Institute of Virology, University Medical Center Ulm, Ulm, Germany.
Max von Pettenkofer-Institute for Virology, Ludwig-Maximilians-Universität, Munich, Germany.
J Virol. 2019 May 15;93(11). doi: 10.1128/JVI.00138-19. Print 2019 Jun 1.
The human cytomegalovirus (HCMV) glycoprotein complex gH/gL/gO is required for the infection of cells by cell-free virions. It was recently shown that entry into fibroblasts depends on the interaction of gO with the platelet-derived growth factor receptor alpha (PDGFRα). This interaction can be blocked with soluble PDGFRα-Fc, which binds to HCMV virions and inhibits entry. The aim of this study was to identify parts of gO that contribute to PDGFRα binding. In a systematic mutational approach, we targeted potential interaction sites by exchanging conserved clusters of charged amino acids of gO with alanines. To screen for impaired interaction with PDGFRα, virus mutants were tested for sensitivity to inhibition by soluble PDGFRα-Fc. Two mutants with mutations within the N terminus of gO (amino acids 56 to 61 and 117 to 121) were partially resistant to neutralization. To validate whether these mutations impair interaction with PDGFRα-Fc, we compared binding of PDGFRα-Fc to mutant and wild-type virions via quantitative immunofluorescence analysis. PDGFRα-Fc staining intensities were reduced by 30% to 60% with mutant virus particles compared to wild-type particles. In concordance with the reduced binding to the soluble receptor, virus penetration into fibroblasts, which relies on binding to the cellular PDGFRα, was also reduced. In contrast, PDGFRα-independent penetration into endothelial cells was unaltered, demonstrating that the phenotypes of the gO mutant viruses were specific for the interaction with PDGFRα. In conclusion, the mutational screening of gO revealed that the N terminus of gO contributes to efficient spread in fibroblasts by promoting the interaction of virions with its cellular receptor. The human cytomegalovirus is a highly prevalent pathogen that can cause severe disease in immunocompromised hosts. Currently used drugs successfully target the viral replication within the host cell, but their use is restricted due to side effects and the development of resistance. An alternative approach is the inhibition of virus entry, for which understanding the details of the initial virus-cell interaction is desirable. As binding of the viral gH/gL/gO complex to the cellular PDGFRα drives infection of fibroblasts, this is a potential target for inhibition of infection. Our mutational mapping approach suggests the N terminus as the receptor binding portion of the protein. The respective mutants were partially resistant to inhibition by PDGFRα-Fc but also attenuated for infection of fibroblasts, indicating that such mutations have little if any benefit for the virus. These findings highlight the potential of targeting the interaction of gH/gL/gO with PDGFRα for therapeutic inhibition of HCMV.
人类巨细胞病毒（HCMV）糖蛋白复合物 gH/gL/gO 是细胞游离病毒感染细胞所必需的。最近的研究表明，成纤维细胞的进入依赖于 gO 与血小板衍生生长因子受体α（PDGFRα）的相互作用。这种相互作用可以被可溶性 PDGFRα-Fc 阻断，它与 HCMV 病毒结合并抑制进入。本研究的目的是鉴定 gO 中有助于 PDGFRα 结合的部分。在系统的突变方法中，我们通过用丙氨酸交换 gO 的保守带电氨基酸簇来靶向潜在的相互作用位点。为了筛选与 PDGFRα 相互作用受损的病毒突变体，我们通过测定对可溶性 PDGFRα-Fc 的抑制作用来测试病毒突变体的敏感性。gO N 端（氨基酸 56 至 61 和 117 至 121）内突变的两个突变体对中和作用具有部分抗性。为了验证这些突变是否会损害与 PDGFRα-Fc 的相互作用，我们通过定量免疫荧光分析比较了 PDGFRα-Fc 与突变型和野生型病毒颗粒的结合。与野生型病毒颗粒相比，突变病毒颗粒与 PDGFRα-Fc 的结合减少了 30%至 60%。与结合可溶性受体减少一致，依赖于与细胞 PDGFRα 结合的病毒进入成纤维细胞也减少。相比之下，PDGFRα 不依赖的进入内皮细胞没有改变，表明 gO 突变病毒的表型特异性地针对与 PDGFRα 的相互作用。总之，gO 的突变筛选表明，gO 的 N 端通过促进病毒与细胞受体的相互作用，有助于在成纤维细胞中有效传播。人类巨细胞病毒是一种高度流行的病原体，可在免疫功能低下的宿主中引起严重疾病。目前使用的药物可成功靶向宿主细胞内的病毒复制，但由于副作用和耐药性的发展，其使用受到限制。另一种方法是抑制病毒进入，为此需要了解初始病毒-细胞相互作用的细节。由于病毒 gH/gL/gO 复合物与细胞 PDGFRα 的结合驱动成纤维细胞感染，因此这是抑制感染的潜在靶标。我们的突变作图方法表明 N 端是该蛋白的受体结合部分。相应的突变体对 PDGFRα-Fc 的抑制有一定的抗性，但对成纤维细胞的感染也减弱，表明这种突变对病毒几乎没有益处。这些发现强调了针对 gH/gL/gO 与 PDGFRα 相互作用进行治疗性抑制 HCMV 的潜力。
