Sars-cov-2 NSP10 protein activates the p38 MAPK signaling pathway by downregulating DUSP2 to trigger cytokine storm

Abstract Background Novel Coronavirus Infections (COVID-19) is a global epidemic caused by the SARS-CoV-2 virus. Due to the high variability of the virus, despite billions of doses of vaccine having been successfully developed and administered and multiple antiviral drugs having been developed, the number of infected individuals remains high, with a significantly increased risk of exacerbation of the disease, especially in groups with underlying medical conditions. Among them, cytokine storm is considered to be one of the central mechanisms leading to severe complications and even death in infected individuals. Therefore, it is important to develop specific anti-inflammatory drugs targeting this pathological process. Methods In this study, a multilevel experimental design was used with the aim of finding the virulence factors that induce cytokine storm, clarifying their mechanisms of action and finding specific anti-inflammatory drugs. First, the virulence effects of different gene fragments of SARS-CoV-2 on A549 (lung-derived), Vero E6 (kidney-derived) and Caco-2 (colon-derived) cells were assessed by CCK-8 assay. Subsequently, the up-regulation of these gene fragments on immune cells such as HL60 and RAW264.7, as well as on key inflammatory factors (including IL-6, IL-10 and TNF-α) and non-specific markers CRP in mouse serum was detected with the help of ELISA technique, while the degree of pathological damage of mouse lung tissues was observed using H&E staining. After screening the major virulence genes based on these results, they were expressed as recombinant proteins and further intervened in RAW264.7 cells and mouse models. The inflammatory effect was verified by ELISA assay with H&E staining. In addition, transcriptome sequencing analysis was used to predict the possible pathways of action and key differential genes involved in the virulence factors, which were confirmed by Western blot experiments. Finally, based on the results of transcriptome sequencing, MAPK pathway inhibitors were used to intervene in the model mice, and the relevant indexes were detected to evaluate the therapeutic effects. Results The experimental results showed that compared with other genes of SARS-CoV-2, the NSP10 gene significantly reduced the survival of three cell lines, A549, Vero E6 and Caco-2 (P < 0.05), and significantly up-regulated the inflammatory factors IL-6, IL-10 and TNF-α secreted by two immune cells, HL60 and RAW264.7 (P < 0.05). In vivo experiments further showed that the SARS-CoV-2 NSP10 gene and its recombinant protein significantly up-regulated the serum levels of IL-6, IL-10, TNF-α, and the non-specific marker CRP in mice (P < 0.05), and at the same time induced significant inflammatory pathological features such as alveolar septal congestion, edema, and neutrophil infiltration in the lung tissues. The Western blot results showed that NSP10 decreased the expression of DUSP2 and p38 MAPK proteins and increased the expression of phospho-p38 MAPK protein in RAW264.7 cells and mouse lung tissues. Notably, all the assays were normalized when intervened with MAPK pathway inhibitors, suggesting that this pathway plays an important role in NSP10-mediated inflammatory response. Conclusion In summary, SARS-CoV-2 NSP10 protein is a key virulence factor, which can activate the p38 MAPK signaling pathway by inhibiting the expression of DUSP2, thus triggering cytokine storm. Inhibitors targeting the p38 MAPK signaling pathway showed promising therapeutic potential to alleviate the excessive inflammatory response triggered by SARS-CoV-2 infection. This finding not only deepens our understanding of the viral pathogenesis, but also provides an important theoretical basis for the development of novel anti-inflammatory therapies in the future.