Efficient interaction of the vesicular stomatitis virus P protein with the L protein or the N protein in cells expressing the recombinant proteins.

Specific in vivo interaction between the phosphoprotein (P) and the large polymerase protein (L) from the Indiana serotype of vesicular stomatitis virus was studied using a two-hybrid system. Transfection of CHO cells with plasmids encoding GALPIND and VPLIND fusion proteins resulted in an easily detectable level of CAT activity, indicating that PIND and LIND associate in vivo in the absence of other viral proteins. Mutational studies of PIND demonstrated that both domains I and II of PIND are important for PIND-LIND association. In addition, casein kinase II (CKII)-mediated phosphorylation within domain I of PIND was necessary for efficient association with LIND. We have also used the two-hybrid system to show PIND interaction with NIND in vivo. PIND and NIND associated more strongly than PIND and LIND. A similar strong association was observed in heterologous interaction studies between Indiana and New Jersey serotype P and N proteins. Mutational studies of PIND demonstrated that, unlike what was found for PNJ-NNJ association, only the C-terminal region of the P protein was important for efficient association with NIND. Like PNJ, CKII-mediated phosphorylation within domain I of PIND was not required for P-N association and, like NNJ, the C-terminal five amino acids of the NIND protein were critical for P association with N. These results demonstrate the importance of phosphorylation and specific domains of the P protein in its interaction with the L and N proteins, which are necessary for viral transcription and replication, respectively.