Interaction between the nucleocapsid protein and the phosphoprotein of human parainfluenza virus 3. Mapping of the interacting domains using a two-hybrid system.
A two-hybrid system was used to study interaction in vivo between the nucleocapsid protein (NP) and the phosphoprotein (P) of human parainfluenza virus type 3 (HPIV-3). Two plasmids, one containing the amino terminus of P fused to the DNA-binding domain of the yeast transactivator, GAL4, and the other containing the amino terminus of NP fused to the herpesvirus transactivator, VP16, were transfected in COS-1 cells along with a chloramphenicol acetyltransferase (CAT) reporter plasmid containing GAL4 DNA-binding sites. A specific and high-affinity interaction between NP and P was observed as measured by the activation of the CAT gene. Mapping of the domains in P (603 amino acids) involved in the association with NP revealed that NH2-terminal 40 and COOH-terminal 20 amino acids are important for such association. Interestingly, a stretch of NH2-terminal amino acids as short as 63-403 interacted with NP more than the wild type, reaching greater than 2.5-fold as measured by the CAT assay. These results suggest that a domain is present in P that negatively regulates its interaction with NP. Deletion of NH2-terminal 40 and COOH-terminal 160 amino acids of NP reduced the CAT activity by more than 95%. These results underscore the important differences between negative strand RNA viruses with respect to interactions between these two viral proteins involved in gene expression.
Zhao
H
H
Department of Biology, Case Western Reserve University, Cleveland, Ohio 44106, USA.
Banerjee
A K
AK
D000818:Animals; D002478:Cells, Cultured; D004252:DNA Mutational Analysis; D004268:DNA-Binding Proteins; D005656:Fungal Proteins / Q000235:genetics; D017405:Herpes Simplex Virus Protein Vmw65 / Q000235:genetics; D006801:Humans; D019590:Nucleocapsid Proteins; D009698:Nucleoproteins; D010224:Parainfluenza Virus 3, Human / Q000235:genetics / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism*; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D017384:Sequence Deletion; D013329:Structure-Activity Relationship; D014157:Transcription Factors; D014758:Viral Core Proteins / Q000235:genetics / Q000378:metabolism*; D014764:Viral Proteins / Q000378:metabolism*
D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C052130:GAL4 protein, S cerevisiae; D017405:Herpes Simplex Virus Protein Vmw65; D019590:Nucleocapsid Proteins; D009698:Nucleoproteins; C095185:P protein, Human parainfluenza virus 3; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D014758:Viral Core Proteins; D014764:Viral Proteins