title: The nucleocapsid protein of coronavirus mouse hepatitis virus interacts with the cellular heterogeneous nuclear ribonucleoprotein A1 in vitro and in vivo.
abstract: The nucleocapsid (N) protein of mouse hepatitis virus (MHV) and the cellular heterogeneous nuclear ribonucleoprotein A1 (hnRNP-A1) are RNA-binding proteins, binding to the leader RNA and the intergenic sequence of MHV negative-strand template RNAs, respectively. Previous studies have suggested a role for both N and hnRNP-A1 proteins in MHV RNA synthesis. However, it is not known whether the two proteins can interact with each other. Here we employed a series of methods to determine their interactions both in vitro and in vivo. Both N and hnRNP-A1 genes were cloned and expressed in Escherichia coli as glutathione S-transferase (GST) fusion proteins, and their interactions were determined with a GST-binding assay. Results showed that N protein directly and specifically interacted with hnRNP-A1 in vitro. To dissect the protein-binding domain on the N protein, 15 deletion constructs were made by PCR and expressed as GST fusion proteins. Two hnRNP-A1-binding sites were identified on N protein: site A is located at amino acids 1 to 292 and site B at amino acids 392 to 455. In addition, we found that N protein interacted with itself and that the self-interacting domain coincided with site A but not with site B. Using a fluorescence double-staining technique, we showed that N protein colocalized with hnRNP-A1 in the cytoplasm, particularly in the perinuclear region, of MHV-infected cells, where viral RNA replication/transcription occurs. The N protein and hnRNP-A1 were coimmunoprecipitated from the lysates of MHV-infected cells either by an N- or by an hnRNP-A1-specific monoclonal antibody, indicating a physical interaction between N and hnRNP-A1 proteins. Furthermore, using the yeast two-hybrid system, we showed that N protein interacted with hnRNP-A1 in vivo. These results thus establish that MHV N protein interacts with hnRNP-A1 both in vitro and in vivo.
authors.affiliation: Department of Microbiology and Immunology, University of Arkansas for Medical Sciences, Little Rock, Arkansas, 72205, USA.
authors.forename: Y
authors.initials: Y
authors.lastname: Wang
authors.forename: X
authors.initials: X
authors.lastname: Zhang
mesh_terms: D000818:Animals; D000086462:Coronavirus Nucleocapsid Proteins; D003593:Cytoplasm / Q000378:metabolism / Q000821:virology; D004591:Electrophoresis, Polyacrylamide Gel; D005455:Fluorescent Antibody Technique; D000076245:Heterogeneous Nuclear Ribonucleoprotein A1; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D051379:Mice; D006517:Murine hepatitis virus; D019251:Nucleocapsid / Q000378:metabolism*; D019590:Nucleocapsid Proteins; D011485:Protein Binding; D014176:Protein Biosynthesis; D012261:Ribonucleoproteins / Q000378:metabolism*; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured; D014779:Virus Replication; D015003:Yeasts
chemical_list: D000086462:Coronavirus Nucleocapsid Proteins; D000076245:Heterogeneous Nuclear Ribonucleoprotein A1; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B; D034441:Heterogeneous-Nuclear Ribonucleoproteins; C000619657:Hnrnpa1 protein, mouse; D019590:Nucleocapsid Proteins; D012261:Ribonucleoproteins; C099600:nucleocapsid protein, Hepatitis virus