title: Mutagenesis of hepatitis E virus helicase motifs: effects on enzyme activity.
abstract: Hepatitis E virus (HEV), the causative agent of hepatitis E, is a non-enveloped RNA virus. The open reading frame 1 encoded non-structural polyprotein has putative domains for methyltransferase, cysteine protease, helicase and RNA-dependent RNA polymerase, however processing of this polyprotein is still uncertain. HEV helicase belongs to superfamily 1 and has all seven conserved motifs typical of the family. NTPase and RNA duplex unwinding activities of HEV helicase domain were recently demonstrated by us. A non-radioactive RNA unwinding assay was developed using biotin and digoxigenin labeled duplex RNA substrate with 5' overhangs for measuring strand displacement activity of the helicase. A series of deletion mutants were constructed to investigate role of individual motifs in the enzymatic activities. Deletion mutants for motif M I and M IV showed increase in ATPase activity. Deletion mutant M VI retained ATPase activity comparable to wild type protein. Mutant M II showed reduced ATPase activity (P=0.003) with no significant decrease in unwinding activity while mutants M Ia and M III showed major reduction of both ATPase and unwinding activities indicating crucial role of these motifs in the helicase function. Overall analysis of deletion mutants showed that Motif I, IV, V and VI have alternative motifs to carry out enzymatic functions of the protein while motifs Ia and III are critical as well as unique motifs in the protein. Knowing the important role of helicase protein during positive sense RNA virus replication, these unique motifs could be good antiviral targets.
authors: Hepatitis Division, National Institute of Virology, Microbial Containment Complex, Sus Road, Pashan, Pune 411021, India.
authors: Vaibhav
authors: V
authors: Mhaindarkar
authors: Hepatitis Division, National Institute of Virology, Microbial Containment Complex, Sus Road, Pashan, Pune 411021, India.
authors: Kavyanjali
authors: K
authors: Sharma
authors: Hepatitis Division, National Institute of Virology, Microbial Containment Complex, Sus Road, Pashan, Pune 411021, India. Electronic address: lolekavita37@yahoo.com.
authors: Kavita S
authors: KS
authors: Lole
mesh_terms: D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020816:Amino Acid Motifs; D016752:Hepatitis E virus / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D016296:Mutagenesis; D009154:Mutation; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
chemical_list: D012367:RNA, Viral; D014764:Viral Proteins; D000251:Adenosine Triphosphatases
keywords: Hepatitis E virus; Kinetics; Motifs; ORF1; RNA helicase