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Molecular and Cellular Biology, March 2000, p. 1583-1595, Vol. 20, No. 5
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Transient Expression of Cellular Polypyrimidine-Tract Binding Protein Stimulates Cap-Independent Translation Directed by Both Picornaviral and Flaviviral Internal Ribosome Entry Sites In Vivo

Rainer Gosert,1 Ki Ha Chang,1,dagger Rene Rijnbrand,2 MinKyung Yi,2 David V. Sangar,2 and Stanley M. Lemon1,2,*

Department of Medicine, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7030,1 and Department of Microbiology and Immunology, The University of Texas Medical Branch, Galveston, Texas 77555-10192

Received 23 August 1999/Returned for modification 29 October 1999/Accepted 30 November 1999

The regulation of cap-independent translation directed by the internal ribosome entry sites (IRESs) present in some viral and cellular RNAs is poorly understood. Polypyrimidine-tract binding protein (PTB) binds specifically to several viral IRESs. IRES-directed translation may be reduced in cell-free systems that are depleted of PTB and restored by reconstitution of lysates with recombinant PTB. However, there are no data concerning the effects of PTB on IRES-directed translation in vivo. We transfected cells with plasmids expressing dicistronic transcripts in which the upstream cistron encoded PTB or PTB deletion mutants (including a null mutant lacking amino acid residues 87 to 531). The downstream cistron encoded a reporter protein (chloramphenicol acetyltransferase [CAT]) under translational control of the poliovirus IRES which was placed within the intercistronic space. In transfected BS-C-1 cells, transcripts expressing wild-type PTB produced 12-fold more reporter protein than similar transcripts encoding the PTB null mutant. There was a 2.4-fold difference in CAT produced from these transcripts in HeLa cells, which contain a greater natural abundance of PTB. PTB similarly stimulated CAT production from transcripts containing the IRES of hepatitis A virus or hepatitis C virus in BS-C-1 cells and Huh-7 cells (37- to 44-fold increase and 5 to 5.3-fold increase, respectively). Since PTB had no quantitative or qualitative effect on transcription from these plasmids, we conclude that PTB stimulates translation of representative picornaviral and flaviviral RNAs in vivo. This is likely to reflect the stabilization of higher ordered RNA structures within the IRES and was not observed with PTB mutants lacking RNA recognition motifs located in the C-terminal third of the molecule.

* Corresponding author. Mailing address: Department of Microbiology and Immunology, The University of Texas Medical Branch, 301 University Blvd., Galveston, Texas 77555-1019. Phone: (409) 772-2324. Fax: (409) 772-3757. E-mail: smlemon{at}utmb.edu.

dagger Deceased.

Molecular and Cellular Biology, March 2000, p. 1583-1595, Vol. 20, No. 5
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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