Coronavirus Papain-like Proteases Negatively Regulate Antiviral Innate Immune Response through Disruption of STING-Mediated Signaling

Viruses have evolved elaborate mechanisms to evade or inactivate the complex system of sensors and signaling molecules that make up the host innate immune response. Here we show that human coronavirus (HCoV) NL63 and severe acute respiratory syndrome (SARS) CoV papain-like proteases (PLP) antagonize innate immune signaling mediated by STING (stimulator of interferon genes, also known as MITA/ERIS/MYPS). STING resides in the endoplasmic reticulum and upon activation, forms dimers which assemble with MAVS, TBK-1 and IKKε, leading to IRF-3 activation and subsequent induction of interferon (IFN). We found that expression of the membrane anchored PLP domain from human HCoV-NL63 (PLP2-TM) or SARS-CoV (PLpro-TM) inhibits STING-mediated activation of IRF-3 nuclear translocation and induction of IRF-3 dependent promoters. Both catalytically active and inactive forms of CoV PLPs co-immunoprecipitated with STING, and viral replicase proteins co-localize with STING in HCoV-NL63-infected cells. Ectopic expression of catalytically active PLP2-TM blocks STING dimer formation and negatively regulates assembly of STING-MAVS-TBK1/IKKε complexes required for activation of IRF-3. STING dimerization was also substantially reduced in cells infected with SARS-CoV. Furthermore, the level of ubiquitinated forms of STING, RIG-I, TBK1 and IRF-3 are reduced in cells expressing wild type or catalytic mutants of PLP2-TM, likely contributing to disruption of signaling required for IFN induction. These results describe a new mechanism used by CoVs in which CoV PLPs negatively regulate antiviral defenses by disrupting the STING-mediated IFN induction

Effect of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker initiation on organ support-free days in patients hospitalized with COVID-19

IMPORTANCE Overactivation of the renin-angiotensin system (RAS) may contribute to poor clinical outcomes in patients with COVID-19. Objective To determine whether angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) initiation improves outcomes in patients hospitalized for COVID-19. DESIGN, SETTING, AND PARTICIPANTS In an ongoing, adaptive platform randomized clinical trial, 721 critically ill and 58 non–critically ill hospitalized adults were randomized to receive an RAS inhibitor or control between March 16, 2021, and February 25, 2022, at 69 sites in 7 countries (final follow-up on June 1, 2022). INTERVENTIONS Patients were randomized to receive open-label initiation of an ACE inhibitor (n = 257), ARB (n = 248), ARB in combination with DMX-200 (a chemokine receptor-2 inhibitor; n = 10), or no RAS inhibitor (control; n = 264) for up to 10 days. MAIN OUTCOMES AND MEASURES The primary outcome was organ support–free days, a composite of hospital survival and days alive without cardiovascular or respiratory organ support through 21 days. The primary analysis was a bayesian cumulative logistic model. Odds ratios (ORs) greater than 1 represent improved outcomes. RESULTS On February 25, 2022, enrollment was discontinued due to safety concerns. Among 679 critically ill patients with available primary outcome data, the median age was 56 years and 239 participants (35.2%) were women. Median (IQR) organ support–free days among critically ill patients was 10 (–1 to 16) in the ACE inhibitor group (n = 231), 8 (–1 to 17) in the ARB group (n = 217), and 12 (0 to 17) in the control group (n = 231) (median adjusted odds ratios of 0.77 [95% bayesian credible interval, 0.58-1.06] for improvement for ACE inhibitor and 0.76 [95% credible interval, 0.56-1.05] for ARB compared with control). The posterior probabilities that ACE inhibitors and ARBs worsened organ support–free days compared with control were 94.9% and 95.4%, respectively. Hospital survival occurred in 166 of 231 critically ill participants (71.9%) in the ACE inhibitor group, 152 of 217 (70.0%) in the ARB group, and 182 of 231 (78.8%) in the control group (posterior probabilities that ACE inhibitor and ARB worsened hospital survival compared with control were 95.3% and 98.1%, respectively). CONCLUSIONS AND RELEVANCE In this trial, among critically ill adults with COVID-19, initiation of an ACE inhibitor or ARB did not improve, and likely worsened, clinical outcomes. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT0273570

Deubiquitinating and Interferon Antagonism Activities of Coronavirus Papain-Like Proteases▿

Coronaviruses encode multifunctional proteins that are critical for viral replication and for blocking the innate immune response to viral infection. One such multifunctional domain is the coronavirus papain-like protease (PLP), which processes the viral replicase polyprotein, has deubiquitinating (DUB) activity, and antagonizes the induction of type I interferon (IFN). Here we characterized the DUB and IFN antagonism activities of the PLP domains of human coronavirus NL63 and severe acute respiratory syndrome (SARS) coronavirus to determine if DUB activity mediates interferon antagonism. We found that NL63 PLP2 deconjugated ubiquitin (Ub) and the Ub-line molecule ISG15 from cellular substrates and processed both lysine-48- and lysine-63- linked polyubiquitin chains. This PLP2 DUB activity was dependent on an intact catalytic cysteine residue. We demonstrated that in contrast to PLP2 DUB activity, PLP2-mediated interferon antagonism did not require enzymatic activity. Furthermore, addition of an inhibitor that blocks coronavirus protease/DUB activity did not abrogate interferon antagonism. These results indicated that a component of coronavirus PLP-mediated interferon antagonism was independent of protease and DUB activity. Overall, these results demonstrate the multifunctional nature of the coronavirus PLP domain as a viral protease, DUB, and IFN antagonist and suggest that these independent activities may provide multiple targets for antiviral therapies

Model depicting the mechanisms used by CoV PLPs to block STING from signaling the activation of the IFN-β induction pathway.

<p>(<b>A</b>) Activation of sensors such as RIG-I induces interaction with the signaling complex including MAVS, STING, IRF-3 and TBK-1. Activated MAVS interacts with STING, which dimerizes, leading to the activation of IKK complex, TBK1 and IKKε <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030802#pone.0030802-Zhong1" target="_blank">[8]</a>. The activation of this complex leads to the ubiquitination of RIG-I, STING, IRF-3 and TBK1 and the phosphorylatin of STING and IRF-3. Activated the transcription factor IRF-3 translocates to the nucleus inducing production of IFN. (<b>B</b>) Coronavirus papain-like protease domains (depicted here as PLP) interact with STING to block signaling by blocking assembly or stability of STING dimers and preventing the ubiquitination of signaling proteins, such as RIG-I, TBK1, and IRF-3.</p

Coronavirus NL63 PLP2-TM associates with STING and nsp3 co-localizes with STING in virus-infected cells.

<p>(<b>A</b>) HEK293T cells were cotransfected with plasmid DNAs expressing STING-Flag and either wild type or catalytic mutants of NL63-PLP2-TM-V5. Cell lysates were prepared at 28 hrs post-transfection and subjected to immunoprecipitation (IP) with anti-Flag antibody. The products of the immunoprecipitation were separated by SDS-PAGE and subjected to immunoblotting (IB). STING-Flag, PLP2-TM-V5 and the catalytic mutant expression were selectively detected from whole cell lysates (WCL) using anti-Flag and anti-V5 antibodies. (<b>B</b>) HEK293-ACE2 cells were transfected with STING-V5 for 4 hours and then infected with HCoV-NL63 for 24 hrs and evaluated for expression of and localization of replicase product nsp3 (anti-nsp3, red) and STING-V5 (anti-V5, green).</p

NL63 PLP2-TM disrupts signaling complex formation.

<p>HEK293T cells were co-transfected with STING-HA together with either Flag-tagged RIG-I (<b>A</b>), Flag-tagged MAVS (<b>B</b>) or Flag-tagged IKKε (<b>C</b>), and PLP2-TM-V5. At 28 h after transfection, cell lysates were prepared and subjected to immunoprecipitate (IP) and immunoblot (IB) with the indicated antibodies. The asterisk indicates the nonspecific band.</p

Reduction of ubiquitinated forms of RIG-I, STING, TBK1 and IRF-3 in the presence of NL63 PLP2-TM.

<p>HEK293 cells were transfected with Flag-tagged RIG-I(<b>A</b>), TBK1(<b>B</b>), myc-IRF-3(<b>C</b>), or STING-Flag (<b>D</b>) together with plasmid DNA expressing HA-tagged Ub in the presence or absence of V5-tagged PLP2-TM-V5. Cells were incubated for 24 hours after transfection and treated with 25 µM MG132 for 4 hours prior to harvesting lysates. Lysates were immunoprecipitated with the indicated antibody and the products were subjected to immunoblotting with anti-HA to evaluate ubiquitinated proteins (upper panels). The whole cell lysates (WCL) were blotted to evaluate expression of each epitope-tagged product (bottom panels).</p

Expression of coronavirus PLPs blocks STING-mediated activation of the interferon stimulated response element (ISRE).

<p>(<b>A</b>) Schematic diagram of human coronaviruses (HCoV) NL63 illustrating the processing of replicase polyproteins to generate nonstructural proteins (nsp's). The papain-like protease domains, the catalytic residues that essential for protease catalytic activity <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030802#pone.0030802-Chen1" target="_blank">[35]</a>, and the transmembrane (TM) domain within nsp3 are indicated. (<b>B</b> and <b>C</b>) HEK293T cells were transfected with the STING-HA, ISRE-luc reporter and either wild-type or catalytic mutants of HCoV-NL63 PLP2-TM or SARS-CoV PLpro-TM. Asterisks indicate statistical significance (P<0.05) in comparison with ISRE-reporter activity stimulated with STING. (<b>D</b>) Immunofluorescence microscopy of HEK-293T cells expressing STING-HA and PLP2-TM-V5. Cells were fixed at 24 hrs post-transfection and the localization of endogenous IRF-3 (anti-IRF-3, green) and the epitope-tagged products was visualized by confocal microscopy.</p

NL63 PLP2-TM interacts with STING and disrupts STING dimers.

<p>(<b>A</b>) HEK293T cells were co-transfected with plasmid DNA expressing STING-Flag, and/or PLP2-TM and/or infected with Sendai virus (SeV) as indicated above. The cell lysates were separated by SDS-PAGE and subjected to immunoblotting with antibodies as indicated on the left. (<b>B</b>) HEK293-ACE2 cells were transfected with plasmid DNA expressing STING-HA and infected with SARS-CoV as indicated and cell lysates were subjected to immunoprecipitation with anti-HA. The immunoprecipitated products were analyzed by SDS-PAGE and immunoblotted to access STING monomers and dimers. Whole cell lysates were immunoblotted to detected SARS-CoV replicase protein nsp3 and STING-HA. (<b>C</b>) Cells were co-transfected with STING-HA and STING-Flag with either wild-type or the indicated catalytic mutant of PLP2-TM and lysates were immunoprecipitated (IP) and immunoblotted (IB) to detect expression of each product.</p