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-dependent RNA polymerization monitored by real-time 1D 1H NMR spectroscopy. Product formation (double-stranded RNA)by the HCV-polymerase was monitored with the native 3′(−)SLRNA template following the addition of NTPs. (A) Proton spectra sensingdouble-stranded RNA were recorded at different time points of thepolymerization reaction: 0–8 h, magenta; 8–16 h, red;16–24 h, orange; 25–46 h, yellow; 46–67 h, green;and 67–88 h, blue after the addition of NTP to the polymerase–RNAcomplex (binary complex) (black). Upon binding of NTPs to the binarycomplex, the intensity of the RNA-related 1H NMR signalsdecreased. During an initial period of ca. 24 h, a constant signalpattern of the RNA was observed. After about 25 h, newly developedimino proton NMR signals were detectable that corresponded to thereleased double-stranded RNA product. (B) The enzymatic progress curveillustrates the HCV-polymerase interacting with the partially double-stranded3′(−)SL RNA as well as the subsequent product formationand release process of the double-stranded RNA. The initial decreaseof the integrated imino proton NMR signals upon addition of NTPs indicatesan increased affinity for the RNA template in the catalysis-competentternary complex compared to the binary polymerase–RNA complex.After a significant lag phase, new imino proton NMR signals developed,corresponding to dsRNA product formation and release. The kcat ≈ 0.1 min–1 (NTP)was determined assuming that 20 μM RNA product (21 bp) was producedby 20 μM polymerase–RNA complex within 5 h. The horizontalbars reflect the time of accumulation of the respective spectra. (C)Comparison of the 1D 1H NMR spectrum of the product ofthe HCV-polymerase-catalyzed polymerization reaction (solid blackline) with a chemically synthesized and annealed 21 bp double-strandedRNA (dashed red line). The integral of the signal at 13.75 ppm inthe 1H NMR spectrum (marked by an asterisk) is equal to0.05 of the total integral of all imino proton signals, as expectedfor 21 paired bases.
© Copyright Policy
Related In: Results  -  Collection

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

fig6: RNA-dependent RNA polymerization monitored by real-time 1D 1H NMR spectroscopy. Product formation (double-stranded RNA)by the HCV-polymerase was monitored with the native 3′(−)SLRNA template following the addition of NTPs. (A) Proton spectra sensingdouble-stranded RNA were recorded at different time points of thepolymerization reaction: 0–8 h, magenta; 8–16 h, red;16–24 h, orange; 25–46 h, yellow; 46–67 h, green;and 67–88 h, blue after the addition of NTP to the polymerase–RNAcomplex (binary complex) (black). Upon binding of NTPs to the binarycomplex, the intensity of the RNA-related 1H NMR signalsdecreased. During an initial period of ca. 24 h, a constant signalpattern of the RNA was observed. After about 25 h, newly developedimino proton NMR signals were detectable that corresponded to thereleased double-stranded RNA product. (B) The enzymatic progress curveillustrates the HCV-polymerase interacting with the partially double-stranded3′(−)SL RNA as well as the subsequent product formationand release process of the double-stranded RNA. The initial decreaseof the integrated imino proton NMR signals upon addition of NTPs indicatesan increased affinity for the RNA template in the catalysis-competentternary complex compared to the binary polymerase–RNA complex.After a significant lag phase, new imino proton NMR signals developed,corresponding to dsRNA product formation and release. The kcat ≈ 0.1 min–1 (NTP)was determined assuming that 20 μM RNA product (21 bp) was producedby 20 μM polymerase–RNA complex within 5 h. The horizontalbars reflect the time of accumulation of the respective spectra. (C)Comparison of the 1D 1H NMR spectrum of the product ofthe HCV-polymerase-catalyzed polymerization reaction (solid blackline) with a chemically synthesized and annealed 21 bp double-strandedRNA (dashed red line). The integral of the signal at 13.75 ppm inthe 1H NMR spectrum (marked by an asterisk) is equal to0.05 of the total integral of all imino proton signals, as expectedfor 21 paired bases.

Mentions: During the polymerizationprocess, the viral RdRp catalyzes RNAsynthesis in a nucleotidyl-transfer reaction. Thus, besides bindingof the RNA template, the enzyme also associates nucleotides in a secondhalf-reaction. First, we applied UV circular dichroism at the negativelocal extremum at 241 nm to measure the formation of polymerase–NTPcomplexes, (Supporting Information Figure S1). The obtained data revealed dissociation constants in a low micromolarrange of the respective NTPs as well as a positive cooperativity,which was indicated by a Hill coefficient of ∼1.5 (summarizedin Table 4). In the absence of template RNA,two nucleotide molecules associated with the polymerase (see Discussion). In a second approach, the initiationof binary complex formation of the HCV-polymerase with the 3′(−)SLRNA was monitored by 1D 1H NMR spectroscopy. The data revealedan overall line-broadening of the imino proton signals in the substratepart of the spectrum. This was explained by an equilibrium formedbetween the unbound nucleic acid, the signals of which remained detectable,and the high molecular weight complex where the RNA-related signalsdisappeared due to the proton’s restricted tumbling motion.To further explore the impact of NTP binding on the binary complexconsisting of the HCV-polymerase and the 3′(−)SL RNAtemplate, we again applied time-resolved 1D 1H NMR. Interestingly,the addition of NTPs led to an intensity loss of the RNA template’simino proton signals (Figure 6A,B), which indicateda decrease in the concentration of unbound RNA and an increase inthe amount of RNA that bound to the polymerase. Hence, the formationof the binary complex increased the affinity of the HCV-polymerasefor the RNA template (KM < KD), leading to the catalysis-competent ternarycomplex.


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-dependent RNA polymerization monitored by real-time 1D 1H NMR spectroscopy. Product formation (double-stranded RNA)by the HCV-polymerase was monitored with the native 3′(−)SLRNA template following the addition of NTPs. (A) Proton spectra sensingdouble-stranded RNA were recorded at different time points of thepolymerization reaction: 0–8 h, magenta; 8–16 h, red;16–24 h, orange; 25–46 h, yellow; 46–67 h, green;and 67–88 h, blue after the addition of NTP to the polymerase–RNAcomplex (binary complex) (black). Upon binding of NTPs to the binarycomplex, the intensity of the RNA-related 1H NMR signalsdecreased. During an initial period of ca. 24 h, a constant signalpattern of the RNA was observed. After about 25 h, newly developedimino proton NMR signals were detectable that corresponded to thereleased double-stranded RNA product. (B) The enzymatic progress curveillustrates the HCV-polymerase interacting with the partially double-stranded3′(−)SL RNA as well as the subsequent product formationand release process of the double-stranded RNA. The initial decreaseof the integrated imino proton NMR signals upon addition of NTPs indicatesan increased affinity for the RNA template in the catalysis-competentternary complex compared to the binary polymerase–RNA complex.After a significant lag phase, new imino proton NMR signals developed,corresponding to dsRNA product formation and release. The kcat ≈ 0.1 min–1 (NTP)was determined assuming that 20 μM RNA product (21 bp) was producedby 20 μM polymerase–RNA complex within 5 h. The horizontalbars reflect the time of accumulation of the respective spectra. (C)Comparison of the 1D 1H NMR spectrum of the product ofthe HCV-polymerase-catalyzed polymerization reaction (solid blackline) with a chemically synthesized and annealed 21 bp double-strandedRNA (dashed red line). The integral of the signal at 13.75 ppm inthe 1H NMR spectrum (marked by an asterisk) is equal to0.05 of the total integral of all imino proton signals, as expectedfor 21 paired bases.
© Copyright Policy
Related In: Results  -  Collection

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

fig6: RNA-dependent RNA polymerization monitored by real-time 1D 1H NMR spectroscopy. Product formation (double-stranded RNA)by the HCV-polymerase was monitored with the native 3′(−)SLRNA template following the addition of NTPs. (A) Proton spectra sensingdouble-stranded RNA were recorded at different time points of thepolymerization reaction: 0–8 h, magenta; 8–16 h, red;16–24 h, orange; 25–46 h, yellow; 46–67 h, green;and 67–88 h, blue after the addition of NTP to the polymerase–RNAcomplex (binary complex) (black). Upon binding of NTPs to the binarycomplex, the intensity of the RNA-related 1H NMR signalsdecreased. During an initial period of ca. 24 h, a constant signalpattern of the RNA was observed. After about 25 h, newly developedimino proton NMR signals were detectable that corresponded to thereleased double-stranded RNA product. (B) The enzymatic progress curveillustrates the HCV-polymerase interacting with the partially double-stranded3′(−)SL RNA as well as the subsequent product formationand release process of the double-stranded RNA. The initial decreaseof the integrated imino proton NMR signals upon addition of NTPs indicatesan increased affinity for the RNA template in the catalysis-competentternary complex compared to the binary polymerase–RNA complex.After a significant lag phase, new imino proton NMR signals developed,corresponding to dsRNA product formation and release. The kcat ≈ 0.1 min–1 (NTP)was determined assuming that 20 μM RNA product (21 bp) was producedby 20 μM polymerase–RNA complex within 5 h. The horizontalbars reflect the time of accumulation of the respective spectra. (C)Comparison of the 1D 1H NMR spectrum of the product ofthe HCV-polymerase-catalyzed polymerization reaction (solid blackline) with a chemically synthesized and annealed 21 bp double-strandedRNA (dashed red line). The integral of the signal at 13.75 ppm inthe 1H NMR spectrum (marked by an asterisk) is equal to0.05 of the total integral of all imino proton signals, as expectedfor 21 paired bases.
Mentions: During the polymerizationprocess, the viral RdRp catalyzes RNAsynthesis in a nucleotidyl-transfer reaction. Thus, besides bindingof the RNA template, the enzyme also associates nucleotides in a secondhalf-reaction. First, we applied UV circular dichroism at the negativelocal extremum at 241 nm to measure the formation of polymerase–NTPcomplexes, (Supporting Information Figure S1). The obtained data revealed dissociation constants in a low micromolarrange of the respective NTPs as well as a positive cooperativity,which was indicated by a Hill coefficient of ∼1.5 (summarizedin Table 4). In the absence of template RNA,two nucleotide molecules associated with the polymerase (see Discussion). In a second approach, the initiationof binary complex formation of the HCV-polymerase with the 3′(−)SLRNA was monitored by 1D 1H NMR spectroscopy. The data revealedan overall line-broadening of the imino proton signals in the substratepart of the spectrum. This was explained by an equilibrium formedbetween the unbound nucleic acid, the signals of which remained detectable,and the high molecular weight complex where the RNA-related signalsdisappeared due to the proton’s restricted tumbling motion.To further explore the impact of NTP binding on the binary complexconsisting of the HCV-polymerase and the 3′(−)SL RNAtemplate, we again applied time-resolved 1D 1H NMR. Interestingly,the addition of NTPs led to an intensity loss of the RNA template’simino proton signals (Figure 6A,B), which indicateda decrease in the concentration of unbound RNA and an increase inthe amount of RNA that bound to the polymerase. Hence, the formationof the binary complex increased the affinity of the HCV-polymerasefor the RNA template (KM < KD), leading to the catalysis-competent ternarycomplex.

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