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Structural and functional studies of the 3' untranslated region of mouse hepatitis virus strain JHM genomic RNA
Structural and functional studies of the three' untranslated region of mouse hepatitis virus strain JHM genomic RNA
The 3' untranslated region (3' UTR) of mouse hepatitis virus (MHV) is a crucial cis-acting element for viral replication. It contains two host protein binding elements, 3'+(42) and 3'+(166-129), that were previously identified by our laboratory. One of those elements, 3'+(42), binds to four host proteins, mitochondrial aconitase, mitochondrial-HSP70 (mtHSP70), HSP60 and HSP40 in MHV infected mammalian cells. Site directed mutagenesis followed by defective interfering (DI) RNA replication assays demonstrated that both 3'+(42) and 3'+(166-129) elements are necessary for MHV DI RNA replication. To explore the mechanism of viral RNA-host protein interaction, we probed the secondary structure of the 3' 166 nucleotides of MHV genomic RNA that contains the two host protein elements. Repeated limited ribonuclease digestion assays indicated that multiple stem-loop structures exist in this region. These enzymatic mapping data are supported by phylogenetic analyses and computer aided secondary structure modeling. Comparison of the predicted structures of the 3' terminal 166 nucleotides of MHV and bovine coronavirus revealed the existence of a conserved 23 nucleotide stem-loop structure. Such a structure is required for DI RNA replication. The mutations that disrupted this conserved stem-loop structure decreased the replication of DI RNA to 17% and 30% of wild type, while the complementary mutation that restored this conserved stem-loop structure replicated to 90% of wild type DI RNA levels. Mutagenesis studies were also conducted in the other two stems within the 3' terminal 166 RNA and their functions in DI RNA replication are discussed. The specific interactions of the 3' UTR of MHV RNA and mtHSP70, HSP60 and HSP40 were examined by immunoprecipitation (IP) and RT-PCR assays. Our data demonstrated that mtHSP70, HSP60 and HSP40 specifically interact with the 3' UTR of MHV in MHV infected cell. When IP-RT-PCR was performed using irrelevant monoclonal antibodies, MHV RNA specific sequence was not detected. The highly expressed GAPDH RNA also does not associate with mitochondrial HSP70, HSP60 and HSP40. Further studies of the interaction of HSP40 and 3'+(42) host protein element using fluorescence resonance energy transfer should reveal the in vivo viral RNA-host protein association.
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Includes bibliographical references (leaves 121-155).