The Kaposi's sarcoma-associated herpesvirus viral genome packaging accessory factor ORF68 forms cytoplasmic puncta dependent on the viral tyrosine kinase

Assembly of Kaposis sarcoma-associated herpesvirus (KSHV) virions is dependent on ORF68, a viral genome packaging accessory factor of unknown function. We used confocal fluorescence imaging to monitor ORF68 localization throughout the KSHV lytic cycle. ORF68 localizes to nuclear viral replication compartments, the site of viral genome packaging. Unexpectedly, ORF68 also localized to cytoplasmic puncta. Using proximity labeling mass spectrometry during infection, we identified ORF68 interaction partners, including the viral tyrosine kinase ORF21. We show that ORF21 colocalizes with ORF68 in cytoplasmic puncta and draws ORF68 to the cytoplasm during infection. This interaction is mediated by the disordered N-terminal region of ORF21. We propose that ORF68 possesses a novel secondary function independent from virion assembly. IMPORTANCEKaposis sarcoma-associated herpesvirus (KSHV) is the underlying cause of multiple human malignancies including Kaposis sarcoma, primary effusion lymphoma, and multicentric Castlemans disease. Encapsidation of the viral genome is necessary to produce new infectious virus. Viral genome packaging can be targeted with small molecules in human cytomegalovirus, a related herpesvirus, but we lack a sufficiently detailed mechanistic understanding to develop additional therapeutics. The molecular role of the essential packaging accessory factor, encoded by ORF68 in KSHV, has remained unclear. We studied the interactions and localization of ORF68 during KSHV infection and find that, in addition to its nuclear role in packaging, ORF68 forms cytoplasmic puncta through its interaction with the viral tyrosine kinase ORF21. This study demonstrates how conserved, essential herpesvirus proteins can play multiple roles in distinct subcellular compartments during viral infection, with secondary functions that may be herpesvirus species-specific.