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NTPase and 5'-RNA triphosphatase activities of Chikungunya virus nsP2 protein.

Karpe YA, Aher PP, Lole KS - PLoS ONE (2011)

Bottom Line: ATP was the most preferred substrate by the enzyme.RTPase activity was inhibited by ATP showing sharing of the binding motif by NTP and RNA.Both enzymatic activities were drastically reduced by mutations in the NTP binding motif (GKT) and co-factor, Mg(2+) ion binding motif (DEXX) suggesting that they have a common catalytic site.

View Article: PubMed Central - PubMed

Affiliation: Hepatitis Division, National Institute of Virology, Microbial Containment Complex, Pashan, Pune, India.

ABSTRACT
Chikungunya virus (CHIKV) is an insect borne virus (genus: Alphavirus) which causes acute febrile illness in humans followed by a prolonged arthralgic disease that affects the joints of the extremities. Re-emergence of the virus in the form of outbreaks in last 6-7 years has posed a serious public health problem. CHIKV has a positive sense single stranded RNA genome of about 12,000 nt. Open reading frame 1 of the viral genome encodes a polyprotein precursor, nsP1234, which is processed further into different non structural proteins (nsP1, nsP2, nsP3 and nsP4). Sequence based analyses have shown helicase domain at the N-terminus and protease domain at C-terminus of nsP2. A detailed biochemical analysis of NTPase/RNA helicase and 5'-RNA phosphatase activities of recombinant CHIKV-nsP2T protein (containing conserved NTPase/helicase motifs in the N-terminus and partial papain like protease domain at the C-terminus) was carried out. The protein could hydrolyze all NTPs except dTTP and showed better efficiency for ATP, dATP, GTP and dGTP hydrolysis. ATP was the most preferred substrate by the enzyme. CHIKV-nsP2T also showed 5'-triphosphatase (RTPase) activity that specifically removes the γ-phosphate from the 5' end of RNA. Both NTPase and RTPase activities of the protein were completely dependent on Mg(2+) ions. RTPase activity was inhibited by ATP showing sharing of the binding motif by NTP and RNA. Both enzymatic activities were drastically reduced by mutations in the NTP binding motif (GKT) and co-factor, Mg(2+) ion binding motif (DEXX) suggesting that they have a common catalytic site.

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ATPase activity of CHIKV-nsP2T.At different enzyme concentrations: CHIKV-nsP2T protein was incubated at different concentrations (5 ng to 50 ng) in a 50 µl reaction containing 50 mM MOPS at pH 7.25, 1 mM ATP, 1 mM MgCl2, at 37°C for 30 min. Released phosphate was quantitated as described in the experimental procedures. Effect of pH on ATPase activity: CHIKV-nsP2T protein was incubated in a 50 µl reaction containing 50 mM MOPS at pH 6.25–8.0, 1 mM ATP, 1 mM MgCl2, at 37°C for 30 min. Released phosphate was quantitated as described in the experimental procedures. Effect of MgCl2 concentration on ATPase activity: CHIKV-nsP2T protein was incubated in a 50 µl reaction containing 50 mM MOPS at pH 7.25, 0–5 mM MgCl2, 1 mM ATP, at 37°C for 30 min. Released phosphate was quantitated as described in the experimental procedures. Analysis of released phosphate on TLC: Reaction was carried out in 20 µl containing 1 nM CHIKV-nsP2T, 50 mM MOPS (pH 7.25), 1 mM MgCl2, 1 mM ATP, 1 µCi of [γ-32P] ATP, incubated at 37°C for 30 min and 1 µl of the mixture was analyzed by TLC and processed for autoradiography.
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pone-0022336-g002: ATPase activity of CHIKV-nsP2T.At different enzyme concentrations: CHIKV-nsP2T protein was incubated at different concentrations (5 ng to 50 ng) in a 50 µl reaction containing 50 mM MOPS at pH 7.25, 1 mM ATP, 1 mM MgCl2, at 37°C for 30 min. Released phosphate was quantitated as described in the experimental procedures. Effect of pH on ATPase activity: CHIKV-nsP2T protein was incubated in a 50 µl reaction containing 50 mM MOPS at pH 6.25–8.0, 1 mM ATP, 1 mM MgCl2, at 37°C for 30 min. Released phosphate was quantitated as described in the experimental procedures. Effect of MgCl2 concentration on ATPase activity: CHIKV-nsP2T protein was incubated in a 50 µl reaction containing 50 mM MOPS at pH 7.25, 0–5 mM MgCl2, 1 mM ATP, at 37°C for 30 min. Released phosphate was quantitated as described in the experimental procedures. Analysis of released phosphate on TLC: Reaction was carried out in 20 µl containing 1 nM CHIKV-nsP2T, 50 mM MOPS (pH 7.25), 1 mM MgCl2, 1 mM ATP, 1 µCi of [γ-32P] ATP, incubated at 37°C for 30 min and 1 µl of the mixture was analyzed by TLC and processed for autoradiography.

Mentions: Initially, ATPase activity of the CHIKV-nsP2T was carried out at different protein concentrations (5–50 ng/reaction), MgCl2 concentrations (0–10 mM) and at different pH (6.25–8.0) to optimize the NTPase assay conditions. There was a gradual increase in the released phosphate with increase in the protein concentration from 10–50 ng/reaction (Figure 2a). Enzyme showed similar activity in 7.0–8.0 pH range, with a minor peak at pH 7.25 (Figure 2b). There was a gradual enhancement in the ATPase activity with increasing MgCl2 concentrations from 0.1 to 1 mM. Released phosphate levels did not increase significantly with further increase in the Mg2+ ion concentrations (Figure 2c). All further NTPase reactions were carried out at pH 7.25 in presence of 1 mM MgCl2. Optimum temperature of the reaction was 37°C (data not shown). ATPase activity of the CHIKV-nsP2T was absolutely dependent on Mg2+, as no detectable ATPase activity was observed without MgCl2. There was no Pi release seen when maltose binding protein alone was incubated with γ-32P labeled ATP (Figure 2d).


NTPase and 5'-RNA triphosphatase activities of Chikungunya virus nsP2 protein.

Karpe YA, Aher PP, Lole KS - PLoS ONE (2011)

ATPase activity of CHIKV-nsP2T.At different enzyme concentrations: CHIKV-nsP2T protein was incubated at different concentrations (5 ng to 50 ng) in a 50 µl reaction containing 50 mM MOPS at pH 7.25, 1 mM ATP, 1 mM MgCl2, at 37°C for 30 min. Released phosphate was quantitated as described in the experimental procedures. Effect of pH on ATPase activity: CHIKV-nsP2T protein was incubated in a 50 µl reaction containing 50 mM MOPS at pH 6.25–8.0, 1 mM ATP, 1 mM MgCl2, at 37°C for 30 min. Released phosphate was quantitated as described in the experimental procedures. Effect of MgCl2 concentration on ATPase activity: CHIKV-nsP2T protein was incubated in a 50 µl reaction containing 50 mM MOPS at pH 7.25, 0–5 mM MgCl2, 1 mM ATP, at 37°C for 30 min. Released phosphate was quantitated as described in the experimental procedures. Analysis of released phosphate on TLC: Reaction was carried out in 20 µl containing 1 nM CHIKV-nsP2T, 50 mM MOPS (pH 7.25), 1 mM MgCl2, 1 mM ATP, 1 µCi of [γ-32P] ATP, incubated at 37°C for 30 min and 1 µl of the mixture was analyzed by TLC and processed for autoradiography.
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pone-0022336-g002: ATPase activity of CHIKV-nsP2T.At different enzyme concentrations: CHIKV-nsP2T protein was incubated at different concentrations (5 ng to 50 ng) in a 50 µl reaction containing 50 mM MOPS at pH 7.25, 1 mM ATP, 1 mM MgCl2, at 37°C for 30 min. Released phosphate was quantitated as described in the experimental procedures. Effect of pH on ATPase activity: CHIKV-nsP2T protein was incubated in a 50 µl reaction containing 50 mM MOPS at pH 6.25–8.0, 1 mM ATP, 1 mM MgCl2, at 37°C for 30 min. Released phosphate was quantitated as described in the experimental procedures. Effect of MgCl2 concentration on ATPase activity: CHIKV-nsP2T protein was incubated in a 50 µl reaction containing 50 mM MOPS at pH 7.25, 0–5 mM MgCl2, 1 mM ATP, at 37°C for 30 min. Released phosphate was quantitated as described in the experimental procedures. Analysis of released phosphate on TLC: Reaction was carried out in 20 µl containing 1 nM CHIKV-nsP2T, 50 mM MOPS (pH 7.25), 1 mM MgCl2, 1 mM ATP, 1 µCi of [γ-32P] ATP, incubated at 37°C for 30 min and 1 µl of the mixture was analyzed by TLC and processed for autoradiography.
Mentions: Initially, ATPase activity of the CHIKV-nsP2T was carried out at different protein concentrations (5–50 ng/reaction), MgCl2 concentrations (0–10 mM) and at different pH (6.25–8.0) to optimize the NTPase assay conditions. There was a gradual increase in the released phosphate with increase in the protein concentration from 10–50 ng/reaction (Figure 2a). Enzyme showed similar activity in 7.0–8.0 pH range, with a minor peak at pH 7.25 (Figure 2b). There was a gradual enhancement in the ATPase activity with increasing MgCl2 concentrations from 0.1 to 1 mM. Released phosphate levels did not increase significantly with further increase in the Mg2+ ion concentrations (Figure 2c). All further NTPase reactions were carried out at pH 7.25 in presence of 1 mM MgCl2. Optimum temperature of the reaction was 37°C (data not shown). ATPase activity of the CHIKV-nsP2T was absolutely dependent on Mg2+, as no detectable ATPase activity was observed without MgCl2. There was no Pi release seen when maltose binding protein alone was incubated with γ-32P labeled ATP (Figure 2d).

Bottom Line: ATP was the most preferred substrate by the enzyme.RTPase activity was inhibited by ATP showing sharing of the binding motif by NTP and RNA.Both enzymatic activities were drastically reduced by mutations in the NTP binding motif (GKT) and co-factor, Mg(2+) ion binding motif (DEXX) suggesting that they have a common catalytic site.

View Article: PubMed Central - PubMed

Affiliation: Hepatitis Division, National Institute of Virology, Microbial Containment Complex, Pashan, Pune, India.

ABSTRACT
Chikungunya virus (CHIKV) is an insect borne virus (genus: Alphavirus) which causes acute febrile illness in humans followed by a prolonged arthralgic disease that affects the joints of the extremities. Re-emergence of the virus in the form of outbreaks in last 6-7 years has posed a serious public health problem. CHIKV has a positive sense single stranded RNA genome of about 12,000 nt. Open reading frame 1 of the viral genome encodes a polyprotein precursor, nsP1234, which is processed further into different non structural proteins (nsP1, nsP2, nsP3 and nsP4). Sequence based analyses have shown helicase domain at the N-terminus and protease domain at C-terminus of nsP2. A detailed biochemical analysis of NTPase/RNA helicase and 5'-RNA phosphatase activities of recombinant CHIKV-nsP2T protein (containing conserved NTPase/helicase motifs in the N-terminus and partial papain like protease domain at the C-terminus) was carried out. The protein could hydrolyze all NTPs except dTTP and showed better efficiency for ATP, dATP, GTP and dGTP hydrolysis. ATP was the most preferred substrate by the enzyme. CHIKV-nsP2T also showed 5'-triphosphatase (RTPase) activity that specifically removes the γ-phosphate from the 5' end of RNA. Both NTPase and RTPase activities of the protein were completely dependent on Mg(2+) ions. RTPase activity was inhibited by ATP showing sharing of the binding motif by NTP and RNA. Both enzymatic activities were drastically reduced by mutations in the NTP binding motif (GKT) and co-factor, Mg(2+) ion binding motif (DEXX) suggesting that they have a common catalytic site.

Show MeSH
Related in: MedlinePlus