Thiopurines activate an antiviral unfolded protein response that blocks viral glycoprotein accumulation in cell culture infection model

  1. Patrick Slaine
  2. Mariel Kleer
  3. Brett Duguay
  4. Eric S. Pringle
  5. Eileigh Kadijk
  6. Shan Ying
  7. Aruna D. Balgi
  8. Michel Roberge
  9. Craig McCormick
  10. Denys A. Khaperskyy

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Abstract

Enveloped viruses, including influenza A viruses (IAVs) and coronaviruses (CoVs), utilize the host cell secretory pathway to synthesize viral glycoproteins and direct them to sites of assembly. Using an image-based high-content screen, we identified two thiopurines, 6-thioguanine (6-TG) and 6-thioguanosine (6-TGo), that selectively disrupted the processing and accumulation of IAV glycoproteins hemagglutinin (HA) and neuraminidase (NA). Selective disruption of IAV glycoprotein processing and accumulation by 6-TG and 6-TGo correlated with unfolded protein response (UPR) activation and HA accumulation could be partially restored by the chemical chaperone 4-phenylbutyrate (4PBA). Chemical inhibition of the integrated stress response (ISR) restored accumulation of NA monomers in the presence of 6-TG or 6-TGo, but did not restore NA glycosylation or oligomerization. Thiopurines inhibited replication of the human coronavirus OC43 (HCoV-OC43), which also correlated with UPR/ISR activation and diminished accumulation of ORF1ab and nucleocapsid (N) mRNAs and N protein, which suggests broader disruption of coronavirus gene expression in ER-derived cytoplasmic compartments. The chemically similar thiopurine 6-mercaptopurine (6-MP) had little effect on the UPR and did not affect IAV or HCoV-OC43 replication. Consistent with reports on other CoV Spike (S) proteins, ectopic expression of SARS-CoV-2 S protein caused UPR activation. 6-TG treatment inhibited accumulation of full length S0 or furin-cleaved S2 fusion proteins, but spared the S1 ectodomain. DBeQ, which inhibits the p97 AAA-ATPase required for retrotranslocation of ubiquitinated misfolded proteins during ER-associated degradation (ERAD) restored accumulation of S0 and S2 proteins in the presence of 6-TG, suggesting that 6-TG induced UPR accelerates ERAD-mediated turnover of membrane-anchored S0 and S2 glycoproteins. Taken together, these data indicate that 6-TG and 6-TGo are effective host-targeted antivirals that trigger the UPR and disrupt accumulation of viral glycoproteins. Importantly, our data demonstrate for the first time the efficacy of these thiopurines in limiting IAV and HCoV-OC43 replication in cell culture models.

IMPORTANCE

Secreted and transmembrane proteins are synthesized in the endoplasmic reticulum (ER), where they are folded and modified prior to transport. During infection, many viruses burden the ER with the task of creating and processing viral glycoproteins that will ultimately be incorporated into viral envelopes. Some viruses refashion the ER into replication compartments where viral gene expression and genome replication take place. This viral burden on the ER can trigger the cellular unfolded protein response (UPR), which attempts to increase the protein folding and processing capacity of the ER to match the protein load. 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 in a manner that impedes viral glycoprotein accumulation for enveloped influenza viruses and coronaviruses. These drugs may impede the replication of viruses that require precise tuning of the UPR to support viral glycoprotein synthesis for the successful completion of a replication cycle.

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  1. ScreenIT

    SciScore for 10.1101/2020.09.30.319863: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    Institutional Review Board Statementnot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Mouse anti-PABP (Santa Cruz, 10E10), Rabbit anti-TIAR (Cell signaling, 8509), 1:3000 rabbit anti-GM130, 1:100 rabit anti-Calnexin, 1:1000 anti-Spike (GeneTex), 1:100 anti-StrepTag (IBA), and 1:800 anti-eIF3A (Cell signaling, 3411), followed by Alexa-coupled donkey or goat secondary antibodies (Thermo Fisher Scientific) at 1:1000 dilution.
    anti-PABP
    suggested: None
    anti-TIAR ( Cell signaling , 8509)
    suggested: (Cell Signaling Technology Cat# 8509, RRID:AB_10839263)
    anti-GM130
    suggested: (Novus Cat# NB100-2622, RRID:AB_10000741)
    anti-Calnexin
    suggested: None
    anti-Spike ( GeneTex)
    suggested: None
    anti-StrepTag ( IBA) ,
    suggested: None
    anti-eIF3A
    suggested: (Cell Signaling Technology Cat# 3411, RRID:AB_2096523)
    Experimental Models: Cell Lines
    SentencesResources
    Cell lines: Human lung adenocarcinoma A549 cells, human embryonic kidney (HEK) 293T and 293A cells, human colorectal adenocarcinoma HCT-8, green monkey kidney (Vero), baby hamster kidney (BHK-21), and Madin-Darby canine kidney (MDCK) cells were all maintained in Dulbecco’s modified Eagle’s medium (DMEM; Thermo Fisher Scientific,
    A549
    suggested: None
    HEK
    suggested: None
    293T
    suggested: None
    293A
    suggested: None
    MDCK
    suggested: CLS Cat# 602280/p823_MDCK_(NBL-2, RRID:CVCL_0422)
    HCT-8 cells were additionally supplemented with 1X MEM Non-Essential Amino Acids (Gibco)
    HCT-8
    suggested: None
    Baby hamster kidney (BHK-21) were maintained in DMEM supplemented with 5% FBS and 100 U/mL penicillin + 100 μg/mL streptomycin + 20 μg/mL-glutamine.
    BHK-21
    suggested: None
    IAV-PR8 stocks were generated using the 8-plasmid reverse genetic system, which was provided by Dr. Richard Webby (St. Jude Children’s Research Hospital, Memphis USA); IAV-Udorn was rescued from the 12-plasmid system that was provided by Dr. Yoshi Kawaoka (University of Wisconsin-Madison, Madison, USA); IAV-CA/07 was provided by the Public Health Agency of Canada (PHAC) National Microbiology Laboratory
    IAV-CA/07
    suggested: None
    To generate OC43 stocks, Vero cells were infected at a MOI of 0.05 for 1 h at 33°C in serum-free DMEM.
    Vero
    suggested: None
    HEK293T cells were grown on poly-D-lysine coated coverslips and transfected as described above.
    HEK293T
    suggested: NCBI_Iran Cat# C498, RRID:CVCL_0063)
    Software and Algorithms
    SentencesResources
    Statistical analysis: Statistical analysis was performed using PRISM graph pad 8, using a one-way ANOVA multiple comparison test (Tukey) or Two-way ANOVA using the multiple comparison test using Dunnett’s correction.
    PRISM
    suggested: None

    Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

    Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

    Results from TrialIdentifier: No clinical trial numbers were referenced.

    Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

    Results from JetFighter: We did not find any issues relating to colormaps.

    Results from rtransparent:
    • Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
    • Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
    • No protocol registration statement was detected.

    About SciScore

    SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

  2. Version published to 10.1101/2020.09.30.319863 on bioRxiv