Biochemical characterization of a hepatitis C virus RNA-dependent RNA polymerase mutant lacking the C-terminal hydrophobic sequence.
The RNA-dependent RNA polymerase activity of hepatitis C virus is carried out by the NS5B protein. The full-length protein was previously purified as a non-fusion protein from insect cells infected with a recombinant baculovirus. The characterization is now described of a C-terminal hydrophobic domain deletion mutant of NS5B purified from E. coli. In addition to increased solubility, deletion of this sequence also positively affected the polymerase enzymatic activity. The efficiency of nucleotide polymerization of both the full-length and the C-terminal truncated enzymes were compared on homopolymeric template-primer couples as well as on RNA templates with heteropolymeric sequences. The largest difference in the polymerase activity was observed on the latter. On all the templates, the increased activity could be ascribed, at least in part, to enhanced template turnover of the deletion mutant with respect to the full-length enzyme. The elongation rates of the two enzyme forms were compared under single processive cycle conditions. Under these conditions, both the full-length and the deletion mutant were able to incorporate about 700 nt/min.
Tomei
L
L
Istituto di Ricerche di Biologia Molecolare 'P. Angeletti', via Pontina Km 30600, 00040-Pomezia (Roma), Italy. Tomei@IRBM.it
Vitale
R L
RL
Incitti
I
I
Serafini
S
S
Altamura
S
S
Vitelli
A
A
De Francesco
R
R
D000818:Animals; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005814:Genes, Viral; D016174:Hepacivirus / Q000201:enzymology* / Q000235:genetics*; D007700:Kinetics; D012324:RNA-Dependent RNA Polymerase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D013379:Substrate Specificity
D011994:Recombinant Proteins; D012324:RNA-Dependent RNA Polymerase