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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,
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.

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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