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George J. Poggioli, Jr., M.D., Ph.D.

Tyler Lab


 

THESIS


Complete Title of Thesis:

"Reovirus-lnduced G2/M Phase Cell Cycle Arrest"


Prepared under the direction of: Kenneth L. Tyler, M.D.


SUMMARY

Mammalian reovirus infection results in perturbation of host cell cycle progression. The prototype serotype 3 reovirus strains Abney (T3A) and Dearing (T3D) inhibit cellular proliferation to a greater extent than the prototype serotype 1 reovirus strain Lang (T1L). We now show that reovirus-induced inhibition of cellular proliferation results from a G2/M phase cell cycle arrest. Using TIL x T3D reassortant viruses, we found that strainspecific differences in the capacity to induce G2/M arrest are determined by the viral SI gene. The SI gene is bicistronic, encoding the viral attachment protein σl and the non structural protein σ1s. A σ1s-deficient reovirus strain, T3C84-MA, fails to induce G2/M arrest, indicating that als is required for reovirus-induced G2/M arrest. Expression of σ1s in C127 cells increases the percentage of cells in the G2/M phase of the cell cycle, supporting a role for this protein in reovirus-induced G2/M arrest. The G2 to M transition represents a cell cycle checkpoint that is regulated by the kinase p34cdc2. We now report that infection with T3A, but not T1L, is associated with inhibition and hyperphosphorylation of p34cdc2. The als protein is necessary and sufficient for inhibitory phosphorylation of p34cdc2, since a viral mutant lacking als fails to hyperphosphorylate p34cdc2 and inducible expression of als is sufficient for p34cdc2 hyperphosphorylation. Since reovirus infection is known to activate cellular transcription factors, we investigated alterations in cell cycle related gene expression following HEK293 cell infection using the Affymetrix U95A microarray. Serotype 3 reovirus infection resulted in differential expression of 10 genes classified as encoding proteins functioning at the G1 to S transition, 11 genes classified as encoding proteins functioning at G2 to M transition, and 4 genes classified as encoding proteins functioning at the mitotic spindle checkpoint. Of the differentially expressed genes encoding proteins known to regulate the G2 to M transition, chkl, weel, and GADD45 are known to inhibit cdc2 kinase activity. A hypothetical model describing reovirus-induced inhibition of cdc2 kinase is presented and reovirus-induced perturbation of the G1 to S, G2 to M, and mitotic spindle checkpoints are discussed.