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 shown to assemble into a homomultimer upon phosphorylation by casein kinase II. It thus acquired transcriptional activity, including the ability 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 trimer using a His-tag dilution method. Trimer stability was assessed using a variation of this method, by measuring the rate of exchange of monomers between preformed tagged and untagged trimers at different values of pH and ionic strength. Exchange rates increased with increasing ionic strength and were similar at pH 6, 8, and 10, but the trimer was completely dissociated at pH 4. This suggests that the trimer is stabilized by electrostatic interactions, probably involving carboxylate and guanidino groups. Addition of viral L protein stabilized the P trimers, completely preventing subunit exchange under transcription conditions. The association constants (Kass) for trimerization of partially active D and A substitution mutants were also determined by His-tag dilution and found to correlate well with transcriptional activity, further confirming that the active species is the trimer. Circular dichroism spectra were identical for phosphorylated and unphosphorylated wild-type P protein and for D and A mutants known to be predominantly trimeric and monomeric, respectively.
Gao
Y
Y
Department of Physiology and Biophysics, UMDNJ-Robert Wood Johnson Medical School, Piscataway 08854, USA.
Greenfield
N J
NJ
Cleverley
D Z
DZ
Lenard
J
J
D000409:Alanine; D001224:Aspartic Acid; D047390:Casein Kinase II; D002942:Circular Dichroism; D007700:Kinetics; D010750:Phosphoproteins; D010766:Phosphorylation; D011487:Protein Conformation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D055672:Static Electricity; D014158:Transcription, Genetic; D014721:Vesicular stomatitis Indiana virus / Q000235:genetics* / Q000378:metabolism; D015678:Viral Structural Proteins / Q000235:genetics* / Q000378:metabolism
C081250:P protein, Vesicular stomatitis virus; D010750:Phosphoproteins; D015678:Viral Structural Proteins; D001224:Aspartic Acid; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases; D000409:Alanine