1 research outputs found
Initiation of RNA Synthesis by the Hepatitis C Virus RNA-Dependent RNA Polymerase Is Affected by the Structure of the RNA Template
The
hepatitis C virus (HCV) RNA-dependent RNA polymerase NS5B is
a central enzyme of the intracellular replication of the viral (+)ĀRNA
genome. Here, we studied the individual steps of NS5B-catalyzed RNA
synthesis by a combination of biophysical methods, including real-time
1D <sup>1</sup>H NMR spectroscopy. NS5B was found to bind to a nonstructured
and a structured RNA template in different modes. Following NTP binding
and conversion to the catalysis-competent ternary complex, the polymerase
revealed an improved affinity for the template. By monitoring the
folding/unfolding of 3ā²(ā)ĀSL by <sup>1</sup>H NMR, the
base pair at the stemās edge was identified as the most stable
component of the structure. <sup>1</sup>H NMR real-time analysis of
NS5B-catalyzed RNA synthesis on 3ā²(ā)ĀSL showed that
a pronounced lag phase preceded the processive polymerization reaction.
The presence of the double-stranded stem with the edge base pair acting
as the main energy barrier impaired RNA synthesis catalyzed by NS5B.
Our observations suggest a crucial role of RNA-modulating factors
in the HCV replication process