Polypyrimidine-tract-binding protein affects transcription but not translation of mouse hepatitis virus RNA.
Polypyrimidine-tract-binding protein (PTB) has been shown to bind specifically to the 5' ends of mouse hepatitis virus (MHV) RNA and its complementary strand. To further characterize the function of PTB in MHV replication, we generated dominant-negative mutant cell lines that express a full-length PTB or a truncated form of PTB, which includes only the N-terminal half of the protein, retaining its protein-dimerization domain. The truncated form of PTB was localized in the cytoplasm, whereas the full-length PTB was present mainly in the nucleus. The truncated form can interact with the full-length PTB in vitro. We observed that both the full-length and the truncated PTB, when overexpressed, functioned in a dominant-negative manner in MHV replication. However, the truncated form exhibited more severe effects on syncytia formation, virus production, and synthesis of viral RNA and viral proteins. To clarify the precise function of PTB in MHV replication, we dissociated the processes of viral transcription from translation by transfecting different types of MHV defective-interfering (DI) RNA that contain various reporter genes into these stable cell lines. Transcription of the DI RNA during MHV infection was greatly inhibited in these cell lines, indicating that PTB modulates MHV transcription. In contrast, translation of the DI RNA was not affected by PTB depletion in in vitro translation in rabbit reticulocyte lysate or by PTB overexpression in in vivo translation experiments in MHV-infected cells. Given that PTB interacts with the viral N protein, which is one of the components of the MHV replication complex, PTB may exert its function on viral replication/transcription by association with viral RNA as well as other viral and cellular factors in the replication complex.
Choi
Keum S
KS
Department of Molecular Microbiology and Immunology, University of Southern California, Keck School of Medicine, Los Angeles, California 90033, USA.
Huang
Pei yong
Py
Lai
Michael M C
MM
D000818:Animals; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D003673:Defective Viruses; D015536:Down-Regulation; D051379:Mice; D006517:Murine hepatitis virus / Q000235:genetics / Q000502:physiology*; D019251:Nucleocapsid / Q000378:metabolism; D019590:Nucleocapsid Proteins; D038941:Polypyrimidine Tract-Binding Protein / Q000378:metabolism / Q000502:physiology*; D012367:RNA, Viral / Q000096:biosynthesis*; D014158:Transcription, Genetic; D014779:Virus Replication / Q000235:genetics*
D019590:Nucleocapsid Proteins; D012367:RNA, Viral; C099600:nucleocapsid protein, Hepatitis virus; D038941:Polypyrimidine Tract-Binding Protein