Mapping cooperative activity of the hepatitis C virus RNA-dependent RNA polymerase using genotype 1a-1b chimeras.
The nonstructural protein 5B (NS5B) of hepatitis C virus (HCV) encodes an RNA-dependent RNA polymerase (RdRp) which is essential for viral replication. NS5B expression in bacteria generated 20- to 50-fold lower yield and 100-fold less product per mol of enzyme for gentoype 1a RdRp than type 1b. Further, unlike type 1b RdRp, type 1a enzyme failed to exhibit cooperative properties in the assays described herein. Differences in thermal stability may partially account for the inability to efficiently oligomerize. Superose gel filtration analyses confirm differences between these RdRp preparations, although affinity for the column rather than size may account for the differences in migration. To further address this complexity, a panel of RdRp type 1a-type 1b chimeras were evaluated and implicate a role for the thumb subdomain of genotype 1b RdRp as critical for cooperative function.
Gu
Baohua
B
Department of Virology, The Metabolic and Viral Diseases Center of Excellence for Drug Discovery, GlaxoSmithKline Pharmaceuticals, Collegeville, PA, USA.
Gutshall
Lester L
LL
Maley
Derrick
D
Pruss
Cynthia M
CM
Nguyen
Tammy T
TT
Silverman
Carol L
CL
Lin-Goerke
Juili
J
Khandekar
Sanjay
S
Liu
Changbao
C
Baker
Audrey E
AE
Casper
David J
DJ
Sarisky
Robert T
RT
D000595:Amino Acid Sequence; D002850:Chromatography, Gel; D002942:Circular Dichroism; D017931:DNA Primers / Q000235:genetics / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000378:metabolism; D005838:Genotype; D016174:Hepacivirus / Q000201:enzymology*; D006358:Hot Temperature; D007527:Isoenzymes / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D012313:RNA / Q000378:metabolism; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D017361:Viral Nonstructural Proteins / Q000378:metabolism
D017931:DNA Primers; D007527:Isoenzymes; D011993:Recombinant Fusion Proteins; D017361:Viral Nonstructural Proteins; D012313:RNA; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase