PMID- 33762409 OWN - NLM STAT- PubMed-not-MEDLINE LR - 20230228 IS - 1098-5514 (Electronic) IS - 0022-538X (Print) IS - 0022-538X (Linking) VI - 95 IP - 11 DP - 2021 May 10 TI - Thiopurines activate an antiviral unfolded protein response that blocks influenza A virus glycoprotein accumulation. LID - JVI.00453-21 [pii] LID - 10.1128/JVI.00453-21 [doi] LID - e00453-21 AB - Influenza A viruses (IAVs) utilize host shutoff mechanisms to limit antiviral gene expression and redirect translation machinery to the synthesis of viral proteins. Previously, we showed that IAV replication is sensitive to protein synthesis inhibitors that block translation initiation and induce formation of cytoplasmic condensates of untranslated messenger ribonucleoprotein complexes called stress granules (SGs). In this study, using an image-based high-content screen, we identified two thiopurines, 6-thioguanine (6-TG) and 6-thioguanosine (6-TGo), that triggered SG formation in IAV-infected cells and blocked IAV replication in a dose-dependent manner without eliciting SG formation in uninfected cells. 6-TG and 6-TGo selectively disrupted the synthesis and maturation of IAV glycoproteins hemagglutinin (HA) and neuraminidase (NA) without affecting the levels of the viral RNAs that encode them. By contrast, these thiopurines had minimal effect on other IAV proteins or the global host protein synthesis. Disruption of IAV glycoprotein accumulation by 6-TG and 6-TGo correlated with activation of unfolded protein response (UPR) sensors activating transcription factor-6 (ATF6), inositol requiring enzyme-1 (IRE1) and PKR-like endoplasmic reticulum kinase (PERK), leading to downstream UPR gene expression. Treatment of infected cells with the chemical chaperone 4-phenylbutyric acid diminished thiopurine-induced UPR activation and partially restored the processing and accumulation of HA and NA. By contrast, chemical inhibition of the integrated stress response downstream of PERK restored accumulation of NA monomers but did not restore processing of viral glycoproteins. Genetic deletion of PERK enhanced the antiviral effect of 6-TG without causing overt cytotoxicity, suggesting that while UPR activation correlates with diminished viral glycoprotein accumulation, PERK could limit the antiviral effects of drug-induced ER stress. Taken together, these data indicate that 6-TG and 6-TGo are effective host-targeted antivirals that trigger the UPR and selectively disrupt accumulation of viral glycoproteins.IMPORTANCESecreted and transmembrane proteins are synthesized in the endoplasmic reticulum (ER), where they are folded and modified prior to transport. Many viruses rely on the ER for the synthesis and processing of viral glycoproteins that will ultimately be incorporated into viral envelopes. Viral burden on the ER can trigger the unfolded protein response (UPR). Much remains to be learned about how viruses co-opt the UPR to ensure efficient synthesis of viral glycoproteins. Here, we show that two FDA-approved thiopurine drugs, 6-TG and 6-TGo, induce the UPR, which represents a previously unrecognized effect of these drugs on cell physiology. This thiopurine-mediated UPR activation blocks influenza virus replication by impeding viral glycoprotein accumulation. Our findings suggest that 6-TG and 6-TGo may have broad antiviral effect against enveloped viruses that require precise tuning of the UPR to support viral glycoprotein synthesis. CI - Copyright (c) 2021 Slaine et al. FAU - Slaine, Patrick D AU - Slaine PD AD - Department of Microbiology & Immunology, Dalhousie University, 5850 College Street, Halifax, NS, Canada, B3H 4R2. FAU - Kleer, Mariel AU - Kleer M AD - Department of Microbiology, Immunology and Infectious Diseases, University of Calgary, 3330 Hospital Drive NW, Calgary, AB, Canada, T2N 4N1. FAU - Duguay, Brett A AU - Duguay BA AD - Department of Microbiology & Immunology, Dalhousie University, 5850 College Street, Halifax, NS, Canada, B3H 4R2. FAU - Pringle, Eric S AU - Pringle ES AD - Department of Microbiology & Immunology, Dalhousie University, 5850 College Street, Halifax, NS, Canada, B3H 4R2. FAU - Kadijk, Eileigh AU - Kadijk E AD - Department of Microbiology & Immunology, Dalhousie University, 5850 College Street, Halifax, NS, Canada, B3H 4R2. FAU - Ying, Shan AU - Ying S AD - Department of Microbiology & Immunology, Dalhousie University, 5850 College Street, Halifax, NS, Canada, B3H 4R2. FAU - Balgi, Aruna AU - Balgi A AD - Department of Biochemistry and Molecular Biology, 2350 Health Sciences Mall, University of British Columbia, Vancouver, BC, Canada, V6T 1Z3. FAU - Roberge, Michel AU - Roberge M AD - Department of Biochemistry and Molecular Biology, 2350 Health Sciences Mall, University of British Columbia, Vancouver, BC, Canada, V6T 1Z3. FAU - McCormick, Craig AU - McCormick C AUID- ORCID: 0000-0003-2794-3722 AD - Department of Microbiology & Immunology, Dalhousie University, 5850 College Street, Halifax, NS, Canada, B3H 4R2 craig.mccormick@dal.ca d.khaperskyy@dal.ca. FAU - Khaperskyy, Denys A AU - Khaperskyy DA AUID- ORCID: 0000-0003-0583-7179 AD - Department of Microbiology & Immunology, Dalhousie University, 5850 College Street, Halifax, NS, Canada, B3H 4R2 craig.mccormick@dal.ca d.khaperskyy@dal.ca. LA - eng PT - Journal Article DEP - 20210324 PL - United States TA - J Virol JT - Journal of virology JID - 0113724 SB - IM PMC - PMC8139708 EDAT- 2021/03/26 06:00 MHDA- 2021/03/26 06:01 PMCR- 2021/05/10 CRDT- 2021/03/25 05:52 PHST- 2021/03/25 05:52 [entrez] PHST- 2021/03/26 06:00 [pubmed] PHST- 2021/03/26 06:01 [medline] PHST- 2021/05/10 00:00 [pmc-release] AID - JVI.00453-21 [pii] AID - 00453-21 [pii] AID - 10.1128/JVI.00453-21 [doi] PST - ppublish SO - J Virol. 2021 May 10;95(11):e00453-21. doi: 10.1128/JVI.00453-21. Epub 2021 Mar 24.