Mechanisms of human cytomegalovirus capsid maturation

人巨细胞病毒（HCMV）感染可导致免疫功能低下的患者和免疫功能不成熟的新生儿发生危及生命的疾病。目前可用的抗CMV药物主要针对病毒DNA聚合酶和病毒基因组复制，并且与相当大的有害副作用相关。因此，需要开发靶向其他病毒特异性机制的新的抗病毒化合物。HCMV衣壳成熟需要几种必需病毒蛋白的相互作用，因此代表了抗病毒药物干预的有希望的靶点。然而，HCMV衣壳成熟的分子机制尚未完全了解。特别是，四个稀疏表征的HCMV蛋白（pUL 51，pUL 52，pUL 77和pUL 93）的作用，这被认为是参与基因组的衣壳化和随后的衣壳成熟步骤，仍然有待确定。在目前的资助期间，我们已经表明，pUL 51是HCMV基因组切割和包装所必需的，它与末端酶亚基pUL 56和pUL 89相互作用，可能促进末端酶复合物的形成或其易位到病毒复制区室。使用表达标记的UL 52蛋白的HCMV突变体和串联亲和纯化，我们可以富集pUL 52的推定相互作用伴侣。UL 77缺失突变体的初步表征提供了pUL 77对HCMV基因组扩增的贡献的证据。通过构建和分析合适的HCMV突变体，我们将进一步研究在破坏每个相应的开放阅读框，特别是pUL 77及其建议的病毒伴侣蛋白pUL 93的开放阅读框后对病毒感染周期的表型后果。此外，我们还将评估HCMV结合蛋白之间的相互作用，以及与其他病毒蛋白以及病毒感染背景下推定的细胞相互作用伴侣的相互作用，重点是pUL 51和pUL 52。我们希望该项目将产生介导HCMV衣壳成熟的机制和蛋白质的相互作用，有助于这一过程的全面知识。介导这些蛋白质-蛋白质相互作用的蛋白质结构域的定义将奠定基础的发展，筛选分析的化合物破坏这些相互作用。

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.