Payen Shannon Harger, Gutierrez Isaura Vanessa, Andrada Kayla, Verma Subhash C, Rossetto Cyprian C
Department of Microbiology and Immunology, University of Nevada, Reno School of Medicine, Reno, Nevada, USA.
Microbiol Spectr. 2024 Oct 21;12(12):e0116324. doi: 10.1128/spectrum.01163-24.
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.
卡波西肉瘤相关疱疹病毒（KSHV）的DNA聚合酶持续合成因子ORF59是一种裂解蛋白，作为核心复制复合体的一部分，对病毒DNA合成至关重要。ORF59的多功能性质促使人们对其各个功能域进行研究。先前对ORF59的研究已确定了其二聚化、DNA相互作用和聚合酶相互作用域。负责与病毒mRNA转运积累蛋白（MTA/ORF57）和病毒长链非编码聚腺苷酸化核（PAN）RNA相互作用的ORF59区域尚未得到探索。我们使用一系列先前已鉴定特征的ORF59缺失KSHV BACmid突变体，确定了ORF59与ORF57和PAN RNA相互作用的结构域。有趣的是，氨基酸51 - 100对于与ORF57和PAN RNA的相互作用至关重要。利用这一信息，我们构建了一个表达DsRed标记的包含ORF59氨基酸30 - 100的多肽的质粒。当在裂解再激活前将30 - 100氨基酸DsRed标记的多肽表达质粒转染到KSHV野生型iSLK细胞中时，观察到病毒复制的显性负抑制，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的裂解复制很重要。
重要性
为了更好地理解卡波西肉瘤相关疱疹病毒（KSHV）的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）RNA的相互作用。我们使用了先前已鉴定特征的含有ORF59内部缺失的KSHV BACmid来确定ORF59与ORF57和PAN RNA相互作用的结构域。我们的研究揭示了ORF59的多个结构域对于其与PAN RNA的结合至关重要。这些结构域跨越氨基酸51 - 100、251 - 300和351 - 396。进一步的实验证实氨基酸51 - 100对于ORF59与ORF57之间的相互作用至关重要。利用这一信息，我们构建了一个包含ORF59的ORF57和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起显性负抑制剂的作用，并导致病毒产生减少。这些发现为ORF59的关键结构域提供了有价值的见解，这些结构域对其功能以及最终感染性病毒的产生至关重要。
