卡波西肉瘤相关疱疹病毒 (KSHV) DNA 聚合酶持续合成因子 ORF59 是病毒 DNA 合成所必需的裂解蛋白，是核心复制复合体的一部分。 ORF59 的多功能性质促使人们对其各种功能域进行研究。先前对 ORF59 的研究已经确定了二聚化、DNA 相互作用和聚合酶相互作用域。 ORF59 负责与病毒 mRNA 转运积累蛋白 (MTA/ORF57) 和病毒长非编码聚腺苷酸化核 (PAN) RNA 相互作用的区域尚未被探索。使用一系列先前表征的 ORF59 缺失 KSHV BACmid 突变体，我们鉴定了与 ORF57 和 PAN RNA 相互作用的 ORF59 结构域。有趣的是，氨基酸 51-100 对于与 ORF57 和 PAN RNA 相互作用至关重要。利用这些信息，我们生成了表达 DsRed 标记多肽的质粒，该多肽跨越 ORF59 的氨基酸 30-100。当在裂解再激活之前将30-100个aa DsRed标记的多肽表达质粒转染至KSHV野生型iSLK细胞中时，观察到病毒复制的显性失活抑制，导致感染性病毒产生减少。我们的数据表明，ORF59 与 ORF57 和 PAN RNA 之间的相互作用对于成功裂解复制非常重要。重要性为了更好地了解卡波西肉瘤相关疱疹病毒 (KSHV) DNA 聚合酶持续合成因子 ORF59，我们研究了 ORF59 与 ORF57 和聚腺苷酸化核 (PAN) 的相互作用。 )RNA。我们使用先前表征的包含 ORF59 内部缺失的 KSHV BACmid 来鉴定与 ORF57 和 PAN RNA 相互作用的 ORF59 结构域。我们的研究揭示了 ORF59 的多个结构域，这些结构域对于其与 PAN RNA 的关联至关重要。这些结构域跨越氨基酸 51-100、251-300 和 351-396。其他实验证实氨基酸 51-100 对于 ORF59 和 ORF57 之间的相互作用至关重要。利用这些信息，我们生成了包含 ORF57 和 ORF59 的 PAN RNA 相互作用结构域的表达质粒。氨基酸30-100的ORF59多肽表达质粒在病毒再激活过程中充当显性失活抑制剂并导致病毒产量减少。这些发现为 ORF59 的关键领域提供了宝贵的见解，这对于其功能以及最终感染性病毒的产生至关重要。

Kaposi's sarcoma-associated herpesvirus (KSHV) DNA polymerase processivity factor, ORF59, is a lytic protein essential for viral DNA synthesis as part of the core replication complex. The multifunctional nature of ORF59 has prompted the investigation into its various functional domains. Prior studies of ORF59 have identified dimerization, DNA interaction, and polymerase interaction domains. The regions of ORF59 responsible for the interaction with the viral mRNA transport accumulation protein (MTA/ORF57) and the viral long non-coding polyadenylated nuclear (PAN) RNA have not been explored. Using a series of previously characterized ORF59 deletion KSHV BACmid mutants, we identified the domains of ORF59 that interact with ORF57 and PAN RNA. Interestingly, amino acids 51-100 were essential for interacting with both ORF57 and PAN RNA. Using this information, we generated a plasmid that expresses a DsRed-tagged polypeptide spanning amino acids 30-100 of ORF59. When the 30-100 aa DsRed-tagged polypeptide expression plasmid was transfected into KSHV wild-type iSLK cells prior to lytic reactivation, a dominant-negative inhibition of virus replication was observed, resulting in a decrease of infectious virus production. Our data suggest that interactions between ORF59 with ORF57 and PAN RNA are important to successful lytic replication.IMPORTANCETo better understand the Kaposi's sarcoma-associated herpesvirus (KSHV) DNA polymerase processivity factor ORF59, we investigated the interaction of ORF59 with ORF57 and polyadenylated nuclear (PAN) RNA. We used a previously characterized KSHV BACmid containing internal deletions of ORF59 to identify the domains of ORF59 that interact with ORF57 and PAN RNA. Our study revealed multiple domains of ORF59 that are essential for its association with PAN RNA. These domains span amino acids 51-100, 251-300, and 351-396. Additional experiments confirmed amino acids 51-100 are critical for the interaction between ORF59 and ORF57. Using this information, we generated an expression plasmid encompassing the ORF57 and PAN RNA interaction domains of ORF59. The ORF59 polypeptide expression plasmid of amino acids 30-100 functioned as a dominant negative inhibitor during viral reactivation and caused a decrease in virus production. These findings provide valuable insights into the key domains of ORF59, essential for its functionality, and ultimately the production of infectious viruses.