NTPase and 5' to 3' RNA duplex-unwinding activities of the hepatitis E virus helicase domain.
Hepatitis E virus (HEV) is a causative agent of acute hepatitis, and it is the sole member of the genus Hepevirus in the family Hepeviridae. The open reading frame 1 (ORF1) protein of HEV encodes nonstructural polyprotein with putative domains for methyltransferase, cysteine protease, helicase and RNA-dependent RNA polymerase. It is not yet known whether ORF1 functions as a single protein with multiple domains or is processed to form separate functional units. On the basis of amino acid conserved motifs, HEV helicase has been grouped into helicase superfamily 1 (SF-1). In order to examine the RNA helicase activity of the NTPase/helicase domain of HEV, the region (amino acids 960 to 1204) was cloned and expressed as histidine-tagged protein in Escherichia coli (HEV Hel) and purified. HEV Hel exhibited NTPase and RNA unwinding activities. Enzyme hydrolyzed all rNTPs efficiently, dATP and dCTP with moderate efficiency, while it showed less hydrolysis of dGTP and dTTP. Enzyme showed unwinding of only RNA duplexes with 5' overhangs showing 5'-to-3' polarity. We also expressed and purified two HEV Hel mutants. Helicase mutant I, with substitution in the nucleotide-binding motif I (GKS to GAS), showed 30% ATPase activity. Helicase mutant II, with substitutions in the Mg(2+) binding motif II (DEAP to AAAP), showed 50% ATPase activity. Both mutants completely lost ability to unwind RNA duplexes with 5' overhangs. These findings represent the first report demonstrating NTPase/RNA helicase activity of the helicase domain of HEV ORF1.
Karpe
Yogesh A
YA
Hepatitis Division, National Institute of Virology, Microbial Containment Complex, Sus Road, Pashan, Pune, India 411021, USA.
Lole
Kavita S
KS
D000251:Adenosine Triphosphatases / Q000378:metabolism; D016752:Hepatitis E virus / Q000201:enzymology*; D043583:Nucleoside-Triphosphatase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D020365:RNA Helicases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D012367:RNA, Viral / Q000737:chemistry*
D012367:RNA, Viral; D000251:Adenosine Triphosphatases; D043583:Nucleoside-Triphosphatase; D020365:RNA Helicases