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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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Strand displacement activity of CHIKV-nsP2T.With different RNA substrates: Unwinding activity of the protein was checked using different RNA substrates. CHIKV-nsP2 protein was incubated with RNA duplexes with 5′ overhang (lanes 1, 2, 3); with 3′ overhang (lanes 4, 5, 6) and with blunt ends (7, 8, and 9). With different protein concentrations: Unwinding activity was carried out in presence of increasing concentrations of CHIKV-nsP2T using RNA substrate with both 5′ and 3′ overhangs, Lanes (1) control, (2) heat denatured substrate RNA, and (3 to 8) different CHIKV-nsP2T concentrations (1, 10, 50, 100, 500 and 1000 ng).
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pone-0022336-g004: Strand displacement activity of CHIKV-nsP2T.With different RNA substrates: Unwinding activity of the protein was checked using different RNA substrates. CHIKV-nsP2 protein was incubated with RNA duplexes with 5′ overhang (lanes 1, 2, 3); with 3′ overhang (lanes 4, 5, 6) and with blunt ends (7, 8, and 9). With different protein concentrations: Unwinding activity was carried out in presence of increasing concentrations of CHIKV-nsP2T using RNA substrate with both 5′ and 3′ overhangs, Lanes (1) control, (2) heat denatured substrate RNA, and (3 to 8) different CHIKV-nsP2T concentrations (1, 10, 50, 100, 500 and 1000 ng).

Mentions: To characterize unwinding activity of CHIKV-nsP2T, RNA/DNA duplexes with either 3′- or 5′-single stranded overhangs or duplexes with blunt ends were used. The blunt end duplexes were generated by annealing 28 nt oligonucleotides while for duplexes with 5′ and 3′ overhangs, 28 nt and 16 nt long oligonucleotides were used and both had 12 nucleotide single stranded stretches at respective ends [12]. One strand in each of the three RNA or DNA duplexes was 5′-end labeled. Duplex unwinding assays were carried out at similar conditions which were optimized for the ATPase activity. There was no detectable strand displacement by CHIKV-nsP2T with any of the RNA/DNA duplexes with 1 nM protein/reaction (Figure 4a). Further, unwinding assays were carried out using different concentrations of the enzyme from 1 ng-1000 ng/reaction. There was no visible unwinding activity seen even at higher concentrations of enzyme in the reactions (Figure 4b).


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

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

Strand displacement activity of CHIKV-nsP2T.With different RNA substrates: Unwinding activity of the protein was checked using different RNA substrates. CHIKV-nsP2 protein was incubated with RNA duplexes with 5′ overhang (lanes 1, 2, 3); with 3′ overhang (lanes 4, 5, 6) and with blunt ends (7, 8, and 9). With different protein concentrations: Unwinding activity was carried out in presence of increasing concentrations of CHIKV-nsP2T using RNA substrate with both 5′ and 3′ overhangs, Lanes (1) control, (2) heat denatured substrate RNA, and (3 to 8) different CHIKV-nsP2T concentrations (1, 10, 50, 100, 500 and 1000 ng).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3139623&req=5

pone-0022336-g004: Strand displacement activity of CHIKV-nsP2T.With different RNA substrates: Unwinding activity of the protein was checked using different RNA substrates. CHIKV-nsP2 protein was incubated with RNA duplexes with 5′ overhang (lanes 1, 2, 3); with 3′ overhang (lanes 4, 5, 6) and with blunt ends (7, 8, and 9). With different protein concentrations: Unwinding activity was carried out in presence of increasing concentrations of CHIKV-nsP2T using RNA substrate with both 5′ and 3′ overhangs, Lanes (1) control, (2) heat denatured substrate RNA, and (3 to 8) different CHIKV-nsP2T concentrations (1, 10, 50, 100, 500 and 1000 ng).
Mentions: To characterize unwinding activity of CHIKV-nsP2T, RNA/DNA duplexes with either 3′- or 5′-single stranded overhangs or duplexes with blunt ends were used. The blunt end duplexes were generated by annealing 28 nt oligonucleotides while for duplexes with 5′ and 3′ overhangs, 28 nt and 16 nt long oligonucleotides were used and both had 12 nucleotide single stranded stretches at respective ends [12]. One strand in each of the three RNA or DNA duplexes was 5′-end labeled. Duplex unwinding assays were carried out at similar conditions which were optimized for the ATPase activity. There was no detectable strand displacement by CHIKV-nsP2T with any of the RNA/DNA duplexes with 1 nM protein/reaction (Figure 4a). Further, unwinding assays were carried out using different concentrations of the enzyme from 1 ng-1000 ng/reaction. There was no visible unwinding activity seen even at higher concentrations of enzyme in the reactions (Figure 4b).

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