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Journal of Virology, December 2008, p. 11927-11938, Vol. 82, No. 23
0022-538X/08/$08.00+0     doi:10.1128/JVI.00924-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Structural Determinant of Human La Protein Critical for Internal Initiation of Translation of Hepatitis C Virus RNA ^,

Tanmoy Mondal,^1, Upasana Ray,^1, Asit Kumar Manna,² Romi Gupta,¹ Siddhartha Roy,² and Saumitra Das^1*

Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560012, India,¹ Indian Institute of Chemical Biology, 4, Raja S. C. Mullick Road, Kolkata 700 032, India²

Received 4 May 2008/ Accepted 16 September 2008

Human La protein has been implicated in facilitating internal ribosome entry site (IRES)-mediated translation of hepatitis C virus (HCV). Earlier, we demonstrated that the RNA recognition motif (RRM) encompassing residues 112 to 184 of La protein [La (112-184)] interacts with the HCV IRES near the initiator AUG codon. A synthetic peptide, LaR2C (24-mer), derived from La RRM (112-184), retains RNA binding ability, competes with La protein binding to the HCV IRES, and inhibits translation. The peptide interferes with the assembly of 48S complexes, resulting in the accumulation of preinitiation complexes that are incompetent for the 60S ribosomal subunit joining. Here, nuclear magnetic resonance spectroscopy of the HCV IRES-bound peptide complex revealed putative contact points, mutations that showed reduced RNA binding and translation inhibitory activity. The residues responsible for RNA recognition were found to form a turn in the RRM (112-184) structure. A 7-mer peptide comprising this turn showed significant translation inhibitory activity. The bound structure of the peptide inferred from transferred nuclear Overhauser effect experiments suggests that it is a β turn. This conformation is significantly different from that observed in the free RRM (112-184) NMR structure, suggesting paths toward a better-stabilized mimetic peptide. Interestingly, addition of hexa-arginine tag enabled the peptide to enter Huh7 cells and showed inhibition of HCV IRES function. More importantly, the peptide significantly inhibited replication of the HCV monocistronic replicon. Elucidation of the structural determinant of the peptide provides a basis for developing small peptidomimetic structures as potent anti-HCV therapeutics.

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* Corresponding author. Mailing address: Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560012, India. Phone: 91 80 2293 2886. Fax: 91 80 2360 2697. E-mail: sdas{at}mcbl.iisc.ernet.in

Published ahead of print on 1 October 2008.

Supplemental material for this article may be found at http://jvi.asm.org/.

Both of these authors contributed equally and should be considered as joint first authors.

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Journal of Virology, December 2008, p. 11927-11938, Vol. 82, No. 23
0022-538X/08/$08.00+0     doi:10.1128/JVI.00924-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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