The DNase Activity of Kaposi's Sarcoma-Associated Herpesvirus SOX Protein Serves an Important Role in Viral Genome Processing during Lytic Replication.

The Kaposi's sarcoma-associated herpesvirus (KSHV) alkaline exonuclease SOX, encoded by open reading frame 37 (ORF37), is a bifunctional early-lytic-phase protein that possesses alkaline 5'-to-3' DNase activity and promotes host shutoff at the mRNA level during productive lytic infection. While the SOX protein is well characterized for drastically impairing cellular gene expression, little is known about the impact of its DNase activity on the KSHV genome and life cycle and the biology of KSHV infections. Here, we introduced a previously described DNase-inactivating Glu129His (Q129H) mutation into the ORF37 gene of the viral genome to generate ORF37-Q129H recombinant virus (the Q129H mutant) and investigated the effects of loss or inactivation of DNase activity on viral genome replication, cleavage, and packaging. For the first time, we provide experimental evidence that the DNase activity of the SOX protein does not affect viral latent/lytic DNA synthesis but is required for cleavage and processing of the KSHV genome during lytic replication. Interestingly, the Q129H mutation severely impaired intranuclear processing of progeny virions compared to the wild-type ORF37, as assessed by pulsed-field and Gardella gel electrophoresis, electron microscopy, and single-molecule analysis of replicating DNA (SMARD) assays. Complementation with ORF37-wt (wild type) or BGLF5 (the KSHV protein homolog in Epstein-Barr virus) in 293L/Q129H cells restored the viral genome encapsidation defects. Together, these results indicated that ORF37's proposed DNase activity is essential for viral genome processing and encapsidation and, hence, can be targeted for designing antiviral agents to block KSHV virion production. Kaposi's sarcoma (KS)-associated herpesvirus is the causative agent of multiple malignancies, predominantly in immunocompromised individuals, including HIV/AIDS patients. Reduced incidence of KS in HIV/AIDS patients receiving antiherpetic drugs to block lytic replication confirms the role of lytic DNA replication and gene products in KSHV-mediated tumorigenesis. Herpesvirus lytic replication results in the production of complex concatemeric DNA, which is cleaved into unit length viral DNA for packaging into the infectious virions. The conserved herpesviral alkaline exonucleases play an important role in viral genome cleavage and packaging. Here, by using the previously described Q129H mutant virus that selectively lacks DNase activity but retains host shutoff activity, we provide experimental evidence confirming that the DNase function of the KSHV SOX protein is essential for viral genome processing and packaging and capsid maturation into the cytoplasm during lytic replication in infected cells. This led to the identification of ORF37's DNase activity as a potential target for antiviral therapeutics.