Reovirus-induced alterations in gene expression related to cell cycle regulation

Abstract

Mammalian reovirus infection results in perturbation of host cell cycle progression. Since reovirus infection is known to activate cellular transcription factors, we investigated alterations in cell cycle-related gene expression following HEK293 cell infection by using the Affymetrix U95A microarray. Serotype 3 reovirus infection results in differential expression of 10 genes classified as encoding proteins that function at the G(1)-to-S transition, 11 genes classified as encoding proteins that function at G(2)-to-M transition, and 4 genes classified as encoding proteins that function at the mitotic spindle checkpoint. Serotype 1 reovirus infection results in differential expression of four genes classified as encoding proteins that function at the G(1)-to-S transition and three genes classified as encoding proteins that function at G(2)-to-M transition but does not alter any genes classified as encoding proteins that function at the mitotic spindle checkpoint. We have previously shown that serotype 3, but not serotype 1, reovirus infection induces a G(2)-to-M transition arrest resulting from an inhibition of cdc2 kinase activity. Of the differentially expressed genes encoding proteins regulating the G(2)-to-M transition, chk1, wee1, and GADD45 are known to inhibit cdc2 kinase activity. A hypothetical model describing serotype 3 reovirus-induced inhibition of cdc2 kinase is presented, and reovirus-induced perturbations of the G(1)-to-S, G(2)-to-M, and mitotic spindle checkpoints are discussed.

FIG. 1.

wee1, chk1, and GADD45 transcripts are increased following reovirus infection. (a) HEK293 cells were either mock infected or infected with T3A, T1L, or UV-inactivated T3A at an MOI of 100 PFU per cell. mRNA was collected at 24 h postinfection and analyzed for chk1, wee1, and GADD45 transcripts by RT-PCR to confirm microarray analyses and also to assess the importance of replication competence for the observed changes in gene expression. β-Actin was used as a control in each RT-PCR to ensure matched total mRNA and equivalent amplication conditions for each experimental sample. (b) Quantitative densitometric analysis of specific mRNA abundance in each sample.

FIG. 1.

wee1, chk1, and GADD45 transcripts are increased following reovirus infection. (a) HEK293 cells were either mock infected or infected with T3A, T1L, or UV-inactivated T3A at an MOI of 100 PFU per cell. mRNA was collected at 24 h postinfection and analyzed for chk1, wee1, and GADD45 transcripts by RT-PCR to confirm microarray analyses and also to assess the importance of replication competence for the observed changes in gene expression. β-Actin was used as a control in each RT-PCR to ensure matched total mRNA and equivalent amplication conditions for each experimental sample. (b) Quantitative densitometric analysis of specific mRNA abundance in each sample.

FIG. 2.

Proposed model for serotype 3 reovirus-induced G₂-to-M transition arrest. cdc2 kinase activity is required for entry into mitosis. Active cdc2 kinase is complexed with cyclin B and dephosphorylated at Thr14/Tyr15. Following reovirus infection, cdc2 is inhibited, in part, by phosphorylation. The increase in phosphorylated cdc2 may be due to upregulation of the kinases chk1 and wee1 and/or localization to the nucleus of the phosphatase PP2A, which can inhibit the cdc2-activating phosphatase cdc25C. cdc2 kinase activity may also be inhibited by dissociation of cyclin B by GADD45. Arrows indicate activation; blunted lines indicate inhibition.