Virus-host interactome and proteomic survey of PMBCs from COVID-19 patients reveal potential virulence factors influencing SARS-CoV-2 pathogenesis
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Abstract
The ongoing coronavirus disease (COVID-19) pandemic caused by Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) is a global public health concern due to relatively easy person-to-person transmission and the current lack of effective antiviral therapy. However, the exact molecular mechanisms of SARS-CoV-2 pathogenesis remain largely unknown. We exploited an integrated proteomics approach to systematically investigate intra-viral and virus-host interactomes for the identification of unrealized SARS-CoV-2 host targets and participation of cellular proteins in the response to viral infection using peripheral blood mononuclear cells (PBMCs) isolated from COVID-19 patients. Using this approach, we elucidated 251 host proteins targeted by SARS-CoV-2 and more than 200 host proteins that are significantly perturbed in COVID-19 derived PBMCs. From the interactome, we further identified that non-structural protein nsp9 and nsp10 interact with NKRF, a NF-КB repressor, and may precipitate the strong IL-8/IL-6 mediated chemotaxis of neutrophils and overexuberant host inflammatory response observed in COVID-19 patients. Our integrative study not only presents a systematic examination of SARS-CoV-2-induced perturbation of host targets and cellular networks to reflect disease etiology, but also reveals insights into the mechanisms by which SARS-CoV-2 triggers cytokine storms and represents a powerful resource in the quest for therapeutic intervention.
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SciScore for 10.1101/2020.03.31.019216: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement IRB: Ethics statement: This study was approved by the Ethics Committee of Shanghai Public Health Clinical Center (#YJ-2020-S052-02). 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 Cell lines, plasmids, and antibodies: HEK293 and A549 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Gibco-BRL) containing 4 mM glutamine and 10% FBS. antibodiessuggested: NoneHEK293suggested: NonePrimary antibodies were purchased from the following venters: mouse anti-Flag Purification of ZIKV viral protein complexes: … SciScore for 10.1101/2020.03.31.019216: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement IRB: Ethics statement: This study was approved by the Ethics Committee of Shanghai Public Health Clinical Center (#YJ-2020-S052-02). 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 Cell lines, plasmids, and antibodies: HEK293 and A549 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Gibco-BRL) containing 4 mM glutamine and 10% FBS. antibodiessuggested: NoneHEK293suggested: NonePrimary antibodies were purchased from the following venters: mouse anti-Flag Purification of ZIKV viral protein complexes: HEK293 were transfected with 3xFlag-tagged plasmids encoding each SARS-CoV-2 protein by Lipofectamine 3000 (Thermo Fisher Scientific, #L3000015). anti-Flagsuggested: NoneExperimental Models: Cell Lines Sentences Resources Cell lines, plasmids, and antibodies: HEK293 and A549 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Gibco-BRL) containing 4 mM glutamine and 10% FBS. A549suggested: NoneVero cells were cultured in DMEM with 5% FBS and 4 mM glutamine. Verosuggested: CLS Cat# 605372/p622_VERO, RRID:CVCL_0059)3×107 HEK293 cells were harvested in 10 ml lysis buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 0.5% Nonidet P40, 10% glycerol, phosphatase inhibitors and protease inhibitors) at 72 h post transfection, followed by centrifugation and filtration (0.45 μm) to remove debris. HEK293suggested: NoneSoftware and Algorithms Sentences Resources The acquired MS/MS data were analyzed against a UniProtKB Human database (database released on Sept. 30, 2018) containing SARS-CoV-2 viral proteins using Maxquant V1.6.10.43. UniProtKBsuggested: (UniProtKB, RRID:SCR_004426)Primer sequences for qPCR were as follow: IL-6 forward: AACCTGAACCTTCCAAAGATGG, IL-6 reverse: TCTGGCTTGTTCCTCACTACT; IL-8 forward: TCTTGCACAAATATTTGATGC, IL-8 reverse: CCACTGTGCCTTGGTTTC; GAPGH forward: GAGTCAACGGATTTGGTCGT, GAPGH reverse: TTGATTTTGGAGGGATCTCG; TNFα forward: CTCCAGGCGGTGCTTGTTC, TNFα reverse: GGCTACAGGCTTGTCACTCG; NKRF forward: GTAAACATGCAGCTGCCGAC, NKRF reverse: CGTGCACACGGGATTTGAAG. siRNA gene silencing: Small interfering RNA (siRNA) targeting human NKRF (#A10001) and negative control siRNAs were purchased from GenePharma. GenePharmasuggested: 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 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:- No conflict of interest statement was detected. If there are no conflicts, we encourage authors to explicit state so.
- 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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