Identification of protein regions involved in the interaction of human respiratory syncytial virus phosphoprotein and nucleoprotein: significance for nucleocapsid assembly and formation of cytoplasmic inclusions.
We have reported previously that the nucleoprotein (N), the phosphoprotein (P), and the 22-kDa protein of human respiratory syncytial virus (HRSV) are components of the cytoplasmic inclusion bodies observed in HEp-2-infected cells. In addition, coexpression of N and P was sufficient to induce the formation of N-P complexes detectable by either coimmunoprecipitation with anti-P antibodies or generation of cytoplasmic inclusions. We now report the identification of protein regions required for these interactions. Deletion mutant analysis of the P protein gene indicated that its C-terminal end was essential for interacting with N. This conclusion was strengthened by the finding that an anti-P monoclonal antibody (021/12P), reacting with a 21-residue P protein C-terminal peptide, apparently displaced N from N-P complexes. The same effect was observed with high concentrations of the C-terminal peptide. However, sequence requirements for the P protein C-terminal end were not absolute, and mutants with the substitution Ser-237-->Ala or Ser-237-->Thr were as efficient as the wild type in interacting with N. In addition, P and N proteins from strains of different HRSV antigenic groups, with sequence differences in the P protein C-terminal end, were able to coimmunoprecipitate and formed cytoplasmic inclusions. Deletion mutant analysis of the N gene indicated that large segments of this polypeptide were required for interacting with P. The relevance of these interactions for HRSV is discussed in comparison with those of analogous proteins from related viruses.
García-Barreno
B
B
Instituto de Salud Carlos III, Centro Nacional de Biología Celular y Retrovirus, Madrid, Spain.
Delgado
T
T
Melero
J A
JA
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002213:Capsid / Q000737:chemistry / Q000378:metabolism*; D017931:DNA Primers; D015707:HN Protein; D006801:Humans; D007181:Inclusion Bodies, Viral / Q000378:metabolism*; D008969:Molecular Sequence Data; D009698:Nucleoproteins / Q000737:chemistry / Q000378:metabolism*; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D018113:Respiratory Syncytial Virus, Human / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D014758:Viral Core Proteins / Q000737:chemistry / Q000378:metabolism*; D014759:Viral Envelope Proteins; D014764:Viral Proteins / Q000737:chemistry / Q000378:metabolism*; D019065:Virus Assembly / Q000502:physiology
D017931:DNA Primers; D015707:HN Protein; D009698:Nucleoproteins; D010750:Phosphoproteins; D014758:Viral Core Proteins; D014759:Viral Envelope Proteins; D014764:Viral Proteins; C116204:attachment protein G