KSHV activates unfolded protein response sensors but suppresses downstream transcriptional responses to support lytic replication

Author summary Like all viruses, Kaposi's sarcoma-associated herpesvirus (KSHV) uses cellular machinery to create viral proteins. Some of these proteins are folded and modified in the endoplasmic reticulum (ER) and traverse the cellular secretory apparatus. Exceeding ER protein folding capacity activates the unfolded protein response (UPR), which resolves ER stress by putting the brakes on protein synthesis and turning on stress-mitigating genes. We show that KSHV replication activates the three cellular proteins that sense ER stress, which are each required to support efficient viral replication. By contrast, KSHV blocks the UPR gene expression program downstream from each of these activated sensor proteins. The failure to resolve ER stress might normally be expected to put the virus at a disadvantage, but we demonstrate that reversal of this scenario is worse; when we supplement infected epithelial cells with the UPR transcription factor XBP1s to artificially stimulate the production of UPR-responsive gene products, virus replication is blocked at a late stage and very few viruses are released from infected cells. Taken together, these observations suggest that KSHV requires UPR sensor protein activation to replicate but has dramatically altered the outcome to prevent the synthesis of new UPR proteins and sustain stress in the ER compartment. Herpesviruses usurp host cell protein synthesis machinery to convert viral mRNAs into proteins, and the endoplasmic reticulum (ER) to ensure proper folding, post-translational modification and trafficking of secreted and transmembrane viral proteins. Overloading ER folding capacity activates the unfolded protein response (UPR), whereby sensor proteins ATF6, PERK and IRE1 initiate a stress-mitigating transcription program that accelerates catabolism of misfolded proteins while increasing ER folding capacity. Kaposi's sarcoma-associated herpesvirus (KSHV) can be reactivated from latency by chemical induction of ER stress, which causes accumulation of the XBP1s transcription factor that transactivates the viral RTA lytic switch gene. The presence of XBP1s-responsive elements in the RTA promoter suggests that KSHV evolved a mechanism to respond to ER stress. Here, we report that ATF6, PERK and IRE1 were activated upon reactivation from latency and required for efficient KSHV lytic replication; genetic or pharmacologic inhibition of each UPR sensor diminished virion production. Despite UPR sensor activation during KSHV lytic replication, downstream UPR transcriptional responses were restricted; 1) ATF6 was cleaved to activate the ATF6(N) transcription factor but ATF6(N)-responsive genes were not transcribed; 2) PERK phosphorylated eIF2 alpha but ATF4 did not accumulate; 3) IRE1 caused XBP1 mRNA splicing, but XBP1s protein did not accumulate and XBP1s-responsive genes were not transcribed. Ectopic expression of the KSHV host shutoff protein SOX did not affect UPR gene expression, suggesting that alternative viral mechanisms likely mediate UPR suppression during lytic replication. Complementation of XBP1s deficiency during KSHV lytic replication inhibited virion production in a dose-dependent manner in iSLK.219 cells but not in TREx-BCBL1-RTA cells. However, genetically distinct KSHV virions harvested from these two cell lines were equally susceptible to XBP1s restriction following infection of naive iSLK cells. This suggests that cell-intrinsic properties of BCBL1 cells may circumvent the antiviral effect of ectopic XBP1s expression. Taken together, these findings indicate that while XBP1s plays an important role in reactivation from latency, it can inhibit virus replication at a later step, which the virus overcomes by preventing its synthesis. These findings suggest that KSHV hijacks UPR sensors to promote efficient viral replication while sustaining ER stress.