SARS-CoV-2 membrane (M) protein activates TLR4 to induce an inflammatory response

An excessive inflammatory response, also known as "cytokine storm," is a major cause of severe COVID-19. Viral RNA and its structural proteins are key mediators of SARS-CoV-2-induced inflammation. While the immune sensors and host inflammatory responses induced by SARS-CoV-2 RNA and the structural proteins spike (S), envelope (E), and nucleocapsid (N) are well defined, the role of membrane (M) protein in inflammatory/antiviral response and the signaling pathway facilitating M protein-mediated immune response is not well characterized. Here, we show that SARS-CoV-2 M protein induces robust inflammatory cytokine responses in murine and human macrophages and lungs. We also show that the M protein-induced inflammatory response relies on TLR4 signaling and that MyD88 serves as the key adaptor driving inflammation, while TRIF is primarily required for the antiviral IFN response. Detailed evaluation of downstream signaling pathways highlighted the critical roles of mitogen-activated protein kinase (MAPK) molecules JNK and ERK1/2, followed by nuclear factor kappa B (NF-κB) and p38 signaling in the M protein-TLR4-mediated inflammatory response. Finally, we show that abrogation of M protein-induced ERK1/2 and p38 signaling reduced inflammatory cytokine production and rescued type I interferon production in wild-type macrophages, while inhibition of JNK and NF-κB suppressed the M protein-induced inflammatory and antiviral cytokine responses. Collectively, our results demonstrate that SARS-CoV-2 M protein induces pro-inflammatory cytokines and type I interferons via TLR4-MyD88/TRIF activation, with MAPK activation playing a dominant role, and offer specific targeting of MAPK signaling to mitigate SARS-CoV-2-induced excessive inflammation and pathology. IMPORTANCE: Severe COVID-19 is often caused by an excessive inflammatory response, but the role of the SARS-CoV-2 membrane (M) protein in this process has not been well understood. We show that the M protein strongly activates inflammatory and antiviral immune responses through TLR4 signaling and identify the key signaling pathways involved. We also demonstrate that selectively targeting MAPK signaling can reduce harmful inflammation while restoring antiviral interferon responses. These findings improve our understanding of how SARS-CoV-2 causes disease and identify potential therapeutic targets to limit excessive inflammation without compromising antiviral immunity.