Mechanisms of human cytomegalovirus capsid maturation

感染人巨细胞病毒(HCMV)可导致免疫功能低下的患者和免疫功能不成熟的新生儿患上危及生命的疾病。目前已有的抗CMV药物主要针对病毒DNA聚合酶和病毒基因组复制，且存在相当大的不良反应。因此，有必要开发针对其他病毒特异性机制的新的抗病毒化合物。HCMV衣壳的成熟需要几种基本病毒蛋白的相互作用，因此是抗病毒药物干预的一个有前途的靶点。然而，人类巨细胞病毒衣壳成熟的分子机制尚不清楚。特别是，四种特征稀少的HCMV蛋白(pUL51、pUL52、pUL77和pUL93)的作用仍有待确定，它们被认为参与了基因组包裹和随后的衣壳成熟步骤。在目前的资助期间，我们已经证明了pUL51对于HCMV基因组的切割和包装是必不可少的，并且它与终止酶亚单位pUL56和pUL89相互作用，可能促进终止酶复合体的形成或将其移位到病毒复制间隔室。利用表达标记的UL52蛋白的巨细胞病毒突变体和串联亲和纯化，我们可以丰富pUL52可能的相互作用伙伴。对一个UL77缺失突变体的初步鉴定提供了pUL77对HCMV基因组囊化有贡献的证据。通过构建和分析合适的HCMV突变体，我们将进一步研究每个相应的开放阅读框中断时对病毒感染周期的表型影响，特别是pUL77及其建议的病毒伙伴蛋白pUL93的表型后果。此外，我们将评估HCMV包膜蛋白之间的相互作用，以及在病毒感染的背景下与其他病毒蛋白以及可能的细胞相互作用伙伴的相互作用，重点是pUL51和pUL52。我们期望该项目将产生关于介导HCMV衣壳成熟的机制以及促进这一过程的蛋白质之间的相互作用的全面知识。蛋白质结构域的定义将为开发干扰这些相互作用的化合物的筛选方法奠定基础。

Infection with human cytomegalovirus (HCMV) can lead to life-threatening disease in immunocompromised patients and immunologically immature newborns. The currently available medication against CMV mainly targets the viral DNA polymerase and viral genome replication, and is associated with considerable harmful adverse effects. Accordingly, there is a need for the development of new antiviral compounds that target other virus-specific mechanisms. HCMV capsid maturation requires the interaction of several essential viral proteins and thus represents a promising target for intervention with antiviral drugs. However, the molecular mechanisms underlying the maturation of HCMV capsids are not well understood yet. In particular, the role of four sparsely characterized HCMV proteins (pUL51, pUL52, pUL77 and pUL93), which were suggested to be involved in genome encapsidation and subsequent capsid maturation steps, remains to be defined. In the current funding period we have shown that pUL51 is essential for HCMV genome cleavage and packaging, and that it interacts with the terminase subunits pUL56 and pUL89, possibly promoting the formation of the terminase complex or its translocation to viral replication compartments. Using an HCMV mutant expressing a tagged UL52 protein and tandem affinity purification we could enrich a putative interaction partner of pUL52. Preliminary characterization of a UL77 deletion mutant provides evidence of a contribution of pUL77 to HCMV genome encapsidation. By constructing and analyzing suitable HCMV mutants we will further investigate the phenotypic consequences on the viral infection cycle upon disruption of each of the respective open reading frames, especially of those for pUL77 and its suggested viral partner protein pUL93. Moreover, we will evaluate the interactions of the HCMV encapsidation proteins with each other, and with other viral proteins as well as putative cellular interaction partners in the context of viral infection, with a focus on pUL51 and pUL52. We expect that the project will yield comprehensive knowledge of the mechanisms mediating HCMV capsid maturation and of the interactions of the proteins contributing to this process. Definition of the protein domains mediating these protein-protein interactions will lay the basis for the development of screening assays for compounds disrupting these interactions.