2009. (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 ORF37s 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. IMPORTANCE 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 ORF37s DNase activity as a potential target for antiviral therapeutics. within the family and is closely related to Epstein-Barr virus (EBV), a B-lymphotropic member of the subfamily infection, KSHV displays two distinct transcriptional programs: a prolonged dormant latency and intermittent productive lytic reactivation (reviewed in reference 9). The process of herpesvirus DNA replication generates branched/longer-than-unit-length concatemeric DNA in the nuclei of infected cells, which is cleaved into linear/unit length monomeric DNA fragments (10). The newly replicated DNA further undergoes a well-coordinated process of encapsidation into immature capsids in the Mbp nucleus, resulting in the generation of DNA-filled capsids (C capsids) that mature into the cytoplasm after budding through the nuclear membrane. Mature C capsids are Molibresib besylate tegumented and enveloped in the cytoplasm by budding into vesicles of the for successful viral replication (26). The crystal structure of Molibresib besylate the SOX protein bound to a DNA duplex indicates that the DNA strand cleavage likely occurs through a bimetal nuclease mechanism that involves the D221 and E244 carboxylate groups (27, 28). The exact mechanism of SOXs DNase function is still unknown; however, it seems to play a prominent role in processing and packaging of newly synthesized nonlinear DNA into a linear form suitable for encapsidation and nuclear egress. Although KSHV SOX protein-initiated host Molibresib besylate shutoff upon productive infection has been extensively studied for many years, much less is known about the conserved DNase activity, which is considered critical for editing of the viral genome during lytic DNA replication (16, 17). Most knowledge about the SOX proteins intrinsic DNase activity comes from the study of alkaline exonucleases of other herpesviruses, namely, HSV-1 (UL12) and EBV (BGLF5). Recombinant mutant viruses devoid of UL12 (the AN-1 or D340E mutant) and BGLF5 (the BGLF5 mutant) genes have been shown to have effects on infectious virion Molibresib besylate production, to generate complex viral DNA replication intermediates, and to display impaired nuclear egress and progeny virion production (19, 23, 29, 30). Based on these observations and similarities between herpesvirus replication and packaging machineries, we predict that KSHV SOX most likely possesses DNA-processing activity similar to that of UL12 and BGLF5. However, the exact role of KSHV ORF37 in viral DNA replication and genome editing remains uncharacterized. Interestingly, a point mutation of amino acid residue 129 of ORF37 (Q129H, or Glu129His) has been previously reported to preserve wild-type shutoff activity but to completely abolish the DNase activity of the ORF37 protein (17). Hence, use of ORF37-Q129H recombinant virus with DNase activity abolished will be instrumental in determining the molecular role of ORF37 in viral genome editing and maturation during KSHV lytic replication. In order to determine the probable role of the ORF37 protein in the context of the KSHV lytic cycle and how the absence of DNase activity may affect virus growth, we constructed.