Kaposi's Sarcoma-Associated Herpesvirus ORF68 Is a DNA Binding Protein Required for Viral Genome Cleavage and Packaging

Herpesviral DNA packaging into nascent capsids requires multiple conserved viral proteins that coordinate genome encapsidation. Here, we investigated the role of the ORF68 protein of Kaposi's sarcoma-associated herpesvirus (KSHV), a protein required for viral DNA encapsidation whose function remains largely unresolved across the herpesviridae. We found that KSHV ORF68 is expressed with early kinetics and localizes predominantly to viral replication compartments, although it is dispensable for viral DNA replication and gene expression. However, in agreement with its proposed role in viral DNA packaging, KSHV-infected cells lacking ORF68 failed to cleave viral DNA concatemers, accumulated exclusively immature B capsids, and released no infectious progeny virions. ORF68 has no predicted domains aside from a series of putative zinc finger motifs. However, in vitro biochemical analyses of purified ORF68 protein revealed that it robustly binds DNA and is associated with nuclease activity. These activities provide new insights into the role of KSHV ORF68 in viral genome encapsidation. IMPORTANCE Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiologic agent of Kaposi's sarcoma and several B-cell cancers, causing significant morbidity and mortality in immunocompromised individuals. A critical step in the production of infectious viral progeny is the packaging of the newly replicated viral DNA genome into the capsid, which involves coordination between at least seven herpesviral proteins. While the majority of these packaging factors have been well studied in related herpesviruses, the role of the KSHV ORF68 protein and its homologs remains unresolved. Here, using a KSHV mutant lacking ORF68, we confirm its requirement for viral DNA processing and packaging in infected cells. Furthermore, we show that the purified ORF68 protein directly binds DNA and is associated with a metal-dependent cleavage activity on double-stranded DNA in vitro. These activities suggest a novel role for ORF68 in herpesviral genome processing and encapsidation.