The Role of SARS-CoV-2 Protein NSP9 and MID1 in Cytokine Storm During COVID-19


COVID-19, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), emerged in late 2019 and swiftly escalated into a global pandemic.

As of June 2023, the virus has afflicted millions and claimed over six million lives, wreaking havoc on public health systems worldwide.

SARS-CoV-2 belongs to the Betacoronaviruses genus and shares genetic similarities with its predecessors, the severe acute respiratory syndrome coronavirus (SARS-CoV) and the Middle East respiratory syndrome coronavirus (MERS-CoV).

While initial symptoms mimic those of SARS and MERS, SARS-CoV-2 has brought about unique challenges, including the emergence of new variants that pose threats to vaccine efficacy.

The immune system, a complex network of defense mechanisms, plays a crucial role in combatting viral infections. The innate immune system is the first line of defense, activated by pattern recognition receptors (PRRs) recognizing pathogen-associated molecular patterns (PAMPs). Cytokines, signaling molecules secreted by immune cells, are instrumental in orchestrating the immune response.

A well-coordinated immune response can effectively combat the virus, but an uncontrolled response can lead to tissue damage and death, often seen in the form of a cytokine storm—a phenomenon where excessive inflammatory cytokines are produced.

The SARS-CoV-2 virus has been shown to induce cytokine storms in some infected individuals, contributing to severe inflammation and multi-organ failure. However, the exact mechanisms behind the development of these cytokine storms during SARS-CoV-2 infection remain poorly understood.

SARS-CoV-2 Genome and Proteins

The SARS-CoV-2 genome is approximately 29.7 kilobases long, encoding four structural proteins (spike (S), envelope (E), membrane (M), and nucleocapsid (N)), seven accessory proteins (ORF3a, ORF3b, ORF6, ORF7a, ORF7b, ORF8, and ORF9b), and 16 nonstructural proteins (NSP1–16). These proteins have been shown to modulate the host response and influence viral pathogenesis. Some proteins suppress the type I interferon (IFN) response, a critical part of antiviral immunity, contributing to immune evasion.

The Role of NSP9 and TBK1

In recent research, it has been discovered that the SARS-CoV-2 protein NSP9 plays a role in regulating cytokine production by targeting TANK-binding kinase 1 (TBK1). TBK1 is a key player in the innate immune response, particularly in the activation of type I IFNs. NSP9 promotes the K63-linked ubiquitination and phosphorylation of TBK1, leading to increased cytokine production.

MID1 and Its Role

MID1 (midline 1), also known as TRIM18, is an E3 ubiquitin ligase with roles in embryonic development, neurodegeneration, and cancer. It is a microtubule-associated protein involved in cell adhesion, migration, and signal transduction. While its role in various physiological processes has been studied, its involvement in viral infections has been less explored.

The Connection: NSP9, TBK1, and MID1

The study unveiled an intriguing connection between NSP9, TBK1, and MID1. Elevated NSP9 expression was observed due to decreased NSP9 degradation mediated by MID1. This phenomenon resulted in the activation of signaling pathways and increased cytokine production.


The COVID-19 pandemic, initiated by SARS-CoV-2, has persisted as a global public health crisis since its emergence in December 2019. The ongoing evolution of the virus, marked by the emergence of multiple mutants, has intensified the threat to public health. These adaptive mutations not only alter the virus’s pathogenicity but also present formidable challenges to drug and vaccine development.

Despite these mutations, certain hallmark symptoms like fever and cough remain consistent. Nevertheless, the leading cause of death among patients infected with various SARS-CoV-2 mutants continues to be the devastating cytokine storm, necessitating a deeper understanding of its pathogenesis.

Our research has provided crucial insights into the role of the SARS-CoV-2 protein NSP9 in promoting cytokine storm development. We observed that NSP9 continuously activates the type I interferon (IFN) pathway, leading to increased production of IFNs and subsequent activation of inflammatory signaling pathways such as IL-6 and TNF-α. We corroborated these findings using an rVSV-NSP9 virus system, which demonstrated a significant rise in cytokine production, tissue damage, and mortality in mice infected with NSP9-expressing virus. This suggests that the excessive production of inflammatory cytokines induced by NSP9 may lead to tissue damage and mortality in vivo.

Our investigation into how NSP9 regulates the antiviral immune response revealed its specific role in activating the type I IFN pathway. Interestingly, NSP9 did not impact the activation of other major signaling pathways, including NF-κB and MAPK pathways. The precise mechanism by which NSP9 activates the type I IFN pathway was elucidated through the identification of its interaction with TBK1. NSP9 was found to directly bind to TBK1 and promote its K63-linked ubiquitination, consequently expediting TBK1 activation and subsequent phosphorylation of IRF3.

This study sheds light on the intricate dynamics between viruses and host immune systems. We identified MID1 as an E3 ubiquitin ligase responsible for NSP9 degradation via K48-linked ubiquitination. However, virus infection disrupts the interaction between NSP9 and MID1, leading to the accumulation of NSP9 and its continued activation of the type I IFN pathway. We propose that this degradation mechanism may have evolved during host-virus co-evolution as a strategy to rapidly eliminate viruses. Conversely, the virus-induced inhibition of NSP9 degradation serves as a cunning strategy for enhanced viral replication. The cytokine storm induced by NSP9, characterized by robust cytokine production and host cell attack, can lead to cell death and tissue damage.

Our findings also hint at the critical role of Lys59 in NSP9 ubiquitination and degradation. The ubiquitination-defective mutant of NSP9 (K59R) exhibited enhanced activation of IFN-β signaling. However, it’s essential to acknowledge that our analyses were conducted using protein overexpression and a replication-competent recombinant VSV (rVSV) system, which differs from a real SARS-CoV-2 infection, highlighting a limitation of our research.

Several other studies have reported different functions of NSP9 in the context of SARS-CoV-2 infection. It has been shown that NSP9 is crucial for virus replication as it binds to RNA and interferes with protein trafficking to the cell membrane, suppressing the interferon response to viral infection. Additionally, NSP9 has been implicated in the activation of the carbamoyl-phosphate synthetase, aspartate transcarbamoylase, and dihydroorotase (CAD), influencing de novo pyrimidine synthesis, and affecting viral infection. TBK1, a critical player in the antiviral immune response, has been reported to interact with the SARS-CoV-2 membrane (M) protein, leading to K48-linked ubiquitination and degradation and thus negatively regulating the production of IFN-I.

In our study, we did not find any direct connections between TBK1 and other factors such as 7SL RNA, CAD, or the M protein. However, confocal imaging revealed NSP9’s localization in close proximity to the endoplasmic reticulum, where key immune response proteins such as STING and MAVS also localize. This suggests a novel mechanism by which NSP9 regulates the TBK1 pathway on the endoplasmic reticulum.

In summary, our research has unveiled a previously unrecognized role for NSP9 as a positive regulator of the cellular antiviral immune response by controlling TBK1 activation and pro-inflammatory cytokine production. These findings contribute to our understanding of the intricate interactions between SARS-CoV-2 and the host during infection and open new avenues for drug target development in the fight against COVID-19. However, it is essential to recognize that our study has limitations, and further research is needed to fully comprehend the complexities of this host-virus interaction and its implications for therapeutic interventions.


The COVID-19 pandemic has challenged the world’s health systems, causing immense suffering and loss of life. Understanding the mechanisms behind the cytokine storms seen in severe cases is critical for developing effective treatments and therapies. This recent study sheds light on the role of NSP9, TBK1, and MID1 in regulating cytokine production during SARS-CoV-2 infection. Further research in this area could lead to novel therapeutic approaches and a deeper understanding of COVID-19 pathogenesis, ultimately aiding in the battle against this global health crisis.


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