Virology Laboratories, Department of Pharmacology and Molecular Sciences, and The School of Medicine Microscopy Facility, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.
Virology Laboratories, Department of Pharmacology and Molecular Sciences, and The School of Medicine Microscopy Facility, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA
J Virol. 2014 Aug;88(15):8256-67. doi: 10.1128/JVI.00971-14. Epub 2014 May 14.
We report that the human cytomegalovirus (HCMV) high-molecular-weight tegument protein (HMWP, pUL48; 253 kDa) and the HMWP-binding protein (hmwBP, pUL47; 110 kDa) can be recovered as a complex from virions disrupted by treatment with 50 mM Tris (pH 7.5), 0.5 M NaCl, 0.5% NP-40, and 10 mM dithiothreitol [DTT]. The subunit ratio of the complex approximates 1:1, with a shape and structure consistent with an elongated heterodimer. The HMWP/hmwBP complex was corroborated by reciprocal coimmunoprecipitation experiments using antipeptide antibodies and lysates from both infected cells and disrupted virus particles. An interaction of the amino end of pUL48 (amino acids [aa] 322 to 754) with the carboxyl end of pUL47 (aa 693 to 982) was identified by fragment coimmunoprecipitation experiments, and a head-to-tail self-interaction of hmwBP was also observed. The deubiquitylating activity of pUL48 is retained in the isolated complex, which cleaves K11, K48, and K63 ubiquitin isopeptide linkages.
Human cytomegalovirus (HCMV, or human herpesvirus 5 [HHV-5]) is a large DNA-containing virus that belongs to the betaherpesvirus subfamily and is a clinically important pathogen. Defining the constituent elements of its mature form, their organization within the particle, and the assembly process by which it is produced are fundamental to understanding the mechanisms of herpesvirus infection and developing drugs and vaccines against them. In this study, we report isolating a complex of two large proteins encoded by HCMV open reading frames (ORFs) UL47 and UL48 and identifying the binding domains responsible for their interaction with each other and of pUL47 with itself. Our calculations indicate that the complex is a rod-shaped heterodimer. Additionally, we determined that the ubiquitin-specific protease activity of the ORF UL48 protein was functional in the complex, cleaving K11-, K48-, and K63-linked ubiquitin dimers. This information builds on and extends our understanding of the HCMV tegument protein network that is required to interface the HCMV envelope and capsid.
我们报告称，人巨细胞病毒（HCMV）高分子量被膜蛋白（HMWP，pUL48；253 kDa）和 HMWP 结合蛋白（hmwBP，pUL47；110 kDa）可以从用 50 mM Tris（pH 7.5）、0.5 M NaCl、0.5%NP-40 和 10 mM DTT [DTT] 处理破坏的病毒粒子中回收为复合物。复合物的亚基比例近似为 1:1，形状和结构与伸长的异二聚体一致。使用抗肽抗体和来自感染细胞和破坏病毒颗粒的裂解物进行的相互共免疫沉淀实验证实了 HMWP/hmwBP 复合物的存在。pUL48 的氨基末端（氨基酸 [aa] 322 至 754）与 pUL47 的羧基末端（aa 693 至 982）之间的相互作用通过片段共免疫沉淀实验确定，并且还观察到 hmwBP 的头对头自相互作用。分离的复合物保留了 pUL48 的去泛素化活性，该复合物可切割 K11、K48 和 K63 泛素异肽键。
人巨细胞病毒（HCMV，或人类疱疹病毒 5 [HHV-5]）是一种大型 DNA 病毒，属于β疱疹病毒亚科，是一种临床上重要的病原体。定义其成熟形式的组成元素、它们在颗粒中的组织以及产生它的组装过程对于理解疱疹病毒感染的机制以及开发针对它们的药物和疫苗至关重要。在这项研究中，我们报告了分离由 HCMV 开放阅读框（ORF）UL47 和 UL48 编码的两种大蛋白的复合物，并确定了负责它们相互作用以及 pUL47 与其自身相互作用的结合域。我们的计算表明该复合物是一种棒状异二聚体。此外，我们确定 ORF UL48 蛋白的泛素特异性蛋白酶活性在复合物中是功能性的，可切割 K11、K48 和 K63 连接的泛素二聚体。该信息建立并扩展了我们对 HCMV 被膜蛋白网络的理解，该网络是将 HCMV 包膜和衣壳接口所需的。
