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Please use this identifier to cite or link to this item: http://repository.iitr.ac.in/handle/123456789/1334
Title: Development of an ELISA assay for screening inhibitors against divalent metal ion dependent alphavirus capping enzyme
Authors: Kaur R.
Mudgal R.
Narwal M.
Tomar S.
Published in: Virus Research
Abstract: Alphavirus non-structural protein, nsP1 has a distinct molecular mechanism of capping the viral RNAs than the conventional capping mechanism of host. Thus, alphavirus capping enzyme nsP1 is a potential drug target. nsP1 catalyzes the methylation of guanosine triphosphate (GTP) by transferring the methyl group from S-adenosylmethionine (SAM) to a GTP molecule at its N7 position with the help of nsP1 methyltransferase (MTase) followed by guanylylation (GT) reaction which involves the formation of m7GMP-nsP1 covalent complex by nsP1 guanylyltransferase (GTase). In subsequent reactions, m7GMP moiety is added to the 5? end of the viral ppRNA by nsP1 GTase resulting in the formation of cap0 structure. In the present study, chikungunya virus (CHIKV) nsP1 MTase and GT reactions were confirmed by an indirect non-radioactive colorimetric assay and western blot assay using an antibody specific for the m7G cap, respectively. The purified recombinant CHIKV nsP1 has been used for the development of a rapid and sensitive non-radioactive enzyme linked immunosorbent assay (ELISA) to identify the inhibitors of CHIKV nsP1. The MTase reaction is followed by GT reaction and resulted in m7GMP-nsP1 covalent complex formation. The developed ELISA nsP1 assay measures this m7GMP-nsP1 complex by utilizing anti-m7G cap monoclonal antibody. The mutation of a conserved residue Asp63 to Ala revealed its role in nsP1 enzyme reaction. Inductively coupled plasma mass spectroscopy (ICP-MS) was used to determine the presence of magnesium ions (Mg2+) in the purified nsP1 protein. The divalent metal ion selectivity and investigation show preference for Mg2+ ion by CHIKV nsP1. Additionally, using the developed ELISA nsP1 assay, the inhibitory effects of sinefungin, aurintricarboxylic acid (ATA) and ribavirin were determined and the IC50 values were estimated to be 2.69 ?M, 5.72 ?M and 1.18 mM, respectively. © 2018 Elsevier B.V.
Citation: Virus Research(2018), 256(): 209-218
URI: https://doi.org/10.1016/j.virusres.2018.06.013
http://repository.iitr.ac.in/handle/123456789/1334
Issue Date: 2018
Publisher: Elsevier B.V.
Keywords: Alphavirus
Chikungunya virus
ELISA
Guanylyltransferase
Methyltransferase
nsP1
ISSN: 1681702
Author Scopus IDs: 57213732771
57195322398
49461618600
57203506001
Author Affiliations: Kaur, R., Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee, 247667, India
Mudgal, R., Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee, 247667, India
Narwal, M., Department of Biotechnology, Indi
Funding Details: This research was supported by Indian Council of Medical Research (ICMR: Ref no. BIC/12(26)/2013). R.K. thanks University Grant Commission (UGC), India and R.M. thanks Council of Scientific and Industrial Research (CSIR), India for providing the financia
Corresponding Author: Tomar, S.; Department of Biotechnology, Indian Institute of Technology RoorkeeIndia; email: shailfbt@iitr.ac.in
Appears in Collections:Journal Publications [BT]

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