Localization of an RNA-binding domain in the nucleocapsid protein of the coronavirus mouse hepatitis virus

Abstract

The interaction between the nucleocapsid (N) protein of mouse hepatitis virus (MHV) and RNA was studied in an effort to define portions of the N molecule that participate in binding to RNA. N mRNAs transcribed from SP6 and T7 vectors were translated in a rabbit reticulocyte lysate. Analysis of synthesized N protein in a nondenaturing gel system showed that it bound in vitro to an endogenous RNA in the reticulocyte lysate but not to its own mRNA. A set of deletion mutants was constructed in order to localize the RNA-binding activity of the N protein. It was found that removal of as much as 135 amino-terminal or 57 carboxy-terminal amino acids from the molecule had little or no effect on RNA binding. Moreover, deletion mutants lacking both termini still retained RNA-binding ability. By contrast, internal deletions or truncations extending beyond these two limits effectively abolished RNA binding by N protein. Thus, the RNA-binding region of N has been mapped to the second (central) of the three structural domains of the molecule.