识别ORF59的氨基酸结构域在KSHV裂解复制过程中与ORF57和PAN RNA相互作用的氨基酸结构域

Kaposi的肉瘤相关疱疹病毒（KSHV）DNA聚合酶加工性因子ORF59是病毒DNA合成必不可少的裂解蛋白，这是核心复制复合物的一部分。 ORF59的多功能性促使对其各种功能领域进行了研究。对ORF59的先前研究已经确定了二聚化，DNA相互作用和聚合酶相互作用域。尚未探索负责与病毒mRNA转运积累蛋白相互作用的ORF59区域（MTA/ORF57）和病毒长的非编码多腺苷酸化核（PAN）RNA的区域。使用一系列先前表征的ORF59缺失KSHV BACMID突变体，我们确定了与ORF57和PAN RNA相互作用的ORF59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。使用此信息，我们生成了一种表达跨越ORF59氨基酸的DSRED标签多肽的质粒。当30-100 AA DSRED标记的多肽表达质粒被转染至KSHV野生型ISLK细胞之前，在裂解再活化之前，观察到了对病毒复制的显性阴性抑制作用，从而导致感染性病毒产生的降低。我们的数据表明，ORF59与ORF57和PAN RNA之间的相互作用对于成功的裂解复制很重要。ImportanCeto更好地了解Kaposi与肉瘤相关的疱疹病毒（KSHV）DNA聚合酶加工性因子ORF59，我们研究了ORF59与ORF57与ORF57和Polyadeenynylyledeenylylenylylenylylenylyly（Pan）的相互作用。我们使用了先前表征的KSHV BACMID，其中包含ORF59的内部缺失来识别与ORF57和PAN RNA相互作用的ORF59域。我们的研究揭示了ORF59的多个领域，这对于与PAN RNA的关联至关重要。这些域跨越氨基酸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。使用此信息，我们生成了一个包含ORF57 ORF57和PAN RNA相互作用域的表达质粒。氨基酸的ORF59多肽表达质粒30-100在病毒再活化过程中起主要的负抑制剂，并导致病毒产生降低。这些发现为ORF59的关键领域提供了宝贵的见解，这对于其功能和最终产生了传染病。

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.