Researchers show that methylation of arginine in the virus nucleocapsid protein controls the protein's functions and plays a role in the life cycle of the SARS-CoV-2 virus.
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has four structural proteins: the spike, nucleocapsid, envelope, membrane, and nine accessory proteins. Among these, the nucleocapsid or N protein is found at the highest levels in infected cells, making it a potential target for developing strategies to combat the COVID-19 pandemic.
The N protein is involved in packaging the viral genome into helical ribonucleoprotein (RNP) complexes and also plays a role in RNA synthesis. The N protein has five RGG/RG motifs, and arginine residues present on these motifs are methylated by protein arginine methyltransferases (PRMTs). Arginine
methylation controls protein-protein interactions and protein-nucleic acid interactions, which affect fundamental cellular processes like transcription and RNA processing.
Arginine methylation methylates viral and host proteins needed for viral replication. Inhibition of a PRMT is found to prevent host RGG/RG methylation and inhibits HIV-1 internal ribosome entry site (IRES) functions.
In a study published on the bioRxiv* preprint server, researchers from McGill University report the PRMT1 methylates the SARS-CoV-2 N protein and regulates RNA binding activity.
Testing the effect of arginine methylation
The SARS-CoV-2 virus has two RGG and three RG motifs, the preferred sites of PRMT1, PRMT5, and PRMT6. Using in vitro arginine methylation assays, the authors found that the N-terminal fragment and the central region of the expressed virus N-protein fragment were methylated by PRMT1, but not by PRMT5 or PRMT6. Testing methylation in HEK293 cells, the team found methylation was significantly reduced when a PRMT inhibitor, MS023, was used. Furthermore, the substitution of the amino acid residue at R95 and R177 also inhibited interaction with the SARS-CoV-2 genomic DNA, suggesting arginine methylation of the N protein is required for its ability to bind RNA.
In addition, they found increased sensitivity to PRMT1 in patients infected with SARS-CoV-2, suggesting it may play a role in the life cycle of the virus.
Using HEK293 cells, the team identified 119 cellular proteins that interacted with the SARS-CoV-2 N protein. Biological process analysis revealed RNA metabolic pathways were the top 10 pathways enriched.
The team also found suppression of stress granules formed upon virus infection and have antiviral functions, when the N protein is methylated at residue R95. Increasing the expression of a methylarginine reader, TDRD3, could affect how the N protein modulates stress granules.
Since R95 and the residue R177 is present within the RNA binding site of the N-terminal domain of the N protein, with R177 playing a role in N protein RNA binding, arginine and its methylation was likely involved in RNA binding. The experiments performed by the authors confirmed the arginine methylation of R95 and R 177 is necessary for the N protein RNA binding in cells.
Furthermore, VeroE6 cells infected with SARS-CoV-2 showed a significant reduction of virus amounts in the presence of the PRMT1 inhibitor MS023.
Potential new approach to combating COVID-19
Thus, the results show that arginine methylation is a mode of interfering with the formation of stress granules in response to viral infections by the SARS-CoV-2 N protein. In addition, PRMT1 plays a key role in the SARS-CoV-2 life cycle. Studies have shown increased expression of PRMT1 in nose, throat, and bronchial samples of COVID-19 patients. Type I PRMTs are in clinical trials for cancer treatment and might be a potential approach for COVID-19 too.
Antibodies against the SARS-CoV-2 N protein are usually the first to appear after infection. The high immunogenicity of this protein is likely because the protein is fully methylated in infected cells.
One of the strategies SARS-CoV-2 uses to ensure its replication in host cells is destabilizing stress granules using the N protein. The residue R95 was responsible for altering stress granules. Thus, targeting the functioning of the N protein could be a new approach to combating COVID-19.
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.