Coronaviruses are plus sense single stranded RNA viruses that infect a variety of vertebrate hosts. The coronavirus nucleocapsid (N) protein is a multifunctional protein proven to be important in infection, transcription, and virulence. N protein contains three distinct regions, an N terminal domain (NTD), a C terminal dimerization domain (CTD), and a serine and arginine rich linker (SR linker) between the NTD and CTD. The Mouse Hepatitis Virus (MHV) NTD and SR linker (N219) of N protein have previously been demonstrated to bind nsp3a, a part of the replicase-transcriptase complex (RTC). Previously made single mutants did not significantly affect binding. Site-directed mutagenesis was used to make double mutants of nsp3a acidic residues to neutral residues to test binding to wild-type N219. The mutants nsp3a E52A/D55A, nsp3a E52A/D59A, and D88A/Y91A were chosen since the acidic residues are likely to be important in the electrostatic component of binding and the D88 and Y91 were shown to have a large perturbation on NMR studies of the interaction between nsp3a and N219. Proteins were grown in BL21 strain E. coli and structural integrity was checked against wild-type nsp3a via HSQC NMR. Binding was tested with isothermal titration calorimetry (ITC) titrating nsp3a or mutant protein into N219. Decreases in binding to double mutants could provide sites of posttranslational modification to control the interaction between N and nsp3a. This work was funded by the NIH grant AI067416.

Important residues in the interaction between mouse hepatitis virus N protein and nsp3a of the replicase transcriptase complex

246th National Meeting (2013)

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.