ORF3a mediated-incomplete autophagy facilitates SARS-CoV-2 replication
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Abstract
SARS-CoV-2 is the causative agent for the COVID-19 pandemic and there is an urgent need to understand the cellular response to SARS-CoV-2 infection. Beclin-1 is an essential scaffold autophagy protein that forms two distinct subcomplexes with modulators Atg14 and UVRAG, responsible for autophagosome formation and maturation, respectively. In the present study, we found that SARS-CoV-2 infection triggers an incomplete autophagy response, elevated autophagosome formation but impaired autophagosome maturation, and declined autophagy by genetic knockout of essential autophagic genes reduces SARS-CoV-2 replication efficiency. By screening 28 viral proteins of SARS-CoV-2, we demonstrated that expression of ORF3a alone is sufficient to induce incomplete autophagy. Mechanistically, SARS-CoV-2 ORF3a interacts with autophagy regulator UVRAG to facilitate Beclin-1-Vps34-Atg14 complex but selectively inhibit Beclin-1-Vps34-UVRAG complex. Interestingly, although SARS-CoV ORF3a shares 72.7% amino acid identity with the SARS-CoV-2 ORF3a, the former had no effect on cellular autophagy response. Thus, our findings provide the mechanistic evidence of possible takeover of host autophagy machinery by ORF3a to facilitate SARS-CoV-2 replication and raises the possibility of targeting the autophagic pathway for the treatment of COVID-19.
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SciScore for 10.1101/2020.11.12.380709: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization To quantitate LC3-positive autophagosomes per cell, cells from five random fields (>50 cells) were counted. 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 Detection of Autophagy: For the LC3 mobility shift assay, SARS-CoV-2-infected or ORF3a expressing cells were washed with cold PBS, lysed with 1% Triton X-100, and then subjected to immunoblot analysis (15% SDS-PAGE) with antibodies against LC3 (Cell Signaling, #3868) or SQSTM1/p62 (Cell Signaling, #39749). LC3suggested: NonePrimary antibodies included: mouse Flag … SciScore for 10.1101/2020.11.12.380709: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization To quantitate LC3-positive autophagosomes per cell, cells from five random fields (>50 cells) were counted. 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 Detection of Autophagy: For the LC3 mobility shift assay, SARS-CoV-2-infected or ORF3a expressing cells were washed with cold PBS, lysed with 1% Triton X-100, and then subjected to immunoblot analysis (15% SDS-PAGE) with antibodies against LC3 (Cell Signaling, #3868) or SQSTM1/p62 (Cell Signaling, #39749). LC3suggested: NonePrimary antibodies included: mouse Flag (Sigma, #F1804), rat Flag-HRP (Biolegend, #637311), mouse HA (BioLegend, #901515), rabbit SQSTM1/p62 (Cell Signaling, #39749), rabbit LC3 (Cell Signaling, #3868), rabbit Beclin-1 (Cell Signaling, #3495), rabbit UVRAG (Cell Signaling, #13115), rabbit Atg14 (Cell Signaling, #96752), rabbit Rubicon (Cell Signaling, #8465), rabbit Vps34 (Cell Signaling, #4263), rabbit Atg3 (Cell Signaling, #3415), rabbit Atg5 (Cell Signaling, #12994), mouse SARS-CoV-2 N (GeneTex, #GTX635689), and mouse GAPDH (Santa Cruz, #365062). HAsuggested: NoneSQSTM1/p62suggested: (Cell Signaling Technology Cat# 39749, RRID:AB_2799160)Beclin-1 (Cell Signaling,suggested: (Cell Signaling Technology Cat# 3495, RRID:AB_1903911)UVRAGsuggested: (Cell Signaling Technology Cat# 13115, RRID:AB_2687988)Atg14 (Cell Signaling, #96752), rabbit Rubicon (Cell Signaling, #8465)suggested: NoneVps34suggested: NoneAtg3suggested: NoneAtg5suggested: (Cell Signaling Technology Cat# 12994, RRID:AB_2630393)#GTX635689suggested: (GeneTex Cat# GTX635689, RRID:AB_2888561)GAPDHsuggested: (Santa Cruz Biotechnology Cat# sc-365062, RRID:AB_10847862)Experimental Models: Cell Lines Sentences Resources SARS-CoV-2 stocks were propagated in Vero-E6 cells and the titer of SARS-CoV-2 stocks were determined by standard plaque assay on Vero-E6 cells as described previously39. Vero-E6suggested: NoneHEK293T (ATCC, #CRL-11268), Hela (ATCC, #CCL-2), A549 (ATCC, #CCL-185), and MEF (wild-type, Atg3 KO, or Atg5 KO, gift from Dr. Qing Zhong at Shanghai Jiao Tong University School of Medicine, China) cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Gibco-BRL) containing 4 mM glutamine and 10% FBS. HEK293Tsuggested: ATCC Cat# CRL-11268, RRID:CVCL_1926)A549suggested: NoneCalu-3 cells (gift from Dr. Rong Zhang at Fudan University, China) were cultured in DMEM with 20% FBS and 4 mM glutamine. Calu-3suggested: NoneSoftware and Algorithms Sentences Resources The number of LC3 puncta per cells was determined by NIH ImageJ software in three independent experiments. ImageJsuggested: (ImageJ, RRID:SCR_003070)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.
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