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Title: trans-protease activity and structural insights into the active form of the alphavirus capsid protease
Authors: Aggarwal M.
Dhindwal S.
Kumar P.
Kuhn R.J.
Tomar S.
Published in: Journal of Virology
Abstract: The alphavirus capsid protein (CP) is a serine protease that possesses cis-proteolytic activity essential for its release from the nascent structural polyprotein. The released CP further participates in viral genome encapsidation and nucleocapsid core formation, followed by its attachment to glycoproteins and virus budding. Thus, protease activity of the alphavirus capsid is a potential antialphaviral target to arrest capsid release, maturation, and structural polyprotein processing. However, the discovery of capsid protease inhibitors has been hampered due to the lack of a suitable screening assay and of the crystal structure in its active form. Here, we report the development of a trans-proteolytic activity assay for Aura virus capsid protease (AVCP) based on fluorescence resonance energy transfer (FRET) for screening protease inhibitors. Kinetic parameters using fluorogenic peptide substrates were estimated, and the Km value was found to be 2.63±0.62 ?M while the kcat/Km value was 4.97 × 104M-1 min-1. Also, the crystal structure of the trans-active form of AVCP has been determined to 1.81-Å resolution. Structural comparisons of the active form with the crystal structures of available substrate-bound mutant and inactive blocked forms of the capsid protease identify conformational changes in the active site, the oxyanion hole, and the substrate specificity pocket residues, which could be critical for rational drug design. © 2014, American Society for Microbiology.
Citation: Journal of Virology(2014), 88(21): 12242-12253
Issue Date: 2014
Publisher: American Society for Microbiology
ISSN: 0022538X
Author Scopus IDs: 54411866100
Author Affiliations: Aggarwal, M., Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee, India
Dhindwal, S., Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee, India
Kumar, P., Department of Biotechnology, Indian Institut
Corresponding Author: Tomar, S.; Department of Biotechnology, Indian Institute of Technology RoorkeeIndia
Appears in Collections:Journal Publications [BT]

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