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Journal of Virology, November 1999, p. 9110-9116, Vol. 73, No. 11
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Polypyrimidine Tract-Binding Protein Binds to the Complementary Strand of the Mouse Hepatitis Virus 3' Untranslated Region, Thereby Altering RNA Conformation

Peiyong Huang1 and Michael M. C. Lai1,2,*

Department of Molecular Microbiology and Immunology1 and Howard Hughes Medical Institute,2 University of Southern California School of Medicine, Los Angeles, California 90033-1054

Received 23 February 1999/Accepted 28 June 1999

Mouse hepatitis virus (MHV) RNA transcription is regulated mainly by the leader and intergenic (IG) sequences. However, a previous study has shown that the 3' untranslated region (3'-UTR) of the viral genome is also required for subgenomic mRNA transcription; deletion of nucleotides (nt) 270 to 305 from the 3'-UTR completely abolished subgenomic mRNA transcription without affecting minus-strand RNA synthesis (Y.-J. Lin, X. Zhang, R.-C. Wu, and M. M. C. Lai, J. Virol. 70:7236-7240, 1996), suggesting that the 3'-UTR affects positive-strand RNA synthesis. In this study, by UV-cross-linking experiments, we found that several cellular proteins bind specifically to the minus-strand 350 nucleotides complementary to the 3'-UTR of the viral genome. The major protein species, p55, was identified as the polypyrimidine tract-binding protein (PTB, also known as heterogeneous nuclear RNP I) by immunoprecipitation of the UV-cross-linked protein and binding of the recombinant PTB. A strong PTB-binding site was mapped to nt 53 to 149, and another weak binding site was mapped to nt 270 to 307 on the complementary strand of the 3'-UTR (c3'-UTR). Partial substitutions of the PTB-binding nucleotides reduced PTB binding in vitro. Furthermore, defective interfering (DI) RNAs harboring these mutations showed a substantially reduced ability to synthesize subgenomic mRNA. By enzymatic and chemical probing, we found that PTB binding to nt 53 to 149 caused a conformational change in the neighboring RNA region. Partial deletions within the PTB-binding sequence completely abolished the PTB-induced conformational change in the mutant RNA even when the RNA retained partial PTB-binding activity. Correspondingly, the MHV DI RNAs containing these deletions completely lost their ability to transcribe mRNAs. Thus, the conformational change in the c3'-UTR caused by PTB binding may play a role in mRNA transcription.

* Corresponding author. Mailing address: Howard Hughes Medical Institute, Department of Molecular Microbiology and Immunology, University of Southern California School of Medicine, 2011 Zonal Ave., HMR-401, Los Angeles, CA 90033-1054. Phone: (323) 442-1748. Fax: (323) 342-9555. E-mail: michlai{at}hsc.usc.edu.

Journal of Virology, November 1999, p. 9110-9116, Vol. 73, No. 11
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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