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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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Effect of different conditions on the RNA 5′-triphosphatase activity of CHIKV-nsP2T.Effect of AMP, ADP and ATP on RTPase activity: CHIKV-nsP2T was incubated with 5′-[γ-32P]-RNA at 37°C for 30 min in presence of different concentrations of AMP/ADP/ATP independently and products were analyzed by TLC. Activity of CHIKV-nsP2T without AMP/ADP/ATP was taken as 100% and the percent activity of each reaction was calculated separately for each reaction. The effect of MgCl2 on RTPase activity: CHIKV-nsP2T was incubated with 5′-[γ-32P]-RNA at 37°C for 30 min in presence of different concentrations of MgCl2 (0 -5.0 mM). Released radiolabel [32Pi] was quantitated for three independent experiments and mean values were plotted. RTPase activity of nsP2 mutants: CHIKV-nsP2T wild type, mut I and mut II proteins were incubated with 5′-[γ-32P]-RNA at 37°C. Aliquots were removed at different time points (5, 10, 15, 20, 25, 30 and 40 min) and analyzed. Released radiolabel [32Pi] was quantitated for three independent experiments and mean values were plotted.
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pone-0022336-g006: Effect of different conditions on the RNA 5′-triphosphatase activity of CHIKV-nsP2T.Effect of AMP, ADP and ATP on RTPase activity: CHIKV-nsP2T was incubated with 5′-[γ-32P]-RNA at 37°C for 30 min in presence of different concentrations of AMP/ADP/ATP independently and products were analyzed by TLC. Activity of CHIKV-nsP2T without AMP/ADP/ATP was taken as 100% and the percent activity of each reaction was calculated separately for each reaction. The effect of MgCl2 on RTPase activity: CHIKV-nsP2T was incubated with 5′-[γ-32P]-RNA at 37°C for 30 min in presence of different concentrations of MgCl2 (0 -5.0 mM). Released radiolabel [32Pi] was quantitated for three independent experiments and mean values were plotted. RTPase activity of nsP2 mutants: CHIKV-nsP2T wild type, mut I and mut II proteins were incubated with 5′-[γ-32P]-RNA at 37°C. Aliquots were removed at different time points (5, 10, 15, 20, 25, 30 and 40 min) and analyzed. Released radiolabel [32Pi] was quantitated for three independent experiments and mean values were plotted.

Mentions: To see the effect of ATP, ADP and AMP on RNA triphosphatase activity of CHIKV-nsP2T, RNA was incubated in presence of different concentrations of ATP, ADP or AMP. ATP showed 80% inhibition of RTPase, activity indicating that ATP and RNA shared the substrate binding site. ADP and AMP did not affect the RTPase activity even at 5 mM concentration (Figure 6a), suggesting that the 5′-terminal γ-phosphate group in the substrate is the major determinant for competition. These data led us to conclude that CHIKV-nsP2T NTPase and RNA 5′-triphosphatase activities have a common active site and 5′-terminal γ- and β-phosphate groups interact with the NTPase/RNA 5′-triphosphatase activity domain of CHIKV-nsP2T.


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

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

Effect of different conditions on the RNA 5′-triphosphatase activity of CHIKV-nsP2T.Effect of AMP, ADP and ATP on RTPase activity: CHIKV-nsP2T was incubated with 5′-[γ-32P]-RNA at 37°C for 30 min in presence of different concentrations of AMP/ADP/ATP independently and products were analyzed by TLC. Activity of CHIKV-nsP2T without AMP/ADP/ATP was taken as 100% and the percent activity of each reaction was calculated separately for each reaction. The effect of MgCl2 on RTPase activity: CHIKV-nsP2T was incubated with 5′-[γ-32P]-RNA at 37°C for 30 min in presence of different concentrations of MgCl2 (0 -5.0 mM). Released radiolabel [32Pi] was quantitated for three independent experiments and mean values were plotted. RTPase activity of nsP2 mutants: CHIKV-nsP2T wild type, mut I and mut II proteins were incubated with 5′-[γ-32P]-RNA at 37°C. Aliquots were removed at different time points (5, 10, 15, 20, 25, 30 and 40 min) and analyzed. Released radiolabel [32Pi] was quantitated for three independent experiments and mean values were plotted.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3139623&req=5

pone-0022336-g006: Effect of different conditions on the RNA 5′-triphosphatase activity of CHIKV-nsP2T.Effect of AMP, ADP and ATP on RTPase activity: CHIKV-nsP2T was incubated with 5′-[γ-32P]-RNA at 37°C for 30 min in presence of different concentrations of AMP/ADP/ATP independently and products were analyzed by TLC. Activity of CHIKV-nsP2T without AMP/ADP/ATP was taken as 100% and the percent activity of each reaction was calculated separately for each reaction. The effect of MgCl2 on RTPase activity: CHIKV-nsP2T was incubated with 5′-[γ-32P]-RNA at 37°C for 30 min in presence of different concentrations of MgCl2 (0 -5.0 mM). Released radiolabel [32Pi] was quantitated for three independent experiments and mean values were plotted. RTPase activity of nsP2 mutants: CHIKV-nsP2T wild type, mut I and mut II proteins were incubated with 5′-[γ-32P]-RNA at 37°C. Aliquots were removed at different time points (5, 10, 15, 20, 25, 30 and 40 min) and analyzed. Released radiolabel [32Pi] was quantitated for three independent experiments and mean values were plotted.
Mentions: To see the effect of ATP, ADP and AMP on RNA triphosphatase activity of CHIKV-nsP2T, RNA was incubated in presence of different concentrations of ATP, ADP or AMP. ATP showed 80% inhibition of RTPase, activity indicating that ATP and RNA shared the substrate binding site. ADP and AMP did not affect the RTPase activity even at 5 mM concentration (Figure 6a), suggesting that the 5′-terminal γ-phosphate group in the substrate is the major determinant for competition. These data led us to conclude that CHIKV-nsP2T NTPase and RNA 5′-triphosphatase activities have a common active site and 5′-terminal γ- and β-phosphate groups interact with the NTPase/RNA 5′-triphosphatase activity domain of CHIKV-nsP2T.

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