制并非必需。然而，一些US12家族成员的失活会影响病毒在其他细胞类型中的复制；例如，缺失US16或US18分别会消除病毒在内皮细胞、上皮细胞或人类牙龈组织中的生长，这表明一些US12蛋白在HCMV细胞嗜性中发挥作用。在这里，我们提供证据表明，另一个成员US20会影响一株临床HCMV毒株在内皮细胞中的复制能力。通过使用编码带有标签的US20蛋白的重组HCMV，我们研究了US20编码蛋白（pUS20）的表达模式、定位和拓扑结构。我们发现pUS20以部分糖基化的7TMD蛋白形式表达，在感染后期积累在内质网衍生的位于细胞质病毒装配区室（cVAC）之外的周边结构中。在HCMV的TR临床毒株中产生的US20缺陷型突变体在不同类型的内皮细胞中表现出主要的生长缺陷，而它们在成纤维细胞和上皮细胞中能正常复制。虽然在内皮细胞中，缺失US20蛋白对附着和进入阶段没有显著影响，但缺失US20的病毒无法复制病毒DNA，也无法表达代表性的E和L mRNA及蛋白。综上所述，这些结果表明，US20在内皮细胞中在进入后但在E基因表达和病毒DNA合成之前的阶段维持HCMV复制周期。
人类巨细胞病毒（HCMV）是新生儿和免疫功能低下个体中的主要病原体。HCMV发病机制的一个标志是其能够在异常广泛的靶细胞中高效复制，包括内皮细胞，内皮细胞是病毒在宿主中传播和复制的关键靶标，并且对病毒持续存在以及相关的炎症和血管疾病都有影响。在内皮细胞中的复制取决于一组病毒蛋白的活性，这些蛋白以内皮细胞类型特异性的方式调节HCMV复制周期的不同阶段，从而作为病毒嗜性的决定因素。在这里，我们报告了一种HCMV蛋白作为内皮细胞进入后嗜性因子的需求。对HCMV内皮嗜性调节蛋白的鉴定和表征将推进我们对HCMV相关发病机制分子机制的理解，并有助于设计能够利用这些功能的新抗病毒策略。
