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 profilin, and characterized its interactions with actin and polyphosphoinositides. Compared with cellular profilins, this viral profilin has a low affinity (Kd > or = 35 microM) for human platelet actin monomers, a weak effect on the exchange of the nucleotide bound to the actin, and no detectable affinity for poly(L-proline). Vaccinia profilin binds to phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 4-monophosphate in micelles and large unilamellar vesicles, but not to phosphatidylserine or phosphatidylcholine. Kinetic analysis by surface plasmon resonance showed that both vaccinia and amoeba profilins bind slowly to polyphosphoinositides, with association rate constants in the range of (1-4) x 10(4) M-1 s-1. The higher affinity of vaccinia profilin for polyphosphoinositides (Kd = 0.2-8.5 microM) than for actin or poly(L-proline) and the concentration of vaccinia profilin expressed in infected HeLa cells (approximately 20 microM) suggest that vaccinia profilin binds preferentially to PIP and PIP2 in vivo. Consequently, vaccinia profilin is more likely to influence phosphoinositide metabolism than actin assembly. Expression of 7-105 microM vaccinia profilin in a Saccharomyces cerevisiae profilin null mutant did not rescue the null phenotype, so that the affinity of vaccinia profilin for phosphoinositides alone is insufficient for normal profilin function in yeast.
Machesky
L M
LM
Department of Cell Biology and Anatomy, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
Cole
N B
NB
Moss
B
B
Pollard
T D
TD
D000199:Actins / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D003001:Cloning, Molecular; D003285:Contractile Proteins; D005814:Genes, Viral; D006367:HeLa Cells; D006801:Humans; D066298:In Vitro Techniques; D008823:Micelles; D008840:Microfilament Proteins / Q000235:genetics / Q000378:metabolism*; D010716:Phosphatidylinositols / Q000378:metabolism*; D051304:Profilins; D011994:Recombinant Proteins; D013947:Thymosin / Q000378:metabolism; D014615:Vaccinia / Q000378:metabolism; D014616:Vaccinia virus / Q000378:metabolism*; D015678:Viral Structural Proteins / Q000235:genetics
D000199:Actins; D003285:Contractile Proteins; D008823:Micelles; D008840:Microfilament Proteins; D010716:Phosphatidylinositols; D051304:Profilins; D011994:Recombinant Proteins; D015678:Viral Structural Proteins; D013947:Thymosin; D000255:Adenosine Triphosphate