识别KSHV潜伏复制过程中ORF59与ORF57和PAN RNA相互作用的氨基酸域

Kaposi肉瘤相关疱疹病毒（KSHV）DNA聚合酶过程延伸因子ORF59，是病毒复制核心复合物中的一种潜伏蛋白，必不可少。ORF59的多功能性质促使研究其不同功能域。先前的研究已识别出ORF59的二聚化、DNA相互作用和聚合酶相互作用域，但尚未探明其与病毒mRNA运输积累蛋白（MTA/ORF57）以及病毒长链非编码多腺苷酸化核RNA（PAN RNA）之间的相互作用区域。利用一系列已表征的ORF59缺失的KSHV BAC质粒突变体，我们鉴定了与ORF57和PAN RNA相互作用的ORF59结构域。研究发现，氨基酸51-100是与两者都必不可少的关键区域。基于此信息，我们构建了表达带有DsRed标签、覆盖氨基酸30-100的ORF59多肽的质粒。当此表达质粒在潜伏激活前转染到野生型iSLK细胞中时，观察到病毒复制的显性阴性抑制，导致感染性病毒产量下降。我们的数据表明，ORF59与ORF57及PAN RNA的相互作用对于成功的潜伏复制具有重要意义。重要性为了更好理解Kaposi肉瘤相关疱疹病毒（KSHV）DNA聚合酶过程延伸因子ORF59，我们研究了其与ORF57和多腺苷酸化核RNA（PAN RNA）的相互作用。利用带有内部缺失的已表征的KSHV BAC质粒，我们确定了ORF59与这些RNA相互作用的结构域，主要位于氨基酸51-100、251-300和351-396。这些结构域中的氨基酸51-100对ORF59与ORF57的相互作用尤为关键。基于这些信息，我们制备了涵盖ORF59与ORF57及PAN RNA相互作用结构域的表达质粒。该质粒（氨基酸30-100）在病毒激活过程中表现为显性阴性抑制，导致病毒产量下降。这些发现为揭示ORF59功能的关键结构域提供了重要线索，并为未来开发抗病毒策略提供了潜在靶点。

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.