English Journal

Mutagenesis of hepatitis E virus helicase motifs: Effects on enzyme activity.

Mhaindarkar V1, Sharma K1, Lole KS2.Author information 1Hepatitis Division, National Institute of Virology, Microbial Containment Complex, Sus Road, Pashan, Pune 411021, India.2Hepatitis Division, National Institute of Virology, Microbial Containment Complex, Sus Road, Pashan, Pune 411021, India. Electronic address: lolekavita37@yahoo.com.AbstractHepatitis 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.
Virus research.Virus Res.2014 Jan 22;179:26-33. doi: 10.1016/j.virusres.2013.11.022. Epub 2013 Dec 11.
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 s
PMID 24333153

Dom34-Hbs1 mediated dissociation of inactive 80S ribosomes promotes restart of translation after stress.

van den Elzen AM, Schuller A, Green R, Séraphin B.Author information Equipe Labellisée La Ligue, Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC) Centre National de Recherche Scientifique (CNRS) UMR 7104/Institut National de Santé et de Recherche Médicale (INSERM) U964/Université de Strasbourg, Illkirch, France.AbstractFollowing translation termination, ribosomal subunits dissociate to become available for subsequent rounds of protein synthesis. In many translation-inhibiting stress conditions, e.g. glucose starvation in yeast, free ribosomal subunits reassociate to form a large pool of non-translating 80S ribosomes stabilized by the 'clamping' Stm1 factor. The subunits of these inactive ribosomes need to be mobilized for translation restart upon stress relief. The Dom34-Hbs1 complex, together with the Rli1 NTPase (also known as ABCE1), have been shown to split ribosomes stuck on mRNAs in the context of RNA quality control mechanisms. Here, using in vitro and in vivo methods, we report a new role for the Dom34-Hbs1 complex and Rli1 in dissociating inactive ribosomes, thereby facilitating translation restart in yeast recovering from glucose starvation stress. Interestingly, we found that this new role is not restricted to stress conditions, indicating that in growing yeast there is a dynamic pool of inactive ribosomes that needs to be split by Dom34-Hbs1 and Rli1 to participate in protein synthesis. We propose that this provides a new level of translation regulation.
The EMBO journal.EMBO J.2014 Jan 14. [Epub ahead of print]
Following translation termination, ribosomal subunits dissociate to become available for subsequent rounds of protein synthesis. In many translation-inhibiting stress conditions, e.g. glucose starvation in yeast, free ribosomal subunits reassociate to form a large pool of non-translating 80S ribosom
PMID 24424461

Functional Crosstalk between Distant Domains of Chikungunya Virus Non-Structural Protein 2 Is Decisive For Its RNA-Modulating Activity.

Das PK, Merits A, Lulla A.Author information University of Tartu, Estonia.AbstractChikungunya virus (CHIKV) non-structural protein 2 (nsP2) is a multifunctional protein that is considered as a master regulator of the viral lifecycle and a main viral factor responsible for cytopathic effects and subversion of antiviral defense. The C-terminal part of nsP2 possesses protease activity, while the N-terminal part exhibits NTPase and RNA triphosphatase activity, and is proposed to have helicase activity. Bioinformatic analysis classified CHIKV nsP2 into helicase superfamily 1 (SF1). However, the biochemical significance of a coexistence of two functionally unrelated modules in this single protein remains unknown. In this study recombinant nsP2 demonstrated the unwinding of double-stranded RNA in a 5'-3' directionally biased manner and RNA strand-annealing activity. Comparative analysis of NTPase and helicase activities of wild type nsP2 with enzymatic capabilities of different truncated or N-terminally extended variants of nsP2 revealed that the C-terminal part of protein is indispensable for helicase functionality and presumably provides platform for RNA binding, whereas the N-terminal-most region is apparently involved in obtaining conformation of nsP2 that allows for its maximal enzymatic activities. The establishment of the protocols for the production of biochemically active CHIKV nsP2 and optimization of the parameters for helicase and NTPase assays is expected to provide the starting point for further search of possibilities for therapeutic interventions to suppress alphaviral infections.
The Journal of biological chemistry.J Biol Chem.2014 Jan 9. [Epub ahead of print]
Chikungunya virus (CHIKV) non-structural protein 2 (nsP2) is a multifunctional protein that is considered as a master regulator of the viral lifecycle and a main viral factor responsible for cytopathic effects and subversion of antiviral defense. The C-terminal part of nsP2 possesses protease activi
PMID 24407286

Japanese Journal

Overexpression of Fap7, a NTPase involved in ribosome biogenesis, confers resistance to arsenite on yeast cells

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日本毒性学会学術年会 39.2(0), AP-50, 2012
… Fap7 is a putative P-loop NTPase essential for the processing of 20S Pre-rRNA in ribosome biogenesis. … To clarify relationship between P-loop NTPase and arsenite toxicity, we examined sensitivity of yeast cells overexpressing one of other P-loop NTPases. …
NAID 130005009211

リボソーム生合成に関わるFap7の高発現による亜ヒ酸耐性

朱俊軒,高橋勉,永沼章
日本毒性学会学術年会 39.1(0), P-102, 2012
… Fap7はそのアミノ酸配列上の特徴からP-loop型NTPaseであると推測されている。 … しかし、他のP-loop型NTPase（Ura6、Adk1）の高発現は酵母に亜ヒ酸耐性を与えなかったこのことから、亜ヒ酸毒性軽減作用はすべてのP-loop型NTPaseに共通した機能ではなく、Fap7に特有のものであると考えられる。 …
NAID 130005008697

Identification of Salt-Inducible Peptide with Putative Kinase Activity in Halophilic Bacterium Virgibacillus halodenitrificans(GENETICS, MOLECULAR BIOLOGY, AND GENE ENGINEERING)

Rafiee Mahmoud-Reza,Sokhansanj Ashrafaddin,Yoosefi Mitra [他],Naghizadeh Mohammad-Ali
Journal of bioscience and bioengineering 104(3), 178-181, 2007-09-25
… Motif analysis of the deduced amino acid sequence indicated that there is a p-loop NTPase domain on the C-terminal of the peptide, which might correlate with its function. …
NAID 110006402607

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