title: NTPase and 5'-RNA triphosphatase activities of Chikungunya virus nsP2 protein.
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.
authors.affiliation: Hepatitis Division, National Institute of Virology, Microbial Containment Complex, Pashan, Pune, India.
authors.forename: Yogesh A
authors.initials: YA
authors.lastname: Karpe
authors.forename: Pankaj P
authors.initials: PP
authors.lastname: Aher
authors.forename: Kavita S
authors.initials: KS
authors.lastname: Lole
mesh_terms: D017766:Acid Anhydride Hydrolases / Q000378:metabolism*; D000595:Amino Acid Sequence; D002646:Chikungunya virus / Q000201:enzymology*; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D004279:DNA, Viral / Q000378:metabolism; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D043583:Nucleoside-Triphosphatase / Q000378:metabolism*; D012367:RNA, Viral / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D004279:DNA, Viral; D050505:Mutant Proteins; D012367:RNA, Viral; D003546:Cysteine Endopeptidases; C078306:nsP2 proteinase; D017766:Acid Anhydride Hydrolases; C023090:RNA triphosphatase; D043583:Nucleoside-Triphosphatase