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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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RNA 5′-triphosphatase activity of CHIKV-nsP2T.MBP-nsP2T and shrimp alkaline phosphatase (SAP) were incubated with 5′-γ-32P/5′-α-32P labeled nonspecific RNA (5′-GGGA24-3′) substrates separately at 37°C for 30 min and analyzed by TLC. Lanes 1) 5′-[α-32P]-RNA, 2) 5′-[α-32P]-RNA with SAP, 3) 5′-[α-32P]-RNA with CHIKV-nsP2T, 4) 5′-[γ-32P]-RNA, 5) 5′-[γ-32P]-RNA with SAP, 6) 5′-[γ-32P]-RNA with CHIKV-nsP2T. CHIKV-nsP2T was incubated with 5′-[γ-32P] labeled CHIKV 5′-NCR RNA at 37°C for 30 min, products was analyzed by TLC and the plate was exposed to X-ray film. Lanes 1) RNA without protein, 2) RNA with MBP, 3) RNA with CHIKV-nsP2T.
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pone-0022336-g005: RNA 5′-triphosphatase activity of CHIKV-nsP2T.MBP-nsP2T and shrimp alkaline phosphatase (SAP) were incubated with 5′-γ-32P/5′-α-32P labeled nonspecific RNA (5′-GGGA24-3′) substrates separately at 37°C for 30 min and analyzed by TLC. Lanes 1) 5′-[α-32P]-RNA, 2) 5′-[α-32P]-RNA with SAP, 3) 5′-[α-32P]-RNA with CHIKV-nsP2T, 4) 5′-[γ-32P]-RNA, 5) 5′-[γ-32P]-RNA with SAP, 6) 5′-[γ-32P]-RNA with CHIKV-nsP2T. CHIKV-nsP2T was incubated with 5′-[γ-32P] labeled CHIKV 5′-NCR RNA at 37°C for 30 min, products was analyzed by TLC and the plate was exposed to X-ray film. Lanes 1) RNA without protein, 2) RNA with MBP, 3) RNA with CHIKV-nsP2T.

Mentions: For RNA triphosphatase activity studies, 5′-[γ-32P]-RNA or 5′-[α-32P]-RNA substrates either with non-specific sequence-(5′-GGGA24-3′) or with 5′NCR of CHIKV genome were generated by in vitro-transcription. Following incubation with the CHIKV-nsP2T at 37°C for 30 min, products were analyzed by TLC. 5′-[γ-32P]-RNA or 5′-[α-32P]-RNA substrates were separately incubated with shrimp alkaline phosphatase (SAP) to see the specificity of CHIKV-nsP2T for phosphate group at the 5′-end of the RNA substrate. As expected, SAP could remove 32P moiety from both RNA substrates, but CHIKV-nsP2T showed no release of label from 5′-α-32P-RNA. CHIKV-nsP2T released labeled 5′-phosphate group from 5′-γ-32P labeled RNA indicating that CHIKV-nsP2T hydrolyzes only γ-β-triphosphate bond and unlike SAP does not have a general phosphohydrolase activity that would also remove the β- and α- phosphate groups (Figure 5a). This suggested that CHIKV-nsP2T has 5′-RNA-triphosphatase (RTPase) activity. Extent of hydrolysis of both CHIKV 5′-NCR and non specific RNA oligo substrates was comparable (Figure 5b). Thus, for the further characterization of 5′-RNA-triphosphatase activity, non-specific RNA substrate was used.


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

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

RNA 5′-triphosphatase activity of CHIKV-nsP2T.MBP-nsP2T and shrimp alkaline phosphatase (SAP) were incubated with 5′-γ-32P/5′-α-32P labeled nonspecific RNA (5′-GGGA24-3′) substrates separately at 37°C for 30 min and analyzed by TLC. Lanes 1) 5′-[α-32P]-RNA, 2) 5′-[α-32P]-RNA with SAP, 3) 5′-[α-32P]-RNA with CHIKV-nsP2T, 4) 5′-[γ-32P]-RNA, 5) 5′-[γ-32P]-RNA with SAP, 6) 5′-[γ-32P]-RNA with CHIKV-nsP2T. CHIKV-nsP2T was incubated with 5′-[γ-32P] labeled CHIKV 5′-NCR RNA at 37°C for 30 min, products was analyzed by TLC and the plate was exposed to X-ray film. Lanes 1) RNA without protein, 2) RNA with MBP, 3) RNA with CHIKV-nsP2T.
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pone-0022336-g005: RNA 5′-triphosphatase activity of CHIKV-nsP2T.MBP-nsP2T and shrimp alkaline phosphatase (SAP) were incubated with 5′-γ-32P/5′-α-32P labeled nonspecific RNA (5′-GGGA24-3′) substrates separately at 37°C for 30 min and analyzed by TLC. Lanes 1) 5′-[α-32P]-RNA, 2) 5′-[α-32P]-RNA with SAP, 3) 5′-[α-32P]-RNA with CHIKV-nsP2T, 4) 5′-[γ-32P]-RNA, 5) 5′-[γ-32P]-RNA with SAP, 6) 5′-[γ-32P]-RNA with CHIKV-nsP2T. CHIKV-nsP2T was incubated with 5′-[γ-32P] labeled CHIKV 5′-NCR RNA at 37°C for 30 min, products was analyzed by TLC and the plate was exposed to X-ray film. Lanes 1) RNA without protein, 2) RNA with MBP, 3) RNA with CHIKV-nsP2T.
Mentions: For RNA triphosphatase activity studies, 5′-[γ-32P]-RNA or 5′-[α-32P]-RNA substrates either with non-specific sequence-(5′-GGGA24-3′) or with 5′NCR of CHIKV genome were generated by in vitro-transcription. Following incubation with the CHIKV-nsP2T at 37°C for 30 min, products were analyzed by TLC. 5′-[γ-32P]-RNA or 5′-[α-32P]-RNA substrates were separately incubated with shrimp alkaline phosphatase (SAP) to see the specificity of CHIKV-nsP2T for phosphate group at the 5′-end of the RNA substrate. As expected, SAP could remove 32P moiety from both RNA substrates, but CHIKV-nsP2T showed no release of label from 5′-α-32P-RNA. CHIKV-nsP2T released labeled 5′-phosphate group from 5′-γ-32P labeled RNA indicating that CHIKV-nsP2T hydrolyzes only γ-β-triphosphate bond and unlike SAP does not have a general phosphohydrolase activity that would also remove the β- and α- phosphate groups (Figure 5a). This suggested that CHIKV-nsP2T has 5′-RNA-triphosphatase (RTPase) activity. Extent of hydrolysis of both CHIKV 5′-NCR and non specific RNA oligo substrates was comparable (Figure 5b). Thus, for the further characterization of 5′-RNA-triphosphatase activity, non-specific RNA substrate was used.

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