Analysis of the interaction sites of SARS-CoV-2

Avani Dave, Jai Hind College.

It has been more than half a century that the Coronaviruses (CoVs) have been rampantly spreading across the globe. This has led to the current pandemic caused by SARS-CoV-2, which extends a momentum to be able to characterize the largely expressed protein, the Nucleocapsid (N) protein. This N protein is one of the four structural proteins along with the envelope (E), the membrane (M), and the spike (S) proteins, but the N protein has relatively higher immunogenicity, allowing it to be a probable vaccine candidate. 

In a new study, NMR was used to start identifying the interactions of the N protein NTD and its flanking regions along with their biological targets, further providing better insight into interactions underlying the viral infections. The motion observed during the NMR is hypothesized to be altered when linked to larger RNA fragments including the full-length N protein. CypA, i.e. the host cell was seen to be engaging with the regions that lead to self-association. This hints towards a probable amplification of viral replication for several varieties of CoVs. However, the mechanism hasn’t been identified so far. The data derived from the study suggests that epitopes from the NTD region can be used as potential vaccine candidates. 

• Protein expression and purification:

For the expression of proteins, the study utilized seven, engineered N protein constructs, mainly involving the full-length N protein of several specific residues.  The terminal step in the purification of the size exclusion chromatography was executed in the same NMR buffer that was used for other N protein constructs.

• Nucleotide reagents:

The RNA fragments essential for the study were procured from Horizon Discovery (Boulder, CO) and the DNA fragments were from IDT (Coralville, IA). 

• Nuclear magnetic resonance spectroscopy:

During the backbone assignment, standard HNCACB and CBCA (co)NH spectra of N 48–178, N 1–178, and N 1–209 were gathered and later processed by utilizing the NMRPipe43 and further analyzed by CCPNmr software.

Discussion

NMR was utilized to experimentally investigate how the N protein N-terminal region of SARS-CoV-2 interacts with different regions of the N protein itself along with RNA and host cell CypA. Although prior studies established that the NTD fails to show any association with the CTD, in sync with the current SAXS data. Any self-association could be credited to the disordered flanking regions, as discovered during the study. This was warranted by the analysis of constructs that contained SARS-CoV-2 N protein NTD accompanied by the titration experiments of independently purified regions. 

A range of dynamic interactions between the N protein NTD and its corresponding flanking regions were reported during the study. For instance, relaxation rates collected on various NTD constructs highlighted μs-ms motions of much of the NTD core domain and elevated ps-ns motions within the β-hairpin of several residues, thereby showing great flexibility. In a comparison of the data with the β-hairpin in the SARS-CoV-1 N protein NTD, similar flexibility was witnessed. Further attention to the serine/arginine-rich (SR) region was under consideration as it is the “hot spot” for evolutionary changes accounting for its dynamic nature and characteristic properties. 

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Source:Jasmina S.R., Eunjeong, L., Alexandra, B, et al. The Inherent Dynamics and Interaction Sites of the SARS-CoV-2 Nucleocapsid N-Terminal Region. Journal of Molecular Biology, Volume 433, Issue 15. 2021,167108, ISSN 0022-2836. https://doi.org/10.1016/j.jmb.2021.167108.

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Author Info: Avani Dave is currently in the final year of her bachelor’s degree, majoring in Life Sciences. Holding a good academic and extra-curricular record, she is on a constant journey of acquiring exposure in her field of interest while simultaneously not limiting herself to just that. Avani likes studying Diseases and Syndromes and everything under this umbrella! That being said, she is adept at working across departments and promises to deliver.