Abstract 1043 Cytokines: Intracellular Signalling Platform, Tuesday, 5/4

Following virus infection, host cells produce cytokines including interferons and tumor necrosis factor-a (TNF-a) to act as signalling molecules for cell-to-cell communication and immune defense mobilization. We have recently demonstrated that TNF-a induces PKR kinase, a double-stranded RNA-dependent and virus-inducible kinase, to mediate the apoptopic effects of TNF-a via the induction of tumor suppressor gene p53 (JBC 1998 & PNAS 1996). In view of the antiviral and apoptogenic effects of PKR, we examined whether suppression of PKR expression results in reduced virus-induced cytopathicity and possibly the establishment of persistent viral infection in susceptible cells.

Promonocytic U937 cells were transfected with control and PKR-antisense expression plasmids to generate stable transfectants designated as U9K-C and U9K-A, respectively. Following infection of these cells with highly cytolytic encephalomyocarditis virus (EMCV), the cells were examined for cell viability and viral TCID50 titers, ultrastructural morphology by electron microscopy, DNA fragmentation, and expression of apoptogenic genes including p53 and ICE by RT-PCR assays. We showed that a deficiency of PKR resulted in a delay in virus-induced apoptosis in EMCV-infected U9K-A cells, in contrast to rapid cell lysis in parental and U9K-C cells. Further culturing of the infected cells resulted in establishment of persistent EMCV infection in these cells (U9K-AV2).

We have demonstrated that this was a bona fide persistent infection by the following experiments: the ability of infected cells to propagate as long-term virus-shedding cultures; electron microscopy studies showing presence of intracellular EMCV virions and chromatin condensation; detection of active EMCV transcription by RT-PCR; and demonstration of virus-induced chromosomal DNA fragmentation and sustained expression of apoptogenic p53 and ICE.

These experiments indicate a role for PKR in virus-induced apoptosis and cytopathicity. This virus-PKR interaction may be used as a paradigm to study the interaction of virus with the host self-suicide system and to investigate the mechanisms for the establishment of persistent virus infection.