The papain-like protease of coronaviruses cleaves ULK1 to disrupt host autophagy
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Abstract
The ongoing pandemic of COVID-19 alongside the outbreaks of SARS in 2003 and MERS in 2012 underscore the significance to understand betacoronaviruses as a global health challenge. SARS-CoV-2, the etiological agent for COVID-19, has infected more than 29 million individuals worldwide with nearly ~1 million fatalities. Understanding how SARS-CoV-2 initiates viral pathogenesis is of the utmost importance for development of antiviral drugs. Autophagy modulators have emerged as potential therapeutic candidates against SARS-CoV-2 but recent clinical setbacks underline the urgent need for better understanding the mechanism of viral subversion of autophagy. Using murine hepatitis virus-A59 (MHV-A59) as a model betacoronavirus , time-course infections revealed a significant loss in the protein level of ULK1, a canonical autophagy regulating serine-threonine kinase, and the concomitant appearance of a possible cleavage fragment. To investigate whether virus-encoded proteases target this protein, we conducted in vitro and cellular cleavage assays and identified ULK1 as a novel bona fide substrate of SARS-CoV-2 papain-like protease (PL pro ). Mutagenesis studies discovered that ULK1 is cleaved at a conserved PL pro recognition sequence (LGGG) after G499, separating its N-terminal kinase domain from the C-terminal substrate recognition region. Consistent with this, over-expression of SARS-CoV-2 PL pro is sufficient to impair starvation-induced canonical autophagy and disrupt formation of ULK1-ATG13 complex. Finally, we demonstrated a dual role for ULK1 in MHV-A59 replication, serving a pro-viral functions during early replication that is inactivated at late stages of infection. In conclusion, our study identified a new mechanism by which PL pro of betacoronaviruses induces viral pathogenesis by targeting cellular autophagic pathway (Word count=250)
IMPORTANCE
The recent COVID-19 global pandemic alongside the 2003 SARS and 2012 MERS outbreaks underscore an urgent need to better understand betacoronaviruses as pathogens that pose global challenge to human health. Studying the underlying biology of how betacoronaviruses subvert innate cellular defense pathways such as autophagy will help to guide future efforts to develop anti-viral therapy. (Word count= 55)
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SciScore for 10.1101/2020.10.23.353219: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources Western blot analysis: Cells were lysed in buffer (10 mM HEPES pH 7.4, 50 mM NaPyrophosphate, 50 mM NaF, 50 mM NaCl, 5 mM EDTA, 5 mM EGTA, 100 μM Na3VO4, 0.1% Triton X-100) and western blotting was conducted using the following primary antibodies: LC3 (Novus Biologicals, NB100-2220) LC3suggested: (Novus Cat# NB100-2220SS, RRID:AB_791015)Experimental Models: Cell Lines Sentences Resources Briefly, HeLa cell were transfected with 3×Flag-ULK1 (WT or mutant) and … SciScore for 10.1101/2020.10.23.353219: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources Western blot analysis: Cells were lysed in buffer (10 mM HEPES pH 7.4, 50 mM NaPyrophosphate, 50 mM NaF, 50 mM NaCl, 5 mM EDTA, 5 mM EGTA, 100 μM Na3VO4, 0.1% Triton X-100) and western blotting was conducted using the following primary antibodies: LC3 (Novus Biologicals, NB100-2220) LC3suggested: (Novus Cat# NB100-2220SS, RRID:AB_791015)Experimental Models: Cell Lines Sentences Resources Briefly, HeLa cell were transfected with 3×Flag-ULK1 (WT or mutant) and either control vector or eGFP-PLpro for 16h. HeLasuggested: NoneResults 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 found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).
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.
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