An Enzymatic Footprinting Analysis of the Interaction of 40S Ribosomal Subunits with the Internal Ribosomal Entry Site of Hepatitis C Virus

doi:10.1128/JVI.74.14.6242-6250.2000

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Journal of Virology, July 2000, p. 6242-6250, Vol. 74, No. 14
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

An Enzymatic Footprinting Analysis of the Interaction of 40S Ribosomal Subunits with the Internal Ribosomal Entry Site of Hepatitis C Virus

Victoria G. Kolupaeva,¹ Tatyana V. Pestova,¹^,² and Christopher U. T. Hellen¹^,^*

Department of Microbiology and Immunology, State University of New York Health Science Center at Brooklyn, Brooklyn, New York 11203,¹ and A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, 119899 Moscow, Russia²

Received 14 December 1999/Accepted 20 April 2000

Hepatitis C virus translation is initiated on a ~330-nucleotide (nt)-long internal ribosomal entry site (IRES) at the 5' endof the genome. In this process, a 43S preinitiation complex (comprisinga 40S ribosomal subunit, eukaryotic initiation factor 3 (eIF3),and a ternary [eIF2-GTP-initiator tRNA] complex) binds the IRESin a precise manner so that the initiation codon is placed atthe ribosomal P site. This binding step involves specific interactionsbetween the IRES and different components of the 43S complex.The 40S subunit and eIF3 can bind to the IRES independently; previousanalyses revealed that eIF3 binds specifically to an apical halfof IRES domain III. Nucleotides in the IRES that are involvedin the interaction with the 40S subunit were identified by RNasefootprinting and mapped to the basal half of domain III and indomain IV. Interaction sites were identified in locations thathave been found to be essential for IRES function, including (i)the apical loop residues GGG_266-268 in subdomain IIId and (ii)the pseudoknot. Extensive protection from RNase cleavage alsooccurred downstream of the pseudoknot in domain IV, flanking bothsides of the initiation codon and corresponding in length to thatof the mRNA-binding cleft of the 40S subunit. These results indicatethat the 40S subunit makes multiple interactions with the IRESand suggest that only nucleotides in domain IV are inserted intothe mRNA-binding cleft of the 40Ssubunit.

^* Corresponding author. Mailing address: State University of New York Health Science Center at Brooklyn, 450 Clarkson Ave.,Brooklyn, NY 11203. Phone: (718) 270-1034. Fax: (718) 270-2656.E-mail: chellen{at}netmail.hscbklyn.edu.

Journal of Virology, July 2000, p. 6242-6250, Vol. 74, No. 14
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

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