Studies on the attenuation phenotype of polio vaccines: Poliovirus RNA polymerase derived from Sabin type 1 sequence is temperature sensitive in the uridylytation of VPg

Determinants of temperature sensitivity and/or attenuation in Sabin type 1 poliovirus reside in the 5' NTR and coding sequences of the capsid proteins and Viral RNA polymerase, 3D(pol). Previous studies have implicated at lea st two mutations in 3D(pol) of Sabin 1 vaccine strain [PV1(S)], including a Y73H change, as contributing to these phenotypes. We have used an in vitro assay to test the first step in RNA synthesis, the uridylylation of the te rminal protein VPg with 3D(pol) isolated from PV1(S). Wt and two mutant 3D( pol) proteins (Y73H, D53N/Y73H) were expressed in Escherichia coil and were purified, and their activities were measured in the synthesis of VPgpU(pU) and of VPg-linked poly(U) at 30 and 39.5 degrees C. Our results show that at 39.5 degrees C the Y73H mutation leads to a defect in the synthesis of V PgpUp(U) and of VPg-poly(U) but not in the elongation of a (dT)(15) primer. The double mutant protein had the same activities as Y73H 3D(pol). Using t he yeast two-hybrid assay, we detected a reduced interaction between 3D(pol ) molecules carrying either the single or double mutations. Tyrosine-73 map s to the finger domain in the three-dimensionat structure or 3D(pol). A mod el will be presented in which a change of Y73 to H73 may interfere with an interaction between two polymerase molecules that, in turn, may interfere w ith VPg uridylylation. Alternative explanations, however, cannot be exclude d at the present time. (C) 2000 Academic Press.