A Human Cytomegalovirus gO-Null Mutant Fails To Incorporate gH/gL into the Virion Envelope and Is Unable To Enter Fibroblasts and Epithelial and Endothelial Cells
A Human Cytomegalovirus gO-Null Mutant Fails To Incorporate gH/gL into the Virion Envelope and Is Unable To Enter Fibroblasts and Epithelial and Endothelial Cells
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DOI：
10.1128/jvi.02249-09
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发表时间：
2009-12
影响因子：
5.4
通讯作者：
Paul T. Wille;A. J. Knoche;J. Nelson;M. Jarvis;David C. Johnson
中科院分区：
文献类型：
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作者：
Paul T. Wille;A. J. Knoche;J. Nelson;M. Jarvis;David C. Johnson
人巨细胞病毒（HCMV）依赖于一种五蛋白复合物gH/gL/UL 128 -131进入上皮和内皮细胞。已经描述了一种单独的含HCMV gH/gL的复合物gH/gL/gO。我们的流行模型是gH/gL/UL 128 -131是进入生物学上重要的上皮和内皮细胞所必需的，并且gH/gL/gO是感染成纤维细胞所必需的。编码UL 128 -131的基因在HCMV在成纤维细胞上的实验室繁殖期间迅速突变，显然与成纤维细胞进入途径的选择压力有关。在B的随附论文中反对这种模式。J. Ryckman等人（J. Virol.，84：2597-2609，2010），我们描述了临床HCMV毒株TR表达gO分子的证据，该gO分子用于促进gH/gL的内质网（ER）输出，并且gO不稳定地掺入病毒包膜中。这与涉及成纤维细胞适应的HCMV毒株AD 169的结果不同，后者将gO掺入病毒体包膜中。在这里，我们构建了一个TR gO无效突变体TRΔgO，其复制至低滴度，在成纤维细胞中传播不良，但产生正常数量的细胞外病毒颗粒。成纤维细胞释放的TRΔgO颗粒不能感染成纤维细胞和上皮及内皮细胞，但化学融合剂聚乙二醇（PEG）可以部分克服感染缺陷。因此，TRΔgO不能进入所有三种细胞类型。与野生型病毒颗粒相比，TRΔgO掺入的gH/gL量约为细胞外病毒颗粒的5%，这解释了进入缺陷。尽管TRΔgO颗粒不能进入细胞，但涉及上皮细胞和内皮细胞的细胞间扩散相对于TR增加，这显然是由于病毒粒子中gH/gL/UL 128 -131数量增加所致。总之，我们的数据表明，TR gO作为一个伴侣，以促进ER的出口和gH/gL复合物纳入HCMV包膜。此外，这些数据表明，它是gH/gL，而不是gH/gL/gO，这是存在于病毒粒子，并需要感染成纤维细胞和上皮细胞和内皮细胞。我们观察到gH/gL和gH/gL/UL 128 -131都是进入上皮/内皮细胞所必需的，这与使用两种不同gH/gL复合物之一来进入不同细胞的其他β-和γ-疱疹病毒的模型不同。
ABSTRACT Human cytomegalovirus (HCMV) depends upon a five-protein complex, gH/gL/UL128-131, to enter epithelial and endothelial cells. A separate HCMV gH/gL-containing complex, gH/gL/gO, has been described. Our prevailing model is that gH/gL/UL128-131 is required for entry into biologically important epithelial and endothelial cells and that gH/gL/gO is required for infection of fibroblasts. Genes encoding UL128-131 are rapidly mutated during laboratory propagation of HCMV on fibroblasts, apparently related to selective pressure for the fibroblast entry pathway. Arguing against this model in the accompanying paper by B. J. Ryckman et al. (J. Virol., 84:2597-2609, 2010), we describe evidence that clinical HCMV strain TR expresses a gO molecule that acts to promote endoplasmic reticulum (ER) export of gH/gL and that gO is not stably incorporated into the virus envelope. This was different from results involving fibroblast-adapted HCMV strain AD169, which incorporates gO into the virion envelope. Here, we constructed a TR gO-null mutant, TRΔgO, that replicated to low titers, spread poorly among fibroblasts, but produced normal quantities of extracellular virus particles. TRΔgO particles released from fibroblasts failed to infect fibroblasts and epithelial and endothelial cells, but the chemical fusogen polyethylene glycol (PEG) could partially overcome defects in infection. Therefore, TRΔgO is defective for entry into all three cell types. Defects in entry were explained by observations showing that TRΔgO incorporated about 5% of the quantities of gH/gL in extracellular virus particles compared with that in wild-type virions. Although TRΔgO particles could not enter cells, cell-to-cell spread involving epithelial and endothelial cells was increased relative to TR, apparently resulting from increased quantities of gH/gL/UL128-131 in virions. Together, our data suggest that TR gO acts as a chaperone to promote ER export and the incorporation of gH/gL complexes into the HCMV envelope. Moreover, these data suggest that it is gH/gL, and not gH/gL/gO, that is present in virions and is required for infection of fibroblasts and epithelial and endothelial cells. Our observations that both gH/gL and gH/gL/UL128-131 are required for entry into epithelial/endothelial cells differ from models for other beta- and gammaherpesviruses that use one of two different gH/gL complexes to enter different cells.