NTPase and 5′-RNA Triphosphatase Activities of Chikungunya Virus nsP2 Protein

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 Mg2+ 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, Mg2+ ion binding motif (DEXX) suggesting that they have a common catalytic site

RNA 5′-Triphosphatase Activity of the Hepatitis E Virus Helicase Domain▿

Hepatitis E virus (HEV) has a positive-sense RNA genome with a 5′-m7G cap. HEV open reading frame 1 (ORF1) encodes a polyprotein with multiple enzyme domains required for replication. HEV helicase is a nucleoside triphosphatase (NTPase) with the ability to unwind RNA duplexes in the 5′-to-3′ direction. When incubated with 5′-[γ-32P]RNA and 5′-[α-32P]RNA, HEV helicase released 32P only from 5′-[γ-32P]RNA, showing specificity for the γ-β-triphosphate bond. Removal of γ-phosphate from the 5′ end of the primary transcripts (pppRNA to ppRNA) by RNA triphosphatase is an essential step during cap formation. It is suggested that HEV employs the helicase to mediate the first step of 5′ cap synthesis

Strand displacement activity of CHIKV-nsP2T.

<p>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).</p

Helicase sequence motifs of the CHIKV-nsP2 identified by using amino acid alignments.

<p>Helicase sequence motifs of the CHIKV-nsP2 identified by using amino acid alignments.</p

ATPase activity of CHIKV-nsP2T.

<p>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 MgCl_2, 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 MgCl_2, 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.</p

NTPase activity of CHIKV-nsP2T.

<p>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.</p

Expression of truncated CHIKV nsP2.

<p>Schematic representation of CHIKV nsP2: N- terminal helicase domain (white) and C-terminal protease domain (gray) are indicated in the figure. The two proteins expressed were, CHIKV-Hel (166–441 a.a.) and CHIKV-nsP2T (166–630 a.a.). Both this proteins spanned all seven signature motifs of SF1 helicases which are conserved among Alphaviruses. SDS-PAGE analysis: HPLC purified proteins were analyzed on 10% SDS-PAGE. Lanes are: (1) Wild type CHIKV-nsP2T, (2) nsP2T-mut I, (3) nsP2T-mut II, (MW) Molecular weight marker.</p

Effect of different conditions on the RNA 5′-triphosphatase activity of CHIKV-nsP2T.

<p>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.</p