Researchers at the Fujita Health University School of Medicine in Japan have been investigating COVID-19 to lead to therapeutic options against the severe form of the disease.
Levels of pro-inflammatory cytokines are dramatically elevated in patients with severe COVID-19, the molecular mechanisms of which are still unclear.
In a recent paper in mBio, researchers elucidated the role of different molecular players involved in the activation of NF-κB signaling, a key mediator in the cytokine pathway.
Most people infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have mild symptoms, but for others, COVID can cause severe pneumonia, acute respiratory distress syndrome, and damage to organs Symptoms are usually triggered by an extreme immune response that causes high levels of cytokines, or a “cytokine storm.” However, the detailed mechanisms of proinflammatory cytokine production are not fully understood.
Previous studies reported that the NF-κB family of transcription factors is an integral component of the process. This makes NF-κB signaling an important therapeutic target related to COVID. Viral nucleocapsid (N) proteins can also interact with the transforming growth factor-activated kinase 1 (TAK1) and IκB kinase (IKK) complexes to activate the NF-κB pathway. Although there has been speculation about the definition of these interactions, scientists are still trying to determine which viral genes are primarily involved.
To unravel these mechanisms, Takayuki Murata and Hironiri Nishitisuji of Fujita Health University School of Medicine and Kunitada Shimotohno of the National Center for Global Health and Medicine, Japan, examined 22 SARS-CoV-2 proteins to identify those that might activate NF. -κB signaling.
Murata said, “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 core 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, 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).
The scientists said this suggests that viral components may be activating NF-κB to trigger the cytokine storm in the later stages of infection, as infected people experience extreme symptoms during severe COVID-19.
CRISPR
Using CRISPR-Cas9 knockout studies, the team further discovered that NSP6 and ORF7a act by transforming growth factor b-activated kinase 1 (TAK1) and NF-κB essential modulator (NEMO), which are players crucial in the NF-κB pathway. Knockdown of TAK1/NEMO significantly reduced NF-κB activation induced by SARS-CoV-2, indicating that these proteins could be considered as potential targets for the treatment of COVID-19.
The researchers then studied the ubiquitination of NSP6 and ORF7a, which could be critical for exerting their downstream effects. Murata and his team found tripartite motifs containing 13 (TRIM 13) and ring finger protein 121 (RNF121) for the ubiquitination of NSP6 and ORF7a, respectively. This process appeared essential to recruit NEMO to the NSP6-TAK1 complex and activate NF-κB.
Since suppression of cytokine production and replication of SARS-CoV-2 is halted by blocking NF-κB signaling, it is possible that the virus hijacked the pathway for its replication. “We propose that suppressing NF-κB from pro-inflammatory cytokine responses while maintaining adequate immunity for viral clearance may be a good strategy to stop the virus,” Murata said.
The team said the study may add to the toolbox of existing treatment approaches and ideas for COVID-19.
“Our findings provide a better understanding of the pathogenesis of SARS-CoV-2 and the host’s immune response to infection,” said Murata.
“Inhibitors of molecules that mediate NF-κB activation can be used to reduce the severity of COVID-19 symptoms.”