Characterization of cellular gene expression by coronavirus infection and the role of cellular proteins in coronavirus replication

Murine coronavirus mouse hepatitis virus (MHV) causes encephalitis and demyelination in the central nervous system (CNS) of susceptible rodents. MHV can establish persistent infection in the CNS. Astrocytes are the major target for viral persistence. However, the mechanisms by which astrocytes survive MHV infection and permit viral persistence are not known. We performed DNA microarray analysis on differential gene expression in astrocytoma DBT cells by MHV infection. The expression of substantial numbers of genes was altered by virus infection. In this dissertation, we specifically analyzed the expression of two genes, the pro-apoptotic BNip3 gene and the transcription factor Egr-1, in response to MHV infection. We found that the expression of BNip3 was significantly decreased, while that of Egr-1 was dramatically increased following MHV infection. Interestingly, infection with ultraviolet light-inactivated viruses not only repressed BNip3 expression but also induced Egr-1 expression, indicating that the down-regulation of BNip3 expression and up-regulation of Egr-1 expression do not require virus replication and are mediated during cell entry. Furthermore, we found that over-expression of Egr-1 suppressed the expression of BNip3, suggesting that down-regulation of BNip3 gene expression by MHV infection may be mediated through the induction of Egr-1 expression. Using various inhibitors of mitogen-activated protein kinases (MAPKs), we identified that the extracellular signal-regulated kinase 1/2 (ERK1/2) was involved in the activation of Egr-1 transcription by MHV infection. Moreover, the ERK signal pathway was found to be activated by MHV infection at an early time point of infection. Interestingly, the persistence/demyelinating-positive strains (JHM and A59) induced Egr-1 expression, whereas the persistence/demyelinating-negative strain (MHV-2) did not. These results indicate that there is a correlation between the ability of MHVs to induce Egr-1 expression and their ability to persist in astrocytes and cause CNS demyelination. These results may provide insights into the mechanisms by which MHV evades host antiviral defense and promotes cell survival, thereby allowing its persistence in the host astrocytes. In this dissertation, we also assessed the biological significance of the ERK signal pathway. We showed that Inhibition of ERK pathway by MEK inhibitor UO126 significantly impaired MHV progeny production as compared to the control DMSO treatment. Moreover, the ability of MHV replication was shown to be correlated with the status of ERK1/2 activation in the cells. We showed that UO126 did not affect either the entry of virus into cells or the viral primary translation. However, we demonstrated that UO126 specifically inhibited the viral RNA synthesis, including genomic RNA replication and sub-genomic mRNA transcription. These findings suggest that the ERK signal pathway or other signaling molecules that are affected by UO126 may play an important role in regulation of MHV replication. A new model for MHV persistence in astrocytes is proposed.