Studies on the attenuation phenotype of polio vaccines: poliovirus RNA polymerase derived from Sabin type 1 sequence is temperature sensitive in the uridylylation 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 least 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 terminal protein VPg with 3D(pol) isolated from PV1(S). Wt and two mutant 3D(pol) proteins (Y73H, D53N/Y73H) were expressed in Escherichia coli 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 VPgpUp(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 the yeast two-hybrid assay, we detected a reduced interaction between 3D(pol) molecules carrying either the single or double mutations. Tyrosine-73 maps to the finger domain in the three-dimensional structure of 3D(pol). A model 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 with VPg uridylylation. Alternative explanations, however, cannot be excluded at the present time.
Paul
A V
AV
Department of Molecular Genetics and Microbiology, State University of New York, Stony Brook, New York 11790, USA. apaul@asterix.bio.sunysb.edu
Mugavero
J
J
Yin
J
J
Hobson
S
S
Schultz
S
S
van Boom
J H
JH
Wimmer
E
E
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D006860:Hydrogen Bonding; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D010641:Phenotype; D017955:Poliovirus / Q000201:enzymology* / Q000235:genetics; D011055:Poliovirus Vaccine, Oral / Q000737:chemistry / Q000235:genetics*; D011072:Poly U / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D014176:Protein Biosynthesis; D011487:Protein Conformation; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D012367:RNA, Viral / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D012324:RNA-Dependent RNA Polymerase; D013696:Temperature; D014158:Transcription, Genetic / Q000235:genetics; D020798:Two-Hybrid System Techniques; D014613:Vaccines, Attenuated / Q000737:chemistry / Q000235:genetics; D014758:Viral Core Proteins / Q000235:genetics / Q000378:metabolism*; D010948:Viral Plaque Assay
D011055:Poliovirus Vaccine, Oral; D012333:RNA, Messenger; D012367:RNA, Viral; D014613:Vaccines, Attenuated; D014758:Viral Core Proteins; D011072:Poly U; C090443:polymerase 3Dpol, poliovirus; D012324:RNA-Dependent RNA Polymerase; D012321:DNA-Directed RNA Polymerases