Caspase cleavage of Kaposi sarcoma-associated herpesvirus proteins: a role for K5 in preventing caspase-mediated cell death during lytic replication

Viral infections lead to caspase activation as a cellular defense response. Some viruses overcome this response by encoding viral proteins that undergo caspase cleavage and, by various mechanisms, aid in cell survival. Kaposi sarcoma-associated herpesvirus (KSHV) latency-associated nuclear antigen acts as a pseudo-substrate for caspases-1 and -3, thereby interfering with the inflammatory and apoptotic response. Here, we screened the KSHV proteome for additional caspase cleavage sites. Using SitePrediction, many KSHV proteins with potential caspase cleavage sites were identified. Among the highest-scoring proteins was the early lytic protein, K5. Treatment of BJAB-K5-FLAG-expressing cells with ⍺Fas led to caspase processing of K5-FLAG. Using mass spectrometry, we determined that caspase cleavage of K5 occurs at D222. K5 was also cleaved by caspases when KSHV was induced to lytic replication. Interestingly, the expression of K5-FLAG significantly inhibited ⍺Fas-induced caspase-mediated cell death. To determine if K5 plays a protective role in KSHV-infected cells, iSLK cells infected with wild-type or K5-knockout BAC16 virus were induced to lytic replication. Although lytic induction showed no significant effect on the viability of wild-type-infected cells, the viability of K5-knockout cells decreased by 25%. Thus, K5 may protect KSHV-infected cells from caspase-mediated cell death during lytic replication. Interestingly, cleavage of K5 by caspases did not affect its previously reported ability to downregulate immune surface markers. These data suggest that K5 not only downregulates immune surface markers to help avoid immune recognition but plays an additional role in mitigating caspase-mediated cell death during lytic replication.IMPORTANCEKaposi sarcoma-associated herpesvirus (KSHV) is the etiological agent for Kaposi sarcoma, primary effusion lymphoma, multicentric Castleman's disease, and KSHV inflammatory cytokine syndrome. Understanding how KSHV thwarts host defense responses is necessary to help develop strategies to treat these rare, yet deadly, diseases. We profiled potential caspase cleavage sites in the viral proteome <i>in silico</i> and found many viral proteins with high-scoring caspase cleavage sites. We follow up on this by demonstrating that K5 is, in fact, a substrate for caspases <i>in vitro</i> and <i>in cellulo</i> and provide data to suggest that K5 plays a role in obviating caspase-mediated cell death during lytic replication. The work described here furthers our understanding of the roles that KSHV proteins, like K5, play to prevent or divert the host apoptotic defense responses that involve host caspase activation that normally would lead to cell death.