ORF48 is required for optimal lytic replication of Kaposi’s sarcoma-associated herpesvirus

Kaposi's sarcoma-associated herpesvirus (KSHV) establishes persistent infection in the host by encoding a vast network of proteins that aid immune evasion. One of these targeted innate immunity pathways is the cGAS-STING pathway, which inhibits the reactivation of KSHV from latency. Previously, we identified multiple cGAS/STING inhibitors encoded by KSHV, suggesting that the counteractions of this pathway by viral proteins are critical for maintaining a successful KSHV life cycle. However, the detailed mechanisms of how these viral proteins block innate immunity and facilitate KSHV lytic replication remain largely unknown. In this study, we report that ORF48, a previously identified negative regulator of the cGAS/STING pathway, is required for optimal KSHV lytic replication. We used both siRNA and deletion-based systems to evaluate the importance of intact ORF48 in the KSHV lytic cycle. In both systems, loss of ORF48 resulted in defects in lytic gene transcription, lytic protein expression, viral genome replication and infectious virion production. ORF48 genome deletion caused more robust and global repression of the KSHV transcriptome, possibly due to the disruption of RTA promoter activity. Mechanistically, overexpressed ORF48 was found to colocalize and interact with endogenous STING in HEK293 cells. Endogenous ORF48 and STING interactions were also detected in reactivated iSLK.219 cells. Compared with the control cell line, HUVEC cells stably expressing ORF48 exhibited repressed STING-dependent innate immune signaling upon ISD or diABZI treatment. However, the loss of ORF48 in our iSLK-based lytic system failed to induce IFNβ production, suggesting a redundant role of ORF48 on STING signaling during the KSHV lytic phase. Thus, ORF48 is required for optimal KSHV lytic replication through additional mechanisms that need to be further explored.