Role of glycans for gO-dependent entry of the human cytomegalovirus

人巨细胞病毒（HCMV）感染是孕妇的一个主要问题，因为即使在预先存在免疫力的条件下，它们也会导致宫内感染儿童的严重疾病。这种免疫保护不足的原因可能是针对糖蛋白O（gO）的抗体的低频率。该蛋白质与糖蛋白gH和gL形成复合物，其不是必需的，但决定子代病毒的感染性。目前，HCMV的gO依赖性进入机制尚未完全了解。超过50%的gO分子量由聚糖组成，这可能有助于感染以及保护病毒免受抗体的侵害。然而，它们的作用尚未得到直接讨论。因此，本项目的目的是确定gO上聚糖对HCMV进入的作用，并阐明聚糖是否有助于保护HCMV免受中和抗体的侵害。为了回答这些问题，第一步是系统地突变gO内的每个预测的糖基化位点。这将允许确定哪些聚糖有助于gH/gL/gO复合物的形成，以及哪些聚糖修饰被抑制，因此可以被去除而不干扰复合物掺入病毒体。基于这些数据，将产生具有降低的gO糖基化的病毒。为了测试聚糖对于病毒进入的重要性，然后将表征该病毒结合并渗透到HCMV宿主细胞中的能力。最近开发的人凝集素微阵列将被用来直接和系统地测试这类聚糖结合蛋白的gO的相互作用。这种方法将确定新的HCMV的细胞相互作用伙伴，从而有助于进一步阐明HCMV的gO依赖性进入的机制。为了检验gO介导的聚糖屏蔽的假设，将分析糖基化降低的病毒是否对中和更敏感以及是否诱导增强的抗体应答。总的来说，该项目将加深我们对HCMV进入的理解，并可能导致针对HCMV感染的改进免疫策略的发展。

Infections with the human cytomegalovirus (HCMV) are a major problem for pregnant women as they can result in severe diseases of the in utero infected child, even under conditions of preexisting immunity. A reason for this insufficient immune protection could be the low frequency of antibodies against the glycoprotein O (gO). This protein forms a complex with the glycoproteins gH and gL which is not essential but determines the infectivity of progeny virus. Currently, the mechanisms of gO-dependent entry of HCMV are not fully understood. More than 50% of the molecular mass of gO are made up by glycans which could contribute to infection as well as to protection of the virus from antibodies. However, their role has not been addressed directly yet. Therefore, the aim of this project is to determine the role of the glycans on gO for HCMV entry and to clarify whether the glycans contribute to shielding of HCMV from neutralizing antibodies. To answer these questions, the first step is to systematically mutate each of the predicted glycosylation sites within gO. This will allow to determine which glycans contribute to formation of the gH/gL/gO complex and which glycan modifications are dispensable and can be therefore removed without interfering with the incorporation of the complex into the virion. Based on these data, a virus with reduced glycosylation of gO will be generated. In order to test the importance of the glycans for virus entry, this virus will then be characterized regarding its ability to bind to and penetrate into HCMV host cells. A recently developed human lectin microarray will be utilized to directly and systematically test the interaction of gO with this class of glycan-binding proteins. This approach will identify novel cellular interaction partners of HCMV and thereby help to further elucidate the mechanisms of gO-dependent entry of HCMV. To test the hypothesis of gO-mediated glycan shielding, it will be analyzed whether the virus with reduced glycosylation is more sensitive to neutralization and if it induces an enhanced antibody response. Overall, this project will deepen our understanding of HCMV entry and potentially lead to the development of improved immunization strategies against HCMV infection.