There are several RNA-binding proteins, including heterogeneous nuclear ribonucleoprotein (hnRNP A1) and polypyrimidine tract-binding protein (PTB/hnRNP 1), that were initially characterized as nuclear proteins but later found to shuttle between the nucleus and the cytoplasm. These proteins play important roles in RNA splicing shown to interact with RNAs of several RNA viruses including mouse hepatitis virus (MHV), and may possibly be involved in viral RNA transcription and translation in the cytoplasm. It has been hypothesized that the discontinuous transcription of MHV RNA may involve RNA splicing, a function that has never been thought to be present in the cytoplasm before. As nuclear splicing factors, hnRNP A1 and PTB may possess similar functions in the cytoplasm. Thus, the purpose of this proposal is to study the potential roles in splicing of these proteins in the cytoplasm using MHV as a model systems. I will first establish the functional importance of hnRNP A1 and PTB in MHV RNA transcription by immunodepletion of these proteins in an in vitro transcription system. I will then focus my study on the possible cytoplasmic RNA splicing as a mechanism by which hnRNP A1 and PTB regulate MHV RNA transcription. Finally, I will also investigate the effects of PTB and hnRNP A1 and PTB regulate MHV RNA transcription. Finally, I will also investigate the effects of PTB and hnRNP A1 on MHV protein translation. By the completion of the proposed studies, I hope to characterize the functions of hnRNP A1 and PTB in the cytoplasm of MHV-infected cells, which may reflect their normal cytoplasmic functions.
