Mapping of interacting domains between the nucleocapsid protein and the phosphoprotein of vesicular stomatitis virus by using a two-hybrid system.
Specific interaction between the nucleocapsid protein (N) and the phosphoprotein (P) of vesicular stomatitis virus (VSV), an important step in the life-cycle of the virus, was studied by using a two-hybrid system. Plasmids encoding P fused with the yeast GAL4 DNA-binding domain (pGALP) and N fused with the herpes simplex virus VP16 transactivating region (pVPN) were transfected into CHO cells along with a reporter plasmid encoding chloramphenicol acetyltransferase (CAT). The ability of N and P to associate in vivo was measured by activation of the CAT gene by the VP16 transactivating region. Transfection of plasmids pGALP and pVPN resulted in a high level of CAT activity, indicating that the N and P portions of the fusion proteins associated very strongly with each other. Progressive C-terminal deletions of the P protein revealed two regions that are important for association with the N protein: the N-terminal acidic domain and the C-terminal basic domain. Phosphorylation of P protein was not required for N-P association. Various deletions and mutations of the N protein revealed the C-terminal 5 amino acids (Val-Glu-Phe-Asp-Lys), in particular the amino acids Val-Glu-Phe, to be critical for N association with P. This two-hybrid system can be used in other viral systems to study the interaction between proteins involved in transcription and replication.
Takacs
A M
AM
Department of Molecular Biology, Cleveland Clinic Foundation, OH 44195.
Das
T
T
Banerjee
A K
AK
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D002213:Capsid / Q000096:biosynthesis* / Q000302:isolation & purification; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis / Q000378:metabolism; D006224:Cricetinae; D016679:Genome, Viral; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010750:Phosphoproteins / Q000096:biosynthesis* / Q000302:isolation & purification; D010957:Plasmids; D017384:Sequence Deletion; D014162:Transfection; D014721:Vesicular stomatitis Indiana virus / Q000235:genetics* / Q000378:metabolism*; D014758:Viral Core Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D014764:Viral Proteins / Q000096:biosynthesis* / Q000302:isolation & purification
D010750:Phosphoproteins; D014758:Viral Core Proteins; D014764:Viral Proteins; D015500:Chloramphenicol O-Acetyltransferase