Limits...
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.

Show MeSH

Related in: MedlinePlus

NTPase activity of CHIKV-nsP2T.Effect of poly (U) RNA on ATPase activity: CHIKV-nsP2T protein was incubated in a 50 µl reaction containing 50 mM MOPS at pH 7.25, 1 mM ATP, 1 mM MgCl2, poly (U) RNA (0–5000 ng), at 37°C for 30 min. Released phosphate was quantitated as described in the experimental procedures. Effect of different nucleic acid oligonucleotides on ATPase activity: CHIKV-nsP2T protein was incubated in a 50 µl reaction containing 50 mM MOPS at pH 7.25, 1 mM ATP, 1 mM MgCl2, and different homopolynucleotides (25 ng/µl of the reaction), at 37°C for 30 min. Released phosphate was quantitated as described in the experimental procedures. ATPase activity of CHIKV-nsP2T mutant proteins (mut I and mut II): CHIKV-nsP2T mutant proteins were incubated in a 50 µl reaction containing 50 mM MOPS at pH 7.25, 1 mM MgCl2, 1 mM ATP, at 37°C for 30 min. Released phosphate was quantitated as given in the experimental procedures. NTPase activity of CHIKV-nsP2T: CHIKV-nsP2T protein was incubated in a 50 µl reaction containing 50mM MOPS at pH 7.25, 1 mM MgCl2, and increasing concentrations of different NTPs, at 37°C for 30 min. Released phosphate was quantitated as described in the experimental procedures.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3139623&req=5

pone-0022336-g003: NTPase activity of CHIKV-nsP2T.Effect of poly (U) RNA on ATPase activity: CHIKV-nsP2T protein was incubated in a 50 µl reaction containing 50 mM MOPS at pH 7.25, 1 mM ATP, 1 mM MgCl2, poly (U) RNA (0–5000 ng), at 37°C for 30 min. Released phosphate was quantitated as described in the experimental procedures. Effect of different nucleic acid oligonucleotides on ATPase activity: CHIKV-nsP2T protein was incubated in a 50 µl reaction containing 50 mM MOPS at pH 7.25, 1 mM ATP, 1 mM MgCl2, and different homopolynucleotides (25 ng/µl of the reaction), at 37°C for 30 min. Released phosphate was quantitated as described in the experimental procedures. ATPase activity of CHIKV-nsP2T mutant proteins (mut I and mut II): CHIKV-nsP2T mutant proteins were incubated in a 50 µl reaction containing 50 mM MOPS at pH 7.25, 1 mM MgCl2, 1 mM ATP, at 37°C for 30 min. Released phosphate was quantitated as given in the experimental procedures. NTPase activity of CHIKV-nsP2T: CHIKV-nsP2T protein was incubated in a 50 µl reaction containing 50mM MOPS at pH 7.25, 1 mM MgCl2, and increasing concentrations of different NTPs, at 37°C for 30 min. Released phosphate was quantitated as described in the experimental procedures.

Mentions: NTPase activity of helicase-like protein is typically stimulated by nucleic acids, particularly by poly (U). To test that, different concentrations of Poly (U) (125 ng to 5000 ng/reaction) were added and released phosphate was measured. There was no increase in the activity of CHIKV-nsP2T in presence of poly (U). Amount of phosphate released remained same even after increasing the oligo concentration up to 5.0 µg/reaction (Figure 3a). Results remained same even when increased amount of the protein was added in the reaction (10 nM, 50 ng/reaction) (data not shown). Similarly, there was no enhancement observed in presence of any of the other RNA/DNA homopolynucleotides (polyA/U/G/C/dA/dT/dG-/dC) (Figure 3b).


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

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

NTPase activity of CHIKV-nsP2T.Effect of poly (U) RNA on ATPase activity: CHIKV-nsP2T protein was incubated in a 50 µl reaction containing 50 mM MOPS at pH 7.25, 1 mM ATP, 1 mM MgCl2, poly (U) RNA (0–5000 ng), at 37°C for 30 min. Released phosphate was quantitated as described in the experimental procedures. Effect of different nucleic acid oligonucleotides on ATPase activity: CHIKV-nsP2T protein was incubated in a 50 µl reaction containing 50 mM MOPS at pH 7.25, 1 mM ATP, 1 mM MgCl2, and different homopolynucleotides (25 ng/µl of the reaction), at 37°C for 30 min. Released phosphate was quantitated as described in the experimental procedures. ATPase activity of CHIKV-nsP2T mutant proteins (mut I and mut II): CHIKV-nsP2T mutant proteins were incubated in a 50 µl reaction containing 50 mM MOPS at pH 7.25, 1 mM MgCl2, 1 mM ATP, at 37°C for 30 min. Released phosphate was quantitated as given in the experimental procedures. NTPase activity of CHIKV-nsP2T: CHIKV-nsP2T protein was incubated in a 50 µl reaction containing 50mM MOPS at pH 7.25, 1 mM MgCl2, and increasing concentrations of different NTPs, at 37°C for 30 min. Released phosphate was quantitated as described in the experimental procedures.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0022336-g003: NTPase activity of CHIKV-nsP2T.Effect of poly (U) RNA on ATPase activity: CHIKV-nsP2T protein was incubated in a 50 µl reaction containing 50 mM MOPS at pH 7.25, 1 mM ATP, 1 mM MgCl2, poly (U) RNA (0–5000 ng), at 37°C for 30 min. Released phosphate was quantitated as described in the experimental procedures. Effect of different nucleic acid oligonucleotides on ATPase activity: CHIKV-nsP2T protein was incubated in a 50 µl reaction containing 50 mM MOPS at pH 7.25, 1 mM ATP, 1 mM MgCl2, and different homopolynucleotides (25 ng/µl of the reaction), at 37°C for 30 min. Released phosphate was quantitated as described in the experimental procedures. ATPase activity of CHIKV-nsP2T mutant proteins (mut I and mut II): CHIKV-nsP2T mutant proteins were incubated in a 50 µl reaction containing 50 mM MOPS at pH 7.25, 1 mM MgCl2, 1 mM ATP, at 37°C for 30 min. Released phosphate was quantitated as given in the experimental procedures. NTPase activity of CHIKV-nsP2T: CHIKV-nsP2T protein was incubated in a 50 µl reaction containing 50mM MOPS at pH 7.25, 1 mM MgCl2, and increasing concentrations of different NTPs, at 37°C for 30 min. Released phosphate was quantitated as described in the experimental procedures.
Mentions: NTPase activity of helicase-like protein is typically stimulated by nucleic acids, particularly by poly (U). To test that, different concentrations of Poly (U) (125 ng to 5000 ng/reaction) were added and released phosphate was measured. There was no increase in the activity of CHIKV-nsP2T in presence of poly (U). Amount of phosphate released remained same even after increasing the oligo concentration up to 5.0 µg/reaction (Figure 3a). Results remained same even when increased amount of the protein was added in the reaction (10 nM, 50 ng/reaction) (data not shown). Similarly, there was no enhancement observed in presence of any of the other RNA/DNA homopolynucleotides (polyA/U/G/C/dA/dT/dG-/dC) (Figure 3b).

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