The COVID-19 pandemic has wreaked havoc worldwide. While most infected individuals experience mild symptoms, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus may cause severe pneumonia, acute respiratory distress syndrome, and organ damage in some patients, particularly those with comorbidities. Typically, the symptoms are triggered by an extreme immune response that causes elevated levels of cytokines, referred to as the “cytokine storm.” However, the detailed mechanisms of proinflammatory cytokine production are not fully understood.
Previous studies have reported that the NF-κB family of transcription factors is an integral component of the process. This makes NF-κB signaling an important COVID-related therapeutic target. Interestingly, viral nucleocapsid (N) proteins can also interact with transforming growth factor b-activated kinase 1 (TAK1) and IκB kinase (IKK) complexes to activate the NF-κB pathway. Although there have been speculations to define these interactions, scientists are still trying to determine which viral genes they primarily involve.
To disentangle these mechanisms, a team of researchers, including Professor Takayuki Murata and Dr. Hironiri Nishitisuji from the Fujita Health University School of Medicine in Japan, has conducted a comprehensive study which was recently published in the journal mBio (available online on 20 July 2022). The team, which also contained Dr. Kunitada Shimotohno from the National Centre for Global Health and Medicine, Japan, screened 22 SARS-CoV-2 proteins to identify the ones that could activate NF-κB signaling. In context of the motivation driving the study, Prof. Murata explains, “We tried to identify the modifiers of cell signals, especially those of inflammatory signals, because inflammation is central to the symptoms of COVID-19.”
The team found that two central SARS-CoV-2 proteins—nonstructural protein 6 (NSP6) and open reading frame 7a (ORF7a)—were essential for activating the NF-κB pathway. Consistent with their hypothesis, the expression of NSP6 and ORF7a increased the levels of proinflammatory cytokines such as interleukin 8 (IL-8) and interferon gamma-induced protein 10 (IP-10). This suggests that the viral components may be activating NF-κB to cause the cytokine storm in the later stages of the infection, since infected individuals experience extreme symptoms during severe COVID-19.
Using CRISPR-Cas9 knockout studies, the team further discovered that NSP6 and ORF7a act via transforming growth factor b-activated kinase 1 (TAK1) and NF-κB essential modulator (NEMO), which are crucial players in the NF-κB pathway. Interestingly, the knockdown of TAK1/NEMO significantly reduced SARS-CoV-2-induced NF-κB activation, indicating that these proteins could be considered as potential targets for COVID-19 treatment.
The researchers then looked into the ubiquitination of NSP6 and ORF7a, which could be critical for exerting their downstream effects. Prof. Murata and his team found that tripartite motif-containing 13 (TRIM 13) and ring finger protein 121 (RNF121) were required for the ubiquitination of NSP6 and ORF7a, respectively. This process seemed to be essential for recruiting NEMO to the NSP6-TAK1 complex and activating NF-κB.
Since the suppression of cytokine production and SARS-CoV-2 replication are both halted by blocking NF-κB signaling, it is possible that the virus hijacks the pathway for its replication. “We propose that NF-κB suppression of proinflammatory cytokine responses while maintaining adequate immunity for viral clearance may be a good strategy to stop the virus,” adds Prof. Murata.
Optimistic about the significance of their research, the team anticipates that the study may add to the tool box of existing approaches and treatment ideas for COVID-19. “Our findings offer a better understanding of SARS-CoV-2 pathogenesis and host immune response to the infection”, says Prof. Murata. “Inhibitors of the molecules that mediate NF-κB activation may be used to reduce the severity of COVID-19 symptoms,” he concludes.
About Fujita Health University
Fujita Health University is a private university situated in Toyoake, Aichi, Japan. It was founded in 1964 and houses one of the largest teaching university hospitals in Japan in terms of the number of beds. With over 900 faculty members, the university is committed to providing various academic opportunities to students internationally. Fujita Health University has been ranked eighth among all universities and second among all private universities in Japan in the 2020 Times Higher Education (THE) World University Rankings. THE University Impact Rankings 2019 visualized university initiatives for sustainable development goals (SDGs). For the “good health and well-being” SDG, Fujita Health University was ranked second among all universities and number one among private universities in Japan. The university became the first Japanese university to host the "THE Asia Universities Summit" in June 2021. The university’s founding philosophy is “Our creativity for the people (DOKUSOU-ICHIRI),” which reflects the belief that, as with the university’s alumni and alumnae, current students also unlock their future by leveraging their creativity.
About Professor Takayuki Murata from Fujita Health University
Prof. Takayuki Murata is a professor at the Department of Virology and Parasitology, Fujita Health University School of Medicine, Aichi, Japan. He received his Doctorate in Medical Sciences from Nagoya University. Currently, he is also a visiting professor at Nagoya University, Japan. His major research interests include Virology, Tumor Biology, Epstein-Barr virus, Hepatitis B Virus and SARS-CoV-2. He is a member of the prestigious American Society for Microbiology and the Fujita Medical Society. In his scientific career spanning over two decades, he has more than 100 publications to his credit and has been awarded numerous research grants.
This work was supported in part by Takeda Science Foundation
Method of Research
Subject of Research
Ubiquitination of SARS-CoV-2 NSP6 and ORF7a Facilitates NF-κB Activation
Article Publication Date
The authors declare no competing interests.