Gammaherpesvirus Tegument Protein ORF33 Is Associated with Intranuclear Capsids at an Early Stage of the Tegumentation Process
Gammaherpesvirus Tegument Protein ORF33 Is Associated with Intranuclear Capsids at an Early Stage of the Tegumentation Process
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伽马疱疹病毒外皮蛋白 ORF33 在外皮过程的早期阶段与核内衣壳相关
DOI：
10.1128/jvi.00079-15
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发表时间：
2015-05-01
影响因子：
5.4
通讯作者：
Deng, Hongyu
中科院分区：
文献类型：
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作者：
Shen, Sheng;Jia, Xing;Deng, Hongyu
疱疹病毒新生衣壳在核内组装后，在成熟过程中必须获得各种被膜蛋白。然而，由于缺乏有效的裂解复制系统，对与核中衣壳相关的被膜蛋白的身份或衣壳进入细胞质的核出口所涉及的分子机制知之甚少，特别是对于两种人类γ疱疹病毒Epstein-Barr病毒（EBV）和卡波西肉瘤相关疱疹病毒（KSHV）。鼠γ疱疹病毒68（MHV-68）与人γ疱疹病毒具有遗传相关性，并且是研究γ疱疹病毒的从头裂解复制的极好模型。我们先前已经证明，MHV-68的开放阅读框33（ORF 33）是成熟病毒体的被膜蛋白，并且对于病毒体组装和排出是必需的。然而，ORF 33是如何整合到病毒粒子中的仍不清楚。在这项研究中，我们首先表明，内源性ORF 33蛋白与衣壳蛋白共定位在离散地区的核病毒感染期间。共沉淀分析以及免疫沉淀试验表明，ORF 33是与核和细胞质衣壳。使用抗ORF 33单克隆抗体的免疫金标记实验显示，细胞核中富含ORF 33的区域被未成熟的衣壳包围。此外，ORF 33与原代表达前的核衣壳以及细胞质中的成熟病毒体相关。最后，我们发现ORF 33与两种衣壳蛋白相互作用，表明核衣壳可能以直接的方式与ORF 33相互作用。综上所述，我们鉴定了ORF 33是一种被膜蛋白，它在初级增殖之前与核内衣壳相关，可能是通过与衣壳蛋白直接相互作用。重要的是，形态发生是病毒增殖中的一个重要步骤，导致子代病毒体的产生。对于疱疹病毒来说，这是一个复杂的过程，始于细胞核。虽然衣壳组装和基因组包装的过程相对较好地理解，但衣壳如何获得被膜（疱疹病毒病毒粒子中衣壳和包膜之间的层）以及最初的被膜过程是否发生在细胞核中仍然不清楚。我们以前的研究表明，MHV-68的ORF 33是一种被膜蛋白，在衣壳的核出口和最终的病毒粒子在细胞质中的成熟中发挥作用。在本研究中，我们表明，ORF 33是与核内衣壳在初级纯化之前，并确定ORF 33和两个衣壳蛋白之间的新的相互作用。我们的工作提供了新的见解之间的关联被蛋白和核衣壳在疱疹病毒的病毒粒子成熟过程的早期阶段。
Herpesvirus nascent capsids, after assembly in the nucleus, must acquire a variety of tegument proteins during maturation. However, little is known about the identity of the tegument proteins that are associated with capsids in the nucleus or the molecular mechanisms involved in the nuclear egress of capsids into the cytoplasm, especially for the two human gammaherpesviruses Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV), due to a lack of efficient lytic replication systems. Murine gammaherpesvirus 68 (MHV-68) is genetically related to human gammaherpesviruses and serves as an excellent model to study the de novo lytic replication of gammaherpesviruses. We have previously shown that open reading frame 33 (ORF33) of MHV-68 is a tegument protein of mature virions and is essential for virion assembly and egress. However, it remains unclear how ORF33 is incorporated into virions. In this study, we first show that the endogenous ORF33 protein colocalizes with capsid proteins at discrete areas in the nucleus during viral infection. Cosedimentation analysis as well as an immunoprecipitation assay demonstrated that ORF33 is associated with both nuclear and cytoplasmic capsids. An immunogold labeling experiment using an anti-ORF33 monoclonal antibody revealed that ORF33-rich areas in the nucleus are surrounded by immature capsids. Moreover, ORF33 is associated with nucleocapsids prior to primary envelopment as well as with mature virions in the cytoplasm. Finally, we show that ORF33 interacts with two capsid proteins, suggesting that nucleocapsids may interact with ORF33 in a direct manner. In summary, we identified ORF33 to be a tegument protein that is associated with intranuclear capsids prior to primary envelopment, likely through interacting with capsid proteins in a direct manner.IMPORTANCEMorphogenesis is an essential step in virus propagation that leads to the generation of progeny virions. For herpesviruses, this is a complicated process that starts in the nucleus. Although the process of capsid assembly and genome packaging is relatively well understood, how capsids acquire tegument (the layer between the capsid and the envelope in a herpesvirus virion) and whether the initial tegumentation process takes place in the nucleus remain unclear. We previously showed that ORF33 of MHV-68 is a tegument protein and functions in both the nuclear egress of capsids and final virion maturation in the cytoplasm. In the present study, we show that ORF33 is associated with intranuclear capsids prior to primary envelopment and identify novel interactions between ORF33 and two capsid proteins. Our work provides new insights into the association between tegument proteins and nucleocapsids at an early stage of the virion maturation process for herpesviruses.