Investigating Herpesvirus Replication and Virus-Host Interactions During SARS-CoV-2 Co-infections

Kaposi’s sarcoma-associated herpesvirus (KSHV) is a gamma-herpesvirus in the Herpesviridae family. KSHV is an oncogenic virus and is the causative agent of Kaposi’s sarcoma (KS), Multicentric Castleman’s Disease (MCD), primary effusion lymphoma (PEL), and Kaposi Sarcoma Inflammatory Cytokine Syndrome (KICS). KSHV, like all herpesviruses, has a bi-phasic replication cycle consisting of a latent phase with periodic episodes of lytic reactivation. Within the replication complex, the DNA polymerase processivity factor plays a critical role in viral DNA synthesis through interactions with both viral and cellular components. Previous studies have described various functional domains of ORF59. However, the regions responsible for interactions with viral mRNA transport accumulation protein ORF57 and viral polyadenylated nuclear (PAN) RNA remained unexplored. Using ORF59 deletion mutants in KSHV BACmid, we identified multiple domains of ORF59 (amino acids 51-100, 251-300, and 351-396) that interact with PAN RNA. Notably, the absence of association with ORF59 led to an aberrant localization pattern of PAN RNA, forming aggregates concentrated into foci. Interestingly, amino acids 51-100 were found to be essential for interaction with ORF57, indicating a shared interaction domain for PAN RNA and ORF57. To further elucidate the role of ORF59 in viral replication, a small DsRed-tagged polypeptide spanning amino acids 30-100 of ORF59 was created. Transfection of this polypeptide into iSLK-WT cells resulted in a dominant-negative inhibition of virus replication, leading to decreased infectious virion production. To understand why nuclear punctate formed within specific ORF59 deletion domains, we investigated the role of paraspeckle nuclear bodies, comprising core ribonucleoproteins SFPQ and NONO during lytic KSHV replication. Using SFPQ immunoprecipitation followed by mass spectrometry, we found interactions between SFPQ and KSHV viral proteins, ORF10, ORF59, ORF61, ORF67, ORF11, ORF21, ORF50, ORF52, ORF57, ORF6, ORF25, and ...