A critical step in viral lytic replication is the assembly of progeny viral particles. Herpesviruses are important pathogens.Tegument, which occupies the space between the nucleocapsid and the envelope, is a unique structure of a herpesvirion. Tegument proteins are major components of tegument and play critical roles in virus life cycle. Murine gammaherpesvirus 68 (MHV-68), a member of the gammaherpesvirus subfamily, is closely related to two human herpesviruses, Kaposi's sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV). We have previously shown that MHV-68 ORF33, conserved among all herpesviruses, encodes a tegument protein that is associated with intranuclear capsids and is essential for virion morphogenesis and egress. Another tegument protein ORF45, which is conserved only among gammaherpesviruses, also plays an essential role in virion morphogenesis of MHV-68. In this study, we investigated the underlying mechanism and showed that these two proteins colocalize and interact with each other during virus infection. We mapped the ORF33-interacting domain to the conserved carboxyl-terminal 23 amino acids (C23) of ORF45. Deletion of the C23 coding sequence in the context of viral genome abolished the production of infectious virions. Transmission electron microscopy results demonstrated that C23 of ORF45 are essential for virion tegumentation in the cytoplasm. We further mapped the ORF45-interacting domain to the N-terminal 17 amino acids (N17) of ORF33. Deletion of the N17 coding sequence in the context of viral genome also abolished production of infectious virions, and N17 of ORF33 are also essential for virion tegumentation in the cytoplasm. Taken together, our data strongly indicate that the interaction between ORF45 and ORF33 plays an essential role in cytoplasmic maturation of MHV-68 virions. IMPORTANCE A critical step in viral lytic replication is the assembly of progeny viral particles. Herpesviruses are important pathogens. A herpesvirus particle comprises, from inside to outside, four layers: DNA core, capsid, tegument, and envelope. The tegument layer contains dozens of virally encoded tegument proteins, which play critical roles in virus assembly. Murine gammaherpesvirus 68 (MHV-68) is a tumor-associated herpesvirus and is closely related to Kaposi's sarcoma-associated herpesvirus and Epstein-Barr virus. We previously found that the absence of either tegument protein ORF33 or ORF45 inhibits the translocation of nucleocapsids to the cytoplasm and blocks virion maturation, but the underlying mechanism remained unclear. Here, we showed that ORF33 interacts with ORF45. We mapped their interaction domains and constructed viral mutants with defects in ORF33-ORF45 interaction. Transmission electron microscopy data demonstrated that the assembly of these viral mutants in the cytoplasm is blocked. Our results indicate that ORF33-ORF45 interaction is essential for gammaherpesvirus replication.

疱疹病毒裂解复制期间病毒颗粒的组装和释放是产生子代病毒粒子的重要步骤，多种病毒蛋白在此过程中发挥关键作用，阐明这一过程的分子机理对于抗病毒手段的开发有重要意义。我们前期的研究成果表明鼠γ疱疹病毒68(MHV-68)编码的间质蛋白ORF45对病毒粒子的形态发生有重要作用。为深入解析ORF45在病毒包装释放中的功能，本课题针对与ORF45存在相互作用的病毒蛋白进行了详细研究，鉴定出ORF45与另一对病毒包装释放有重要功能的间质蛋白ORF33的相互作用区域。通过构建ORF45与ORF33相互作用区域缺失的突变病毒株并结合电子显微镜等成像手段，我们证实这二者的相互作用对MHV-68病毒粒子在细胞质中的成熟和释放到细胞外发挥关键作用。同时我们还鉴定了ORF45与病毒核释放复合体ORF67和ORF69的相互作用区域，这为进一步阐明ORF45在介导病毒衣壳的核释放中发挥的功能打下了基础。

Tegument proteins play critical roles in herpesvirus morphogenesis. ORF45 is a conserved tegument protein of gammaherpes-viruses; however, its role in virion morphogenesis is largely unknown. In this work, we determined the ultrastructural localization of murine gammaherpesvirus 68 (MHV-68) ORF45 and found that this protein was incorporated into virions around the site of host-derived vesicles. Notably, the absence of ORF45 inhibited nucleocapsid egress and blocked cytoplasmic virion maturation, demonstrating that ORF45 is essential for MHV-68 virion morphogenesis.