KAPOSI’S SARCOMA-ASSOCIATED HERPESVIRUS MODULATES THE UNFOLDED PROTEIN RESPONSE DURING LYTIC REPLICATION

Abstract

Kaposi’s sarcoma-associated herpesvirus (KSHV) is the infectious cause of the complex endothelial neoplasm Kaposi’s sarcoma, and two B cell malignancies, primary effusion lymphoma and multicentric Castleman’s disease. KSHV activates multiple cellular stress responses during infection but the impact of stress on viral replication and tumorigenesis remain obscure. One such stress management pathway is the unfolded protein response (UPR), which is activated by endoplasmic reticulum (ER) stress. Activation of the three host cell sensors of ER stress, IRE1, PERK, and ATF6, stimulates synthesis of transcription factors XBP1s, ATF4, and ATF6-N, respectively, which coordinate a transcriptional program to mitigate stress and restore protein homeostasis. Failure to restore homeostasis results causes the UPR to shift from an adaptive response to a pro-apoptotic response. In cells latently infected with KSHV, ER stress initiates lytic reactivation through XBP1s-mediated transcription of the KSHV lytic switch gene, RTA. Thus, it appears that KSHV has evolved a mechanism to respond to ER stress. I investigated the role of the UPR during lytic replication in this thesis. Here, I demonstrate that lytic replication activates all three sensors of the UPR, but the downstream transcription factors are inhibited. RNA silencing or chemical inhibition of any of the UPR sensors inhibits virion production, indicating that their activation is important for some aspect of productive viral replication. Surprisingly, ectopic expression of XBP1s can also inhibit viral production. Therefore, while XBP1s plays an important role in reactivation from latency, it inhibits later steps in lytic replication. This suggests that XBP1s downregulation by viral gene products may effectively remove an anti-viral host factor. To identify potential KSHV proteins that can modulate the UPR, I screened a KSHV lentiviral ORF library with a UPR fluorescent reporter cell line. I found multiple viral proteins that showed UPR-modulating ability. Of these hits, I found that ectopic expression of Kaposin C inhibits IRE1, and that ORF45 upregulates multiple UPR markers, but likely not through ER stress induction. Overall these findings suggest that KSHV modulates UPR signaling during lytic replication to promote virus production instead of resolving ER stress.