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Initiation of RNA synthesis by the hepatitis C virus RNA-dependent RNA polymerase is affected by the structure of the RNA template.

Reich S, Kovermann M, Lilie H, Knick P, Geissler R, Golbik RP, Balbach J, Behrens SE - Biochemistry (2014)

Bottom Line: 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.Our observations suggest a crucial role of RNA-modulating factors in the HCV replication process.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biochemistry and Biotechnology, Section of Microbial Biotechnology, ‡Institute of Physics, Section of Biophysics, §Institute of Biochemistry and Biotechnology, Section of Technical Biochemistry, Martin Luther University Halle-Wittenberg , D-06120 Halle/Saale, Germany.

ABSTRACT
The hepatitis C virus (HCV) RNA-dependent RNA polymerase NS5B is a central enzyme of the intracellular replication of the viral (+)RNA genome. Here, we studied the individual steps of NS5B-catalyzed RNA synthesis by a combination of biophysical methods, including real-time 1D (1)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 3'(-)SL by (1)H NMR, the base pair at the stem's edge was identified as the most stable component of the structure. (1)H NMR real-time analysis of NS5B-catalyzed RNA synthesis on 3'(-)SL 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.

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Structuralanalysis and thermodynamic stability of the stem-loopformed by a 21 nt RNA oligonucleotide corresponding to the 3′-endof the HCV (−)RNA. (A) One-dimensional 1H NMR spectrumof the 3′(−)SL RNA. The RNA displays 5 prominent basepairs that form the stem. The spectral region that is sensitive fordouble-stranded RNA is shown. Each signal corresponds to the respectiveimino proton that contributes to hydrogen bonding of one canonicalbase pair, as indicated. Assignment of the individual signals wasperformed by a NOESY spectrum and by considering some RNA variants(see Figure 5). (B) The thermodynamic stabilityof the stem structure of the 3′(−)SL RNA was determinedfrom thermal unfolding transitions monitored by 1D 1H NMRspectroscopy that are sensitive to individual base pairing withinthe stem. Integrated signal intensities of the respective imino protonswere analyzed according to a two-state folding–unfolding mechanism(G3–C17, filled square; G16–C4, filled circle; G15–C5, open circle; G14–C6, open triangle;G13–C7, filled triangle). The thermodynamicparameters that derived from fitting the transition curves are summarizedin Table 3. The secondary structure of theRNA is schematically shown in the inset.
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fig4: Structuralanalysis and thermodynamic stability of the stem-loopformed by a 21 nt RNA oligonucleotide corresponding to the 3′-endof the HCV (−)RNA. (A) One-dimensional 1H NMR spectrumof the 3′(−)SL RNA. The RNA displays 5 prominent basepairs that form the stem. The spectral region that is sensitive fordouble-stranded RNA is shown. Each signal corresponds to the respectiveimino proton that contributes to hydrogen bonding of one canonicalbase pair, as indicated. Assignment of the individual signals wasperformed by a NOESY spectrum and by considering some RNA variants(see Figure 5). (B) The thermodynamic stabilityof the stem structure of the 3′(−)SL RNA was determinedfrom thermal unfolding transitions monitored by 1D 1H NMRspectroscopy that are sensitive to individual base pairing withinthe stem. Integrated signal intensities of the respective imino protonswere analyzed according to a two-state folding–unfolding mechanism(G3–C17, filled square; G16–C4, filled circle; G15–C5, open circle; G14–C6, open triangle;G13–C7, filled triangle). The thermodynamicparameters that derived from fitting the transition curves are summarizedin Table 3. The secondary structure of theRNA is schematically shown in the inset.

Mentions: RNA synthesis catalyzed by the HCV-polymeraseon the structured3′(−)SL RNA template. (A) The HCV-polymerase NS5B isschematically depicted with thumb (T), palm (P), and finger (F) domains.The 3′(−)SL RNA template forms a stem-loop structure;the inset provides the secondary structures that were determined byNMR (see also Figures 4 and 5 and Table 3). For the synthesis ofprogeny (+)RNA molecules, the HCV-polymerase binds in a first half-reaction(binary complex formation). The interaction with NTPs (as indicated)results in the formation of the catalysis-competent ternary complex.Our data suggest that RNA secondary structures of the template haveto be resolved to accomplish double-stranded RNA product formationand release (second half-reaction). (B) Schematic presentation ofthe primary structure of the 3′(−)SL RNA template. Twopossible structures of the stem-loop were derived by the program mfold.45


Initiation of RNA synthesis by the hepatitis C virus RNA-dependent RNA polymerase is affected by the structure of the RNA template.

Reich S, Kovermann M, Lilie H, Knick P, Geissler R, Golbik RP, Balbach J, Behrens SE - Biochemistry (2014)

Structuralanalysis and thermodynamic stability of the stem-loopformed by a 21 nt RNA oligonucleotide corresponding to the 3′-endof the HCV (−)RNA. (A) One-dimensional 1H NMR spectrumof the 3′(−)SL RNA. The RNA displays 5 prominent basepairs that form the stem. The spectral region that is sensitive fordouble-stranded RNA is shown. Each signal corresponds to the respectiveimino proton that contributes to hydrogen bonding of one canonicalbase pair, as indicated. Assignment of the individual signals wasperformed by a NOESY spectrum and by considering some RNA variants(see Figure 5). (B) The thermodynamic stabilityof the stem structure of the 3′(−)SL RNA was determinedfrom thermal unfolding transitions monitored by 1D 1H NMRspectroscopy that are sensitive to individual base pairing withinthe stem. Integrated signal intensities of the respective imino protonswere analyzed according to a two-state folding–unfolding mechanism(G3–C17, filled square; G16–C4, filled circle; G15–C5, open circle; G14–C6, open triangle;G13–C7, filled triangle). The thermodynamicparameters that derived from fitting the transition curves are summarizedin Table 3. The secondary structure of theRNA is schematically shown in the inset.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4230328&req=5

fig4: Structuralanalysis and thermodynamic stability of the stem-loopformed by a 21 nt RNA oligonucleotide corresponding to the 3′-endof the HCV (−)RNA. (A) One-dimensional 1H NMR spectrumof the 3′(−)SL RNA. The RNA displays 5 prominent basepairs that form the stem. The spectral region that is sensitive fordouble-stranded RNA is shown. Each signal corresponds to the respectiveimino proton that contributes to hydrogen bonding of one canonicalbase pair, as indicated. Assignment of the individual signals wasperformed by a NOESY spectrum and by considering some RNA variants(see Figure 5). (B) The thermodynamic stabilityof the stem structure of the 3′(−)SL RNA was determinedfrom thermal unfolding transitions monitored by 1D 1H NMRspectroscopy that are sensitive to individual base pairing withinthe stem. Integrated signal intensities of the respective imino protonswere analyzed according to a two-state folding–unfolding mechanism(G3–C17, filled square; G16–C4, filled circle; G15–C5, open circle; G14–C6, open triangle;G13–C7, filled triangle). The thermodynamicparameters that derived from fitting the transition curves are summarizedin Table 3. The secondary structure of theRNA is schematically shown in the inset.
Mentions: RNA synthesis catalyzed by the HCV-polymeraseon the structured3′(−)SL RNA template. (A) The HCV-polymerase NS5B isschematically depicted with thumb (T), palm (P), and finger (F) domains.The 3′(−)SL RNA template forms a stem-loop structure;the inset provides the secondary structures that were determined byNMR (see also Figures 4 and 5 and Table 3). For the synthesis ofprogeny (+)RNA molecules, the HCV-polymerase binds in a first half-reaction(binary complex formation). The interaction with NTPs (as indicated)results in the formation of the catalysis-competent ternary complex.Our data suggest that RNA secondary structures of the template haveto be resolved to accomplish double-stranded RNA product formationand release (second half-reaction). (B) Schematic presentation ofthe primary structure of the 3′(−)SL RNA template. Twopossible structures of the stem-loop were derived by the program mfold.45

Bottom Line: 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.Our observations suggest a crucial role of RNA-modulating factors in the HCV replication process.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biochemistry and Biotechnology, Section of Microbial Biotechnology, ‡Institute of Physics, Section of Biophysics, §Institute of Biochemistry and Biotechnology, Section of Technical Biochemistry, Martin Luther University Halle-Wittenberg , D-06120 Halle/Saale, Germany.

ABSTRACT
The hepatitis C virus (HCV) RNA-dependent RNA polymerase NS5B is a central enzyme of the intracellular replication of the viral (+)RNA genome. Here, we studied the individual steps of NS5B-catalyzed RNA synthesis by a combination of biophysical methods, including real-time 1D (1)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 3'(-)SL by (1)H NMR, the base pair at the stem's edge was identified as the most stable component of the structure. (1)H NMR real-time analysis of NS5B-catalyzed RNA synthesis on 3'(-)SL 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.

Show MeSH
Related in: MedlinePlus