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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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Characterization of the binding of the HCV-polymerase to differentRNA templates. Association constants of fluorescently labeled RNAsand the HCV-polymerase were determined as described in the Materials and Methods. (A) Binding of the HCV-polymeraseto ssRNA (white squares) and to the 3′(−)SL RNA (blackcircles) was determined as a function of the concentration of NaClat 22.5 °C. The affinity constants were analyzed according toa linear free energy relationship46,47 with NaClperturbing the binary complex formation. (B) Association constantsof the HCV-polymerase and ssRNA (white squares) and the 3′(−)SLRNA (black circles) were determined as a function of temperature at0.15 M NaCl and analyzed according to the van’t Hoff approximation.The respective thermodynamic parameters are summarized in Table 1.
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fig2: Characterization of the binding of the HCV-polymerase to differentRNA templates. Association constants of fluorescently labeled RNAsand the HCV-polymerase were determined as described in the Materials and Methods. (A) Binding of the HCV-polymeraseto ssRNA (white squares) and to the 3′(−)SL RNA (blackcircles) was determined as a function of the concentration of NaClat 22.5 °C. The affinity constants were analyzed according toa linear free energy relationship46,47 with NaClperturbing the binary complex formation. (B) Association constantsof the HCV-polymerase and ssRNA (white squares) and the 3′(−)SLRNA (black circles) were determined as a function of temperature at0.15 M NaCl and analyzed according to the van’t Hoff approximation.The respective thermodynamic parameters are summarized in Table 1.

Mentions: RNA synthesis catalyzed by the HCV-polymeraseis a two-substrate reaction (Figure 1A). The first half-reaction involves the bindingof the RNA template by the active site of the enzyme. To evaluatethe binding behavior of the HCV-polymerase with differently structuredRNAs, we applied a previously established assay28 utilizing a purified HCV-polymerase (HCV genotype 2a, subtypeJFH-1) and two fluorescently labeled RNA templates, i.e., (1) a randomlycomposed 16 nt single-stranded oligonucleotide (ssRNA) and (2) a 21nt oligonucleotide that forms a stable stem-loop structure (Figure 1B). The latter RNA, termed here as 3′(−)SL,corresponds to the immediate 3′-end of the HCV (−)RNA.It was used to mimic the initiation of the second step of the viralRNA replication process, the synthesis of progeny (+)RNA. Under physiologicalconditions, the HCV-polymerase revealed the highest affinity to therandom ssRNA template (Figure 2A and Table 1). Elevation of the ionic strength by NaCl perturbedthe polymerase–RNA association and decreased the affinity followinga linear free energy relationship (LFER). The dependence on the ionicconditions was less pronounced during binding of the HCV-polymeraseto the 3′(−)SL RNA. This suggested that the formationof the binary complex with 3′(−)SL followed a differentmode involving, for example, additional nonionic contributions thatwere not screened by salt.


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)

Characterization of the binding of the HCV-polymerase to differentRNA templates. Association constants of fluorescently labeled RNAsand the HCV-polymerase were determined as described in the Materials and Methods. (A) Binding of the HCV-polymeraseto ssRNA (white squares) and to the 3′(−)SL RNA (blackcircles) was determined as a function of the concentration of NaClat 22.5 °C. The affinity constants were analyzed according toa linear free energy relationship46,47 with NaClperturbing the binary complex formation. (B) Association constantsof the HCV-polymerase and ssRNA (white squares) and the 3′(−)SLRNA (black circles) were determined as a function of temperature at0.15 M NaCl and analyzed according to the van’t Hoff approximation.The respective thermodynamic parameters are summarized in Table 1.
© Copyright Policy
Related In: Results  -  Collection

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

fig2: Characterization of the binding of the HCV-polymerase to differentRNA templates. Association constants of fluorescently labeled RNAsand the HCV-polymerase were determined as described in the Materials and Methods. (A) Binding of the HCV-polymeraseto ssRNA (white squares) and to the 3′(−)SL RNA (blackcircles) was determined as a function of the concentration of NaClat 22.5 °C. The affinity constants were analyzed according toa linear free energy relationship46,47 with NaClperturbing the binary complex formation. (B) Association constantsof the HCV-polymerase and ssRNA (white squares) and the 3′(−)SLRNA (black circles) were determined as a function of temperature at0.15 M NaCl and analyzed according to the van’t Hoff approximation.The respective thermodynamic parameters are summarized in Table 1.
Mentions: RNA synthesis catalyzed by the HCV-polymeraseis a two-substrate reaction (Figure 1A). The first half-reaction involves the bindingof the RNA template by the active site of the enzyme. To evaluatethe binding behavior of the HCV-polymerase with differently structuredRNAs, we applied a previously established assay28 utilizing a purified HCV-polymerase (HCV genotype 2a, subtypeJFH-1) and two fluorescently labeled RNA templates, i.e., (1) a randomlycomposed 16 nt single-stranded oligonucleotide (ssRNA) and (2) a 21nt oligonucleotide that forms a stable stem-loop structure (Figure 1B). The latter RNA, termed here as 3′(−)SL,corresponds to the immediate 3′-end of the HCV (−)RNA.It was used to mimic the initiation of the second step of the viralRNA replication process, the synthesis of progeny (+)RNA. Under physiologicalconditions, the HCV-polymerase revealed the highest affinity to therandom ssRNA template (Figure 2A and Table 1). Elevation of the ionic strength by NaCl perturbedthe polymerase–RNA association and decreased the affinity followinga linear free energy relationship (LFER). The dependence on the ionicconditions was less pronounced during binding of the HCV-polymeraseto the 3′(−)SL RNA. This suggested that the formationof the binary complex with 3′(−)SL followed a differentmode involving, for example, additional nonionic contributions thatwere not screened by salt.

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