VACCINIA VIRUS EXPRESSES A NOVEL PROFILIN WITH A HIGHER AFFINITY FOR POLYPHOSPHOINOSITIDES THAN ACTIN

We expressed in Escherichia coli the vaccinia virus gene for a protein similar to vertebrate profilins, purified the recombinant viral profi lin, and characterized its interactions with actin and polyphosphoinos itides. Compared with cellular profilins, this viral profilin has a lo w affinity (K-d greater than or equal to 35 mu M) for human platelet a ctin monomers, a weak effect on the exchange of the nucleotide bound t o the actin, and no detectable affinity for poly(L-proline). Vaccinia profilin binds to phosphatidylinositol 4,5-bisphosphate and phosphatid ylinositol 4-monophosphate in micelles and large unilamellar vesicles, but not to phosphatidylserine or phosphatidylcholine. Kinetic analysi s by surface plasmon resonance showed that both vaccinia and amoeba pr ofilins bind slowly to polyphosphoinositides, with association rate co nstants in the range of (1-4) x 10(4) M(-1) s(-1). The higher affinity of vaccinia profilin for polyphosphoinositides (K-d = 0.2-8.5 mu M) t han for actin or poly(L-proline) and the concentration of vaccinia pro filin expressed in infected HeLa cells (similar to 20 mu M) suggest th at vaccinia profilin binds preferentially to PIP and PIP2 in vivo. Con sequently, vaccinia profilin is more likely to influence phosphoinosit ide metabolism than actin assembly. Expression of 7-105 mu M vaccinia profilin in a Saccharomyces cerevisiae profilin null mutant did not re scue the null phenotype, so that the affinity of vaccinia profilin for phosphoinositides alone is insufficient for normal profilin function in yeast.