How does nonstructural protein 1 affect the pathogenicity of COVID-19?

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is composed of four major structural proteins, including spikes, nucleocapsids, membranes, and enveloped proteins. To date, most studies of SARS-CoV-2 have been essential to the survival and etiology of SARS-CoV-2 infections, providing high visibility and excellent drug, and thus the effects and changes present on these proteins. Has focused on. Vaccine target.

However, some other SARS-CoV-2 proteins play a role in the survival of this virus during infection.Recent researchers for this purpose bioRxiv * The study discusses the effects of SARS-CoV-2 nonstructural protein 1 (Nsp1), which binds to the ribosome subunit and inhibits translation.

study: Analysis of the role of NSP1 in SARS-CoV-2 infection. Image Credits: Naeblys / Shutterstock.com

Survey results

Previous studies have shown that SARS-CoV-2 Nsp1 can inhibit translation and promote the degradation of ribonucleic acid (RNA), whereas residues R124 and K125 are required for translational inhibition. .. Two Nsp1 mutants were generated to further assess whether Nsp1 is also required for messenger RNA (mRNA) degradation.

The generation of Nsp1-deltaRB removed amino acids 155-165, but the development of Nsp1-CD required that both R124 and K125 be mutated to alanine. Nsp1-deltaRB failed to bind to the ribosome, while Nsp1-CD was able to bind, but was far less effective than wild-type (WT).

Interestingly, the levels of Nsp1 are lower in WT than in the two mutants, thus indicating that they inhibit themselves. Both WT and Nsp1-CD inhibit GFP expression in the host when expression of the green fluorescent protein (GFP) reporter is fused to either the host betaglobin 5’UTR or the 5’leader sequence of SARS-CoV-2. You can, but the reader sequence.

To further investigate the effect of Nsp1 on internal ribosome entry site (IRES) -mediated translation, expression of two luciferases is mediated by either cap-dependent translation or downstream encephalomyitis virus (EMCV) internal ribosome entry site. We used a bicistron mRNA (IRES) that allows it to be used. As expected, WT Nsp1 was most effective in suppressing both luciferases, Nsp1-CD showed less effect, and Nsp1-deltaRB showed no effect.

To characterize this degradation-sensitive cell transcript, we fused the CoV2-5’leader sequence upstream of the Nsp1 construct to prevent self-suppression. The mutated construct increased expression.

Next, we measured the induction of mRNA decay by Nsp1 using SLAM-seq, an approach that enables the measurement of endogenous mRNA. This assay revealed that the U- to C-mutation rate was 1.5% and then increased to 5.5%.

Cells expressing WT-Nsp1 also showed faster turnover of RNA as both time and portion of the labeled RNA increased. Cells expressing Nsp1-deltaRB or Nsp1-CD did not show a decrease in cellular mRNA half-life.

The SARS-CoV-2 mutant (CoV2mut) was generated with the same amino acid (155-165 of nsp1) removed. Next, Vero cells and Calu3 cells were infected with both WTSARS-CoV-2 and mutants.

After confirming that Nsp1 expression was unchanged, virus transmission was investigated. Both cell types may support both viruses, with similar virus titers in Vero cells, but much lower mutant titers in Calu3 cells.

This finding suggests that Nsp1 helps SARS-CoV-2 survive in immunologically active Calu3 cells, and that Nsp1 interference may affect interferon (IFN) responses. To confirm this, a JAK-STAT inhibitor was used to block IFN signaling and successfully rescue CoV2-mut proliferation in Calu3 cells.

A Syrian hamster model was used to further investigate the role of Nsp1 in the pathogenesis of SARS-CoV-2. Two groups of eight hamsters were infected with either WT-SARS-CoV-2 or CoV2-mut, weighed daily, and virus titers were quantified after 7 days.

Mice infected with CoV2-mut had less weight loss and were statistically significant on day 6. In addition to the reduced weight loss, CoV2-infected mice also showed significantly lower levels of viral lung titers below the detection limit at 7 days post-infection (dpi).

Effect of nsp1 on translation and accumulation of viral and host mRNA. aCoV2-wt or CoV2-mut (MOI = 3) -infected Calu3 cells with 4, 5, 7 hpi flow cytometric protein synthesis measurements, or O-propargyl puromycin (OPP) uptake and fluorescence using clicks Post-labeling non-infection control chemistry b bCumulative frequency of human (line) and viral (point) genes according to relative translation efficiency (TE) in cells infected with CoV2-wt (blue) or CoV2-mut (red) at 4 hpi. TE is calculated from ribosome profiling and mRNA sequences and is defined as the ratio of the ribosome footprint to the mRNA of a particular gene. Each dot represents one of the nine major viral mRNA species.

Conclusion

The authors have successfully shown that nsp1 inhibits both captive and IRES-driven translations. In addition, researchers here have identified residues that are essential to these processes and have shown that Nsp1 can easily inhibit itself and prevent its expression from increasing.

Further analysis showed that Nsp1 RNA degradation is dependent on ribosome binding. Studies in Vero and Calu3 cells have demonstrated that Nsp1 is important for virus survival, its mRNA-degrading effect remains active during infection, and this protein is lost. In vivo It alters the pathogenicity of SARS-CoV-2 infection.

In summary, the results of this study help identify that Nsp1 is an important protein for SARS-CoV-2 survival and efficacy. In the future, this information may assist researchers in developing new therapies to reduce the severity or length of the disease in patients with coronavirus disease 2019 (COVID-19).

*Important Notices

bioRxiv Publish preliminary scientific reports that should not be considered definitive as they have not been peer-reviewed, guide clinical practice / health-related behaviors, and should not be treated as established information.