The KSHV ORF20 Protein Interacts with the Viral Processivity Factor ORF59 and Promotes Viral Reactivation.
The KSHV ORF20 Protein Interacts with the Viral Processivity Factor ORF59 and Promotes Viral Reactivation.
复制标题
KSHV ORF20 蛋白与病毒持续因子 ORF59 相互作用并促进病毒重新激活。
DOI：
10.1128/spectrum.00145-21
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发表时间：
2021
影响因子：
3.7
通讯作者：
Muller,M
中科院分区：
文献类型：
--
作者：
Hoffman,D;Rodriguez,W;Macveigh-Fierro,D;Miles,J;Muller,M
在卡波西肉瘤相关疱疹病毒（KSHV）裂解性再活化后，病毒DNA（vDNA）的快速和广泛扩增触发显著的核重组。作为核结构中这种惊人转变的一部分，病毒复制区室形成为裂解vDNA产生的位点沿着着显著的空间重塑和细胞和病毒蛋白的重新定位。这些病毒复制区室容纳协调病毒基因表达、vDNA复制和核衣壳组装的几种裂解基因产物。在KSHV复制过程中调节这种核景观大修的病毒蛋白和机制在很大程度上仍然未知。KSHV的ORF20是一个在所有疱疹病毒中广泛保守的裂解基因，这表明它可能对疱疹病毒感染的进展具有根本性的贡献。在这里，我们利用混杂的生物素连接酶邻近标记方法来识别ORF20的近端相互作用组，ORF20包括几种复制相关的病毒蛋白，其中之一是ORF59，KSHV DNA持续合成因子。使用免疫共沉淀和免疫荧光测定，我们证实了ORF20和ORF59之间的相互作用，并跟踪这两种蛋白质的本地化KSHV复制隔间。为了进一步表征ORF20的功能，我们产生了ORF20缺陷型KSHV，并将其复制适应性与野生型病毒进行了比较。病毒粒子生产显着减少ORF20缺陷型病毒，观察上清液转移试验。此外，我们将活病毒体形成中的这种缺陷与病毒晚期基因表达的减少联系起来。最后，我们观察到ORF20缺陷型病毒中vDNA复制的总体减少，这意味着ORF20在裂解复制的调节中起关键作用。综上所述，这些结果捕获了KSHV ORF 20通过调节vDNA复制以及KSHV复制区室中的其他关键裂解蛋白在进行病毒裂解感染中的重要作用。重要信息卡波西肉瘤相关疱疹病毒（KSHV）是一种诱导终身感染的疱疹病毒，因此，它的裂解性复制受到仔细控制，以允许从其长期宿主有效传播并将病毒传播到新宿主。病毒DNA复制涉及许多宿主和病毒蛋白质，在时间和空间上协调以成功地通过病毒生命周期。然而，这一过程仍未得到充分理解。我们研究了表征不佳的病毒蛋白ORF20的作用，通过邻近标记，我们发现ORF20与复制区室中的ORF59相互作用，并影响DNA复制和病毒生命周期后期的后续步骤。总的来说，这些结果提供了深入了解的ORF20的复杂的裂解DNA复制过程中可能的贡献，并表明，这种高度保守的蛋白质可能是一个重要的调制器，这一关键的病毒机制。
Upon Kaposi’s Sarcoma-associated herpesvirus (KSHV) lytic reactivation, rapid and widespread amplification of viral DNA (vDNA) triggers significant nuclear reorganization. As part of this striking shift in nuclear architecture, viral replication compartments are formed as sites of lytic vDNA production along with remarkable spatial remodeling and the relocalization of cellular and viral proteins. These viral replication compartments house several lytic gene products that coordinate viral gene expression, vDNA replication, and nucleocapsid assembly. The viral proteins and mechanisms that regulate this overhaul of the nuclear landscape during KSHV replication remain largely unknown. KSHV’sORF20is a widely conserved lytic gene among all herpesviruses, suggesting it may have a fundamental contribution to the progression of herpesviral infection. Here, we utilized a promiscuous biotin ligase proximity labeling method to identify the proximal interactome of ORF20, which includes several replication-associated viral proteins, one of which is ORF59, the KSHV DNA processivity factor. Using coimmunoprecipitation and immunofluorescence assays, we confirmed the interaction between ORF20 and ORF59 and tracked the localization of both proteins to KSHV replication compartments. To further characterize the function of ORF20, we generated an ORF20-deficient KSHV and compared its replicative fitness to that of wild-type virus. Virion production was significantly diminished in the ORF20-deficient virus as observed by supernatant transfer assays. Additionally, we tied this defect in viable virion formation to a reduction in viral late gene expression. Lastly, we observed an overall reduction in vDNA replication in the ORF20-deficient virus, implying a key role for ORF20 in the regulation of lytic replication. Taken together, these results capture the essential role of KSHV ORF20 in progressing viral lytic infection by regulating vDNA replication alongside other crucial lytic proteins within KSHV replication compartments.IMPORTANCEKaposi’s Sarcoma-associated herpesvirus (KSHV) is a herpesvirus that induces lifelong infection, and as such, its lytic replication is carefully controlled to allow for efficient dissemination from its long-term reservoir and for the spread of the virus to new hosts. Viral DNA replication involves many host and viral proteins, coordinating both in time and space to successfully progress through the viral life cycle. Yet, this process is still not fully understood. We investigated the role of the poorly characterized viral protein ORF20, and through proximity labeling, we found that ORF20 interacts with ORF59 in replication compartments and affects DNA replication and subsequent steps of the late viral life cycle. Collectively, these results provide insights into the possible contribution of ORF20 to the complex lytic DNA replication process and suggest that this highly conserved protein may be an important modulator of this key viral mechanism.