Reovirus-induced alteration in expression of apoptosis and DNA repair genes with potential roles in viral pathogenesis

Abstract

Reoviruses are a leading model for understanding cellular mechanisms of virus-induced apoptosis. Reoviruses induce apoptosis in multiple cell lines in vitro, and apoptosis plays a key role in virus-induced tissue injury of the heart and brain in vivo. The activation of transcription factors NF-kappaB and c-Jun are key events in reovirus-induced apoptosis, indicating that new gene expression is critical to this process. We used high-density oligonucleotide microarrays to analyze cellular transcriptional alterations in HEK293 cells after infection with reovirus strain T3A (i.e., apoptosis inducing) compared to infection with reovirus strain T1L (i.e., minimally apoptosis inducing) and uninfected cells. These strains also differ dramatically in their potential to induce apoptotic injury in hearts of infected mice in vivo-T3A is myocarditic, whereas T1L is not. Using high-throughput microarray analysis of over 12,000 genes, we identified differential expression of a defined subset of genes involved in apoptosis and DNA repair after reovirus infection. This provides the first comparative analysis of altered gene expression after infection with viruses of differing apoptotic phenotypes and provides insight into pathogenic mechanisms of virus-induced disease.

FIG. 1.

Expression of genes related to death receptor-mediated apoptotic signaling is altered following reovirus T3A infection. HEK293 cells were either mock (−) or T3A (+) infected at an MOI of 100 PFU per cell. mRNA was collected at 6, 12, and 24 h postinfection and analyzed by semiquantitative RT-PCR for expression of selected transcripts encoding proteins involved in death receptor-mediated apoptotic signaling.

FIG. 2.

Expression of genes related to mitochondrion-, ER stress-, and protease-mediated apoptotic signaling is altered following reovirus T3A infection. HEK293 cells were either mock (−) or T3A (+) infected at an MOI of 100 PFU per cell. mRNA was collected at 6, 12, and 24 h postinfection and analyzed by semiquantitative RT-PCR for expression of selected transcripts encoding proteins involved in mitochondrial (SMN and Par-4) and ER stress (GADD 34)-mediated apoptotic signaling as well as for caspase 3, a cysteine protease involved in apoptosis.

FIG. 3.

Genes encoding DNA repair proteins are differentially expressed following reovirus infection. HEK293 cells were either mock (−) or T3A (+) infected at an MOI of 100 PFU per cell, and mRNA was collected at 6, 12, and 24 h postinfection. HEK293 cells were also infected with T1L at an MOI of 100 PFU per cell, with mRNA collected at 24 h postinfection. Samples were analyzed by semiquantitative RT-PCR for expression of selected transcripts encoding proteins important for DNA repair.

FIG. 4.

SMN expression is increased in reovirus 8B-infected myocardial tissues, coincident with myocardial apoptotic injury. Neonatal Swiss-Webster mice were intramuscularly infected with 1,000 PFU of 8B virus. Myocardial tissues were analyzed on days 1 to 7 postinfection for histologic evidence of myocarditis as well as for expression of SMN by immunohistochemistry, since we have previously shown that apoptotic myocardial injury is detected at 7 days postinfection in this model. SMN protein was detected (brown stain) in infected myocardial tissue beginning on day 7 postinfection (at the time that histologic evidence of myocarditis was detected, within discrete myocardial lesions). Neither SMN nor evidence of myocardial injury was detected at earlier time points postinfection, as demonstrated on days 3 and 5 postinfection. Original magnification, ×40.

FIG. 5.

Schematic of mitochondrion- and death receptor-related transcriptional alterations detected by microarray analysis following reovirus infection. Transcripts that were differentially expressed in HEK293 cells following reovirus infection compared to mock infection are indicated in boldface type. Please see Discussion for details of each indicated transcript.

FIG. 6.

Schematic of ER stress-related transcriptional alterations detected by microarray analysis following reovirus infection. Transcripts that were differentially expressed in HEK293 cells following reovirus infection compared to mock infection are indicated in boldface type. Please see Discussion for details of each indicated transcript.

FIG. 7.

Transcriptional regulation of Bcl-2 modulatory proteins is a central theme in reovirus-induced apoptosis. Expression of eight transcripts encoding proteins known to influence Bcl-2 activity was altered following reovirus infection. The predicted result of the observed transcriptional alterations would be net inhibition of Bcl-2, and thus promotion of apoptosis, in reovirus-infected cells. Please see Discussion for details of each indicated transcript.