Analysis of the RNA Binding Capabilities and Structural Predictions of the C-Terminal Domain of the Nucleocapsid Protein of Mouse Hepatitis Virus

Coronaviruses such as SARS-CoV-2, the pathogen responsible for the recent COVID-19 pandemic, pose a serious threat to human health. Due to its genetic similarities, Mouse Hepatitis Virus (MHV) serves as a good model organism to study these dangerous coronaviruses. The Nucleocapsid (N) protein of MHV is responsible for specifically packaging the viral genome in the presence of the RNA genomic packaging element (PS). The C-terminal domain (CTD) is thought to be involved in this process; however, it is unknown which section(s) of the protein are responsible for RNA binding specificity. Thus, this research sought to identify the protein domains involved in specificity of binding and conduct preliminary structural predictions on the MHV-N-CTD structure. Proteins containing varying domains of MHV-N were prepared and complexed to varying lengths of the previously identified PS element. These complexes were run through native gels and specifically bound groups were run in triplicate and quantified. A prediction of the complex structures was then generated using AlphaFold3. Using protein that had been denatured, purified, then refolded, there was no discernible difference between proteins complexed with the unique tip of the PS element (PS25). However, when complexed to the unique tip and one repeating unit of the PS element (PS45), MHV-N-CTD formed a unique complex (Complex 1) at lower concentrations in comparison to the same RNA element with 7 silent mutations (silPS45) and other protein domains (MHV-N-CTD-BN3 and MHV-N-CTD-BN3-Mt) which formed a higher running complex (Complex 2). This indicates that, although non-specific binding can occur at higher concentrations of protein, there is specificity of binding between the CTD and PS45. Further experimentation should focus on developing the experimental structure of CTD, either through X-ray crystallography or other structural determination techniques.