THE TRANSCRIPTIONAL FORM OF THE PHOSPHOPROTEIN OF VESICULAR STOMATITIS-VIRUS IS A TRIMER - STRUCTURE AND STABILITY

The phosphoprotein (P) of vesicular stomatitis virus was previously sh own to assemble into a homomultimer upon phosphorylation by casein kin ase II. It thus acquired transcriptional activity, including the abili ty to bind to the other two transcriptional components, the polymerase L and the N-RNA template. This multimer has now been found to be a tr imer using a His-tag dilution method. Trimer stability was assessed us ing a variation of this method, by measuring the rate of exchange of m onomers between preformed tagged and untagged trimers at different val ues of pH and ionic strength. Exchange rates increased with increasing ionic strength and were similar at pH 6, 8, and 10, but the trimer wa s completely dissociated at pH 4. This suggests that the trimer is sta bilized by electrostatic interactions, probably involving carboxylate and guanidino groups. Addition of viral L protein stabilized the P tri mers, completely preventing subunit exchange under transcription condi tions. The association constants (K-ass) for trimerization of partiall y active D and A substitution mutants were also determined by His-tag dilution and found to correlate well with transcriptional activity, fu rther confirming that the active species is the trimer. Circular dichr oism spectra were identical for phosphorylated and unphosphorylated wi ld-type P protein and for D and A mutants known to be predominantly tr imeric and monomeric, respectively.