The KSHV protein ORF68 is a proteasome-manipulating protein required for DNA packaging

Herpesviruses are large, double-stranded DNA viruses that have evolved over millions of years to become master manipulators of cellular machinery. Herpesviral research has taught us a great deal not only about strategies of viral replication and pathogenesis, but also about how cellular pathways work in the absence of infection. The end goal of viral infection is the production of progeny virions, which involves the packaging viral genomes into nascent capsids. This process is mechanistically conserved across the three herpesvirus subfamilies, with at least seven viral proteins being required for successful packaging to occur. Of these proteins, six have well characterized functions during the packaging process, while the function of the seventh remains largely unknown. In Kaposi’s sarcoma-associated herpesvirus (KSHV), this protein is encoded by ORF68 and here we aimed to elucidate its role in DNA packaging.We first generated an ORF68 mutant of KSHV that contained a premature termination codon (ORF68PTC) and confirmed that ORF68 was not required for viral gene expression or DNA replication but was essential for infectious virion production. Additionally, the KSHV ORF68PTC virus did not cleave or package viral DNA and exclusively accumulated immature B-capsids. Further biochemical characterization of ORF68 revealed several novel activities for this protein that significantly expand our understanding of its function. Unexpectedly, we found that ORF68 robustly binds DNA and exhibits metal-dependent nuclease activity towards dsDNA in vitro. These observations suggest a role in binding and catalytically processing the viral genome during packaging.Additionally, protein-protein interaction profiling of ORF68 revealed an association with multiple subunits of the proteasome, the main protein degradation apparatus of the cell. This led us to hypothesize that KSHV might control protein abundance, in part, through the ORF68-proteasome interaction. In support of this hypothesis, we revealed that ORF68 contains a canonical gate-opening motif observed in proteasome-interacting proteins. Deletion or blocking of this motif in ORF68 inhibited the degradation of a model substrate in transfected cells, suggesting that ORF68 may assemble on proteasomes to control substrate access. Notably, deletion of this motif from ORF68 in the context of KSHV infection prevented progeny virion production, indicating that this function is central to its role in the viral replication cycle. Finally, we observed proteasomes concentrated within replication compartments together with ORF68. Collectively, these data lead to a model in which ORF68 interactions with the proteasome may control protein turnover to facilitate viral DNA packaging