Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the coronavirus disease 2019 (COVID-19) pandemic.
SARS-CoV-2 has a single-stranded RNA genome encoding both structural and non-structural proteins. Of these, the latter is known to play a variety of roles in viral replication and the assembly of new viral particles, as well as in suppressing cellular immune responses.
A new study reports the activity of one such protein, the non-structural protein 14 (NSP14), which activates the inflammatory mediator NF-κB to trigger intense inflammation.
A preprint version of the study is available on the bioRxiv* server, while the article undergoes peer review.
SARS-CoV-2 has been known to inhibit type I interferon (IFN) pathways while it triggers NF-κB-mediated pathways, thus triggering the production of inflammatory cytokines like interleukin (IL)-6 and IL-8. This is thought to underlie the intense systemic inflammation that is responsible for the extreme multi-system damage and respiratory failure seen in some cases of severe COVID-19.
In order to understand how these inflammatory responses relate to NF-κB, the current study examined the regulatory activity of NSP14 in viral replication. This highly conserved protein has many functions, both in the replication of viral genomic and sub-genomic ribonucleic acid (RNA) and in modifying the latter, once synthesized.
Functions of NSP14
NSP14 is a proofreading exonuclease and thus limits mistakes during RNA synthesis by cutting out wrongly paired base pairs. It is also an RNA methyltransferase, transferring a methyl group to guanine at the N7 position.
This activity, combined with NSP10/16-mediated 2’-O RNA methylation, is essential for the addition of a 5’-methyl cap to the new sgRNA strand to stabilize it against degradation by the ubiquitous host exonucleases, as well as disguise it against recognition by the host cell sensors of foreign RNA, such as RIG-1.
Moreover, NSP14 enhances host ribosomal synthesis of viral proteins while reducing the amount of double-stranded RNA, which triggers the recognition of the pathogen-associated molecular pattern (PAMP) by the host cell receptors. Such recognition would otherwise activate antiviral responses.
Finally, NSP14 encourages the recombination of viral RNAs to create new virus strains.
Activity of NSP14 on NF-κB-mediated inflammation
The current study shows that NSP14 activates NF-κB-mediated inflammatory signaling pathways. Secondly, it increases the expression of IL-6 and IL-8.
In COVID-19 patients, the levels of IL-6 and IL-8 were always higher than in controls. IL-8 is more strongly induced by NSP14 than IL-6 and was found to be expressed at higher levels in lung tissue from non-survivors of COVID-19. As with other viruses, viral replication may be enhanced by IL-8 expression.
IL-8 expression is dependent on the formation of the NSP14-IMPDH2 (inosine-5'-monophosphate dehydrogenase 2) complex. IMPDH2 reduces cellular stress responses via its regulation of nucleotide synthesis within the cell.
It is thought that the interaction of NSP14 with IMPDH2 may result in increased modification of host cellular RNAs, with both the exonuclease and methyltransferase activity of the former coming into play. In addition to promoting the nuclear translocation of the p65 molecule, NSP14 could also enhance its transcription and expression.
Blockade of IMPDH2 prevents viral replication
IMPDH2 inhibition by the drugs ribavirin or mycophenolic acid resulted in a significant reduction of NF-κB activation by Nsp14, over a wide range of doses with these inhibitors.
When IMPDH2 expression was blocked, IL-8 expression was likewise prevented. Conversely, higher levels of IMPDH2 did not affect NSP14-mediated NF-κB activation, irrespective of TNF-α.
In vitro, these inhibitors also reduced the rate of infection of cells in culture by SARS-CoV-2, with both nucleocapsid protein and sgRNA levels showing a steep decline. Simultaneously, IL-8 levels also decreased.
Earlier studies have confirmed that NSP14 suppresses host antiviral defenses by blocking type I IFN signaling and preventing the exit of IRF3 from the nucleus.
What are the implications?
The current study shows that the presence of NSP14 early in infection induces multiple cell pathways and promotes viral replication.
The activation of NF-κB pathways leads to the release of other pro-inflammatory mediators to cause a cytokine storm, leading to acute respiratory distress syndrome (ARDS). Increased NF-κB expression and the resulting inflammation may benefit the virus by increasing host cell proliferation and survival or preventing cell death.
NSP14 induces increased expression of IL-6 and IL-8, both potent immune cell recruiters. The resulting influx of neutrophils and macrophages may lead to further intensification of the hyperactive inflammatory response in the lung, causing severe injury.
Thirdly, the role of the host protein IMPDH2 as a cofactor for NSP14 in the activation of NF-κB was confirmed. Not only was it found to interact with NSP14, as shown in earlier studies, but it promotes NF-κB activation.
Thus, “Nsp14 may hijack IMPDH2 for NF-κB activation, contributing to abnormal inflammatory responses.” This action may be the result of NSP14-mediated inhibition of the immunomodulatory role of IMPDH2.
In all these cases, the use of the inhibitory molecules ribavirin and mycophenolic acid, both of which are already approved and in general clinical use, may be useful in treating COVID-19. The ongoing preclinical studies and clinical trials of these drugs against this illness may benefit from this increased understanding of the mechanism by which they act to disrupt the interactions between IMPDH2 and NSP14.
bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.