Caspase Cleavage of Kaposi Sarcoma-Associated Herpesvirus Proteins: A role for K5 in Preventing Caspase-Mediated Cell Death during Lytic Replication

We previously reported that Kaposi sarcoma-associated herpesvirus (KSHV) latency-associated nuclear antigen (LANA) acts as a pseudo-substrate for caspases-1 and 3, thereby interfering with their inflammatory and apoptotic activity, respectively. To determine if other KSHV proteins undergo caspase cleavage, we screened the KSHV proteome for potential caspase cleavage sites. Using SitePrediction (SP), 30 KSHV proteins with potential caspase-cleavage sites were identified. Among those with highest SP score was an early lytic protein, K5. Treatment of BJAB K5-FLAG cells with ⍺Fas, an apoptotic stimulus, led to caspase-processing of full length K5-FLAG and generation of a C-terminal peptide fragment. Using mass spectrometry, we determined that K5-FLAG undergoes caspase cleavage at D222. K5 was also cleaved by caspases in KSHV infected cells induced to lytic replication. Importantly, 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, iSLKK cells infected with wild type or K5 knockout BAC16 virus were induce to lytic replication to activate caspases. Although lytic induction showed little effect on the viability of WT 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 ability to downregulate MHC-1 surface expression. Overall, these data suggest that K5 not only downregulates immunologic surface marker expression to avoid immune recognition but may also play an additional role in mitigating caspase-mediated cell death during KSHV lytic replication.