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

Abstract

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 viral proteins. Overloading ER folding capacity activates the unfolded protein response (UPR), whereby displacement of the ER chaperone BiP activates UPR sensor proteins ATF6, PERK and IRE1 to initiate transcriptional responses to increase catabolic processes and ER folding capacity, while suppressing bulk protein synthesis. Kaposi’s sarcoma-associated herpesvirus (KSHV) can be reactivated from latency by chemical induction of ER stress, whereby the IRE1 endoribonuclease cleaves XBP1 mRNA, resulting in a ribosomal frameshift that yields the XBP1s transcription factor that transactivates the promoter of K-RTA, the viral lytic switch protein. By incorporating XBP1s responsive elements in the K-RTA promoter KSHV appears to have evolved a mechanism to respond to ER stress. Here, we report that following reactivation from latency, KSHV lytic replication causes activation of ATF6, PERK and IRE1 UPR sensor proteins. UPR sensor activation is required for efficient KSHV lytic replication; genetic or pharmacologic inhibition of each UPR sensor diminishes virion production. Despite strong UPR sensor activation during KSHV lytic replication, downstream UPR transcriptional responses were restricted; 1) ATF6 was cleaved to release the ATF6(N) transcription factor but known ATF6(N)-responsive genes were not transcribed; 2) PERK phosphorylated eIF2α but ATF4 did not accumulate as expected; 3) IRE1 caused XBP1 mRNA splicing, but XBP1s protein failed to accumulate and XBP1s-responsive genes were not transcribed. Remarkably, complementation of XBP1s deficiency during KSHV lytic replication by ectopic expression inhibited the production of infectious virions in a dose-dependent manner. Therefore, while XBP1s plays an important role in reactivation from latency, it inhibits later steps in lytic replication, which the virus overcomes by preventing its synthesis. Taken together, these findings suggest that KSHV hijacks UPR sensors to promote efficient viral replication while sustaining ER stress.Author summaryHuman herpesvirus-8 is the most recently discovered human herpesvirus, and it is the infectious cause of Kaposi’s sarcoma, which is why it’s also known as Kaposi’s sarcoma-associated herpesvirus (KSHV). Like all herpesviruses, KSHV replicates in the cell nucleus and uses host cell machinery to convert viral genes into proteins. Some of these proteins are synthesized, folded and modified in the endoplasmic reticulum (ER) and traverse the cellular secretory apparatus. Because the virus heavily utilizes the ER to make and process proteins, there is potential to overwhelm the system, which could impede viral replication and in extreme cases, kill the cell. Normally, when demands on the protein folding machinery are exceeded then misfolded proteins accumulate and activate the unfolded protein response (UPR). The UPR resolves ER stress by putting the brakes on synthesis of many proteins, while signaling to the nucleus to turn on a program that aims to correct this imbalance. Previous work has shown that KSHV is ‘wired’ to sense ER stress, which it uses to reactivate from a largely inactive state known as latency, in order to make more viruses. Specifically, a UPR sensor protein called IRE1 senses the accumulation of unfolded proteins in the ER and rededicates a gene called XBP1 to the production of a transcription factor called XBP1s through an unconventional cytoplasmic mRNA splicing event. XBP1s travels to the cell nucleus and stimulates the production of a collection of proteins that mitigate ER stress. In latently infected cells, XBP1s also binds to the KSHV genome and causes the production of K-RTA, a viral transcription factor that initiates the switch from latency to productive lytic replication. This achieves stress-induced initiation of KSHV replication, but nothing is known about how ER stress and the UPR affect progress through the KSHV replication cycle. Here we show that as KSHV replication progresses, all three known UPR sensor proteins, IRE1, ATF6 and PERK, are activated, which is required for efficient viral replication. Normally, activation of each of these three sensor proteins communicates a unique signal to the cell nucleus to stimulate the production of ER stress mitigating proteins, but in KSHV lytic replication all downstream communication is stymied. The failure to resolve ER stress would normally be expected to put the virus at a disadvantage, but we demonstrate that reversal of this scenario is worse; when we add extra XBP1s to the system to artificially stimulate the production of UPR responsive genes, virus replication is blocked at a late stage and no progeny 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.

Kaposi’s sarcoma-associated herpesvirus (KSHV) utilizes the NDP52/CALCOCO2 selective autophagy receptor to disassemble processing bodies

“…pBMN mCherry-NDP52(C443K) was a gift from Michael Lazarou (Addgene plasmid #119685; http://n2t.net/addgene:119685; RRID:Addgene_119685) and pBMN mCherry-NDP52(V136S/C443K) was a gift from Michael Lazarou (Addgene plasmid #119686; http://n2t.net/addgene: 119686; RRID:Addgene_119686). The overexpression plasmids, pLJM1 mCh-NDP52 C443K and pLJM1 mCh-NDP52 C443K V136S were made using the primers in Table 4 to clone NDP52 into pLJM1 (Johnston et al , 2019).…”

“…All shRNAs were generated by cloning shRNA hairpin sequences found in Table 2 into 4 to clone NDP52 into pLJM1 (Johnston et al, 2019).…”

“…Johnston et al, 2019) Blasticidin/ Puromycin pLJM1 KapB (pulmonary KS) Overexpression Cloned from pBMNIP-KapB(Corcoran et al, 2015) into pLJM1-BSD Blasticidin doxycycline (1 µg/mL). Cells were fixed 12 h post-induction and immunostained for Hedls (PBs, red) and KapB (blue).…”

Human coronaviruses disassemble processing bodies

“…pLenti-IRES-Puro SARS-CoV2 plasmids were a generous gift from the Krogan Lab (89). pLJM1-GFP-Dcp1a was generated by cloning pT7-EGFP-C1-HsDCP1a (Addgene 25030) into pLJM1-BSD (90)using AgeI and SmaI restriction sites (NEB). pLJM1-KapB-BSD was generated by cloning pBMNIP-KapB (13) into pLJM1-BSD using EcoRI and BamHI restriction sites (NEB).…”

Thiopurines Activate an Antiviral Unfolded Protein Response That Blocks Influenza A Virus Glycoprotein Accumulation

“…The assay was performed as described in (51). Specifically, total RNA was isolated from treated A549 cells with the RNAeasy kit (Qiagen).…”