ANALYSIS OF GENE EXPRESSION
FOLLOWING REOVIRUS T3ABNEY INFECTION
USING HIGH DENSITY OLIGONUCLEOTIDE MICROARRAYS
R.L.DeBiasi 1,2, P.Clarke 2, R. Jotte 3,5, G.L. Johnson 3,5, and K.L. Tyler 2,4,
Depts. Pediatric Infectious Diseases 1, Neurology 2, Pharmacology 3, Microbiology and Immunology 4, and Cancer Center 5, University of Colorado Denver, Denver, CO 80262
Reoviruses induce apoptosis in infected host cells, although the signaling pathways by which this occurs are only partially understood. We used Affymetrix cDNA microarrays, containing 7000-12000 human genes, to characterize transcriptional responses of HEK293 human embryonal kidney and H157 lung carcinoma cells to infection with Reovirus T3 Abney (T3A). Total cellular RNA was extracted from T3A infected (MOI 100, 24 hrs) and control, uninfected H157 monolayers . cRNA biotinylated probes were prepared for hybridization to Affymetrix cDNA microarrays, and probe-binding patterns were analyzed to characterize changes in cellular gene expression following reovirus infection. Preliminary analysis indicated that 399 genes were upregulated 2-fold or greater in infected compared to control H157 cells (5% of the screened gene pool). Affected genes encoded proteins including tumor necrosis factor ligand and receptor superfamily members, oncogenes, transcription factors, proteases, protein kinases and phosphatases. 388 genes were downregulated by 2-fold or more, including a variety of genes encoding ribosomal proteins. Stratification of results revealed that 1% of genes screened were up- or down- regulated > 5 fold and 0.1% > 10 fold. We next used Affymetrix 12,000 human genome arrays to compare the transcriptional response of HEK293 cells to T3A versus mock-infection at 6, 12, and 24 hours post-infection. Results of this multi-replicate, time course experiment will be reviewed. High-density oligonucleotide microarrays provide anew method for analyzing gene expression following reovirus infection, and may provide new insights into the intracellular signaling pathways involved in apoptosis and other virus-cell interactions.