Limits...
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.

Show MeSH

Related in: MedlinePlus

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
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4230328&req=5

fig1: 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

Mentions: HepatitisC virus (HCV), a memberof the Flaviviridae family, is a majorcausative agent for liver cirrhosis and hepatocellular carcinoma inman.1 The infection of a host cell by HCVis followed by the release of the about 9.6 kb single-stranded (+)RNAviral genome that is translated and replicated in the cytoplasm.2 The central enzyme involved in the viral RNAreplication process is the RNA-dependent RNA polymerase (RdRp) NS5B.3,4 As part of an as yet incompletely characterized viral replicationcomplex, the HCV-polymerase initiates RNA polymerization denovo and first copies the (+)RNA, generating (−)RNAmolecules.5,6 In a second step, the 3′-end of the(−)RNA serves as an initiation site for the polymerase, whichthen catalyzes the synthesis of progeny (+)RNAs. The latter step canbe easily recapitulated with the purified enzyme and accordingly servesas a suitable model in studies aimed at understanding the mechanismsthat underlie the initiation of RNA polymerization on an authentictemplate (see scheme in Figure 1A).


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)

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
© Copyright Policy
Related In: Results  -  Collection

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

fig1: 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
Mentions: HepatitisC virus (HCV), a memberof the Flaviviridae family, is a majorcausative agent for liver cirrhosis and hepatocellular carcinoma inman.1 The infection of a host cell by HCVis followed by the release of the about 9.6 kb single-stranded (+)RNAviral genome that is translated and replicated in the cytoplasm.2 The central enzyme involved in the viral RNAreplication process is the RNA-dependent RNA polymerase (RdRp) NS5B.3,4 As part of an as yet incompletely characterized viral replicationcomplex, the HCV-polymerase initiates RNA polymerization denovo and first copies the (+)RNA, generating (−)RNAmolecules.5,6 In a second step, the 3′-end of the(−)RNA serves as an initiation site for the polymerase, whichthen catalyzes the synthesis of progeny (+)RNAs. The latter step canbe easily recapitulated with the purified enzyme and accordingly servesas a suitable model in studies aimed at understanding the mechanismsthat underlie the initiation of RNA polymerization on an authentictemplate (see scheme in Figure 1A).

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