Virus-Host Interactome and Proteomic Survey Reveal Potential Virulence Factors Influencing SARS-CoV-2 Pathogenesis

Abstract

Background: The ongoing coronavirus disease 2019 (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.

Methods: Genome-wide screening was used to establish intraviral and viral-host interactomes. Quantitative proteomics was used to investigate the peripheral blood mononuclear cell (PBMC) proteome signature in COVID-19.

Findings: We elucidated 286 host proteins targeted by SARS-CoV-2 and >350 host proteins that are significantly perturbed in COVID-19-derived PBMCs. This signature in severe COVID-19 PBMCs reveals a significant upregulation of cellular proteins related to neutrophil activation and blood coagulation, as well as a downregulation of proteins mediating T cell receptor signaling. From the interactome, we further identified that non-structural protein 10 interacts with NF-κB-repressing factor (NKRF) to facilitate interleukin-8 (IL-8) induction, which potentially contributes to IL-8-mediated chemotaxis of neutrophils and the overexuberant host inflammatory response observed in COVID-19 patients.

Conclusions: Our study not only presents a systematic examination of SARS-CoV-2-induced perturbation of host targets and cellular networks but it also reveals insights into the mechanisms by which SARS-CoV-2 triggers cytokine storms, representing a powerful resource in the pursuit of therapeutic interventions.

Funding: National Key Research and Development Project of China, National Natural Science Foundation of China, National Science and Technology Major Project, Program for Professor of Special Appointment (Eastern Scholar) at Shanghai Institutions of Higher Learning, Shanghai Science and Technology Commission, Shanghai Municipal Health Commission, Shanghai Municipal Key Clinical Specialty, Innovative Research Team of High-level Local Universities in Shanghai, Interdisciplinary Program of Shanghai Jiao Tong University, SII Challenge Fund for COVID-19 Research, Chinese Academy of Sciences (CAS) Large Research Infrastructure of Maintenance and Remolding Project, and Chinese Academy of Sciences Key Technology Talent Program.

Keywords: COVID-19; IL-8; NKRF; SARS-CoV-2; interactome; nsp10; proteomics.

Graphical abstract

Figure 1

SARS-CoV-2 Intraviral Protein-Protein Interaction Network (A) SARS-CoV-2 intraviral interactions based on Y2H screens and coIP experiments as described in Figure S1. Different types of viral proteins are labeled with the indicated colors. Self-associated viral proteins are circled in blue. (B) Interactions between SARS-CoV-2 structural proteins and other viral proteins. (C) Interaction network among SARS-CoV-2 non-structural proteins. See also Figures S1 and S2.

Figure 2

SARS-CoV-2-Human Protein-Protein Interaction Network Network representation of the high-confidence SARS-CoV-2-host interactome in HEK293 cells. SARS-CoV-2 bait proteins and interacting host proteins are labeled with red squares and circles, respectively. The subcellular localization of host proteins is labeled with the indicated colors. Proteins with known functions in inflammation and immune responses or ubiquitination pathway are circled in purple or red, respectively. See also Figure S3 and Table S1.

Figure 3

Proteome Profile Change in PBMCs of COVID-19 Patients Suggests a Potential Role for the nsp10-NKRF-IL-8 Axis for SARS-CoV-2 Pathogenesis (A–C) Comparative proteomic analysis of COVID-19 cases with mild symptoms (N = 13) compared to healthy controls (N = 3). (A) In the volcano plot, each circle represents a protein that was quantifiable in at least 3 biological replicates. Significantly up- or downregulated proteins (adjusted p ≤ 0.01; |log2(fold change)| ≥ 0.58) are shown in red and blue, respectively. (B) Heatmap of the differential expressed proteins (adjusted p ≤ 0.01; |log2(fold change)| ≥ 0.58) that play important roles in immune responses. (C) Gene Ontology analysis shows the enrichment of biological processes with significantly up- (red bar) or downregulated (blue bar) proteins. (D–F) Comparative proteomic analysis of COVID-19 cases with severe symptoms (N = 10) compared to mild symptoms (N = 13). (D) Each circle represents a protein that was quantifiable in at least 3 biological replicates in the volcano plot. Significantly up- or downregulated proteins (adjusted p ≤ 0.01; |log2(fold change)| ≥ 0.58) are shown in red and blue, respectively. Immune response-related differentially expressed proteins are downregulated and labeled. (E) Heatmap of the differential expressed proteins (adjusted p ≤ 0.01; |log2(fold change)| ≥ 0.58) that play important roles in neutrophil activation or TCR signaling. (F) Gene Ontology analysis shows the enrichment of biological processes with significantly up- (red bar) or downregulated (blue bar) proteins. (G and H) The levels of IL-8 (G) and IL-6 (H) in PBMCs from COVID-19 patients with mild or severe symptoms or recovered patients (viral RNA negative) were determined by fluorescence-activated cell sorting (FACS) analysis. Means ± SEMs; ***p < 0.001 and ****p < 0.0001 by 1-way ANOVA with Bonferroni’s post hoc test. (I) nsp10 interaction with endogenous NKRF in cells. HEK293T cells were transfected with FLAG-nsp10 or vector control. CoIP and immunoblot were performed at 48 h post-transfection with the indicated antibodies. (J) Stable expression of nsp10 from SARS-CoV-2 significantly promotes the mRNA level of IL-8 in the lung epithelial cell line. Means ± SEMs; n = 6; non-significant (ns) and ****p < 0.0001 by 1-way ANOVA with Bonferroni’s post hoc test. (K and L) The promotion of IL-8 mRNA level by nsp10 depends on NKRF. A549-vector or A549-NKRF stable cells were transfected with small interfering-Scramble (si-Scramble) or si-NKRF. NKRF knockdown efficiency by specific siRNA was confirmed (K) and the IL-8 mRNA level was examined (L) by qPCR. Means ± SEMs; n = 4; *p < 0.05 by Student’s t test in (K). ns and ****p < 0.0001 by 1-way ANOVA with Bonferroni’s post hoc test in (L). See also Figure S4 and Tables S2, S3, and S4.

Figure 4

Models of Nsp10-Mediated SARS-CoV-2 Pathogenesis and Potential Design or Repurposing for Specific Inhibitors