RNA



Ribosomal binding to the internal ribosomal entry site of classical swine fever virus


VICTORIA G.  KOLUPAEVA a1, TATYANA V.  PESTOVA a1a2 and CHRISTOPHER U.T.  HELLEN a1c1
a1 Department of Microbiology and Immunology, State University of New York Health Science Center at Brooklyn, Brooklyn, New York 11203, USA
a2 A.N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, 119899 Moscow, Russia

Abstract

Most eukaryotic mRNAs require the cap-binding complex eIF4F for efficient initiation of translation, which occurs as a result of ribosomal scanning from the capped 5′ end of the mRNA to the initiation codon. A few cellular and viral mRNAs are translated by a cap and end-independent mechanism known as internal ribosomal entry. The internal ribosome entry site (IRES) of classical swine fever virus (CSFV) is [similar]330 nt long, highly structured, and mediates internal initiation of translation with no requirement for eIF4F by recruiting a ribosomal 43S preinitiation complex directly to the initiation codon. The key interaction in this process is the direct binding of ribosomal 40S subunits to the IRES to form a stable binary complex in which the initiation codon is positioned precisely in the ribosomal P site. Here, we report the results of analyses done using enzymatic footprinting and mutagenesis of the IRES to identify structural components in it responsible for precise binding of the ribosome. Residues flanking the initiation codon and extending from nt 363–391, a distance equivalent to the length of the 40S subunit mRNA-binding cleft, were strongly protected from RNase cleavage, as were nucleotides in the adjacent pseudoknot and in the more distal subdomain IIId1. Ribosomal binding and IRES-mediated initiation were abrogated by disruption of helix 1b of the pseudoknot and very severely reduced by mutation of the protected residues in IIId1 and by disruption of domain IIIa. These observations are consistent with a model for IRES function in which binding of the region flanking the initiation codon to the decoding region of the ribosome is determined by multiple additional interactions between the 40S subunit and the IRES.

(Received March 22 2000)
(Revised May 31 2000)
(Accepted August 3 2000)


Key Words: classical swine fever virus; eIF3; enzymatic footprinting; initiation; internal ribosomal entry site; ribosome; translation.

Correspondence:
c1 Reprint requests to: Christopher U.T. Hellen, Department of Microbiology and Immunology, State University of New York Health Science Center at Brooklyn, 450 Clarkson Avenue, Box 44, Brooklyn, New York 11203, USA; e-mail: chellen@netmail.hscbklyn.edu.