Polypyrimidine tract-binding protein binds to the complementary strand of the mouse hepatitis virus 3 ' untranslated region, thereby altering RNA conformation

Mouse hepatitis virus (MHV) RNA transcription is regulated mainly by the le ader and intergenic (IG) sequences. However, a previous study has shown tha t 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 affect s positive-strand RNA synthesis. In this study, by UV-cross-linking experim ents, we found that several cellular proteins bind specifically to the minu s-strand 350 nucleotides complementary to the 3'-UTR of the viral genome. T he major protein species, p55, was identified as the polypyrimidine tract-b inding protein (PTB, also known as heterogeneous nuclear RNP I) by immunopr ecipitation of the W-cross-linked protein and binding of the recombinant PT B. A strong PTB-binding site was mapped to nt 53 to 149, and another weak b inding 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 redu ced PTB binding in vitro. Furthermore, defective interfering (DI) RNAs harb oring these mutations showed a substantially reduced ability to synthesize subgenomic mRNA. By enzymatic and chemical probing, we found that PTB bindi ng to nt 53 to 149 caused a conformational change in the neighboring RNA re gion. Partial deletions within the PTB-binding sequence completely abolishe d the PTB-induced conformational change in the mutant RNA even when the RNA retained partial PTB-binding activity, Correspondingly, the MHV DI RNAs co ntaining these deletions completely lost their ability to transcribe mRNAs. Thus, the conformational change in the c3'-UTR caused by PTB binding may p lay a role in mRNA transcription.