The nucleocapsid (N) protein is an essential structural protein of the virus and plays an important role in protecting the viral genome and regulating the process of viral replication. The phosphorylation of N protein of mouse hepatitis virus (MHV), has been proved to be associated with viral transcription, replication and assembly. We found that N protein expressed at late stage rather than early stage in neuro-2a cells infected with MHV-A59 showed a significant charge heterogeneity. Isoelectric focusing electrophoresis showed that the charge of N protein was continuously distributed. Treatment with the protein S-palmitoylation inhibitor 2-BP and the proteasome inhibitor MG-132 showed that viral replication levels were not only associated with N protein electronegativity, but also with the PKCα phosphorylation at S657 and the maintenance of some endoplasmic reticulum components involved in protein modification and degradation. These results suggest that phosphorylation is a factor associated to the heterogeneity and stability of the murine coronavirus N protein.