title: Polypyrimidine tract-binding protein binds to the complementary strand of the mouse hepatitis virus 3' untranslated region, thereby altering RNA conformation.
abstract: 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.
authors.affiliation: Department of Molecular Microbiology, University of Southern California School of Medicine, Los Angeles, California 90033-1054, USA.
authors.forename: P
authors.initials: P
authors.lastname: Huang
authors.forename: M M
authors.initials: MM
authors.lastname: Lai
mesh_terms: D020413:3' Untranslated Regions / Q000737:chemistry*; D000818:Animals; D003673:Defective Viruses; D051379:Mice; D006517:Murine hepatitis virus / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009690:Nucleic Acid Conformation; D016133:Polymerase Chain Reaction; D038941:Polypyrimidine Tract-Binding Protein; D011233:Precipitin Tests; D018075:RNA, Complementary / Q000737:chemistry / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D012367:RNA, Viral / Q000737:chemistry* / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D012261:Ribonucleoproteins / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic
chemical_list: D020413:3' Untranslated Regions; D018075:RNA, Complementary; D012333:RNA, Messenger; D012367:RNA, Viral; D016601:RNA-Binding Proteins; D012261:Ribonucleoproteins; D038941:Polypyrimidine Tract-Binding Protein