Oncolytic ElB-55K-mutant adenoviruses have entered phase III clinical trials. However, the basis for their selective replication in tumor cells is poorly understood. Work of this lab has shown that ElB-55K-mutant viruses selectively replicate in and kill S phase cells better than G1 cells; this is likely to account for the selective replication in tumors. This research seeks to understand the tumor-selective nature of the E1B- 55K-mutant by understanding its S phase-selective and G1-restricted nature. Results from the previous period showed that late viral gene expression is suppressed in G1 cells at the level of translation, apparently in response to the E4orf1 or E4orf2 genes. The polysomal distribution of late viral mRNA in S phase and G1 cells infected with ElB-55K-mutant viruses suggests that late viral translation is restricted in G1 cells at the level of ribosome shunting. The E1B-55K protein of the wild-type virus promotes viral translation in association with the E4orl""""""""6 protein and host cell factors. A candidate host cell factor has been mapped to the RUNX1 gene.
Three specific aims are proposed in this application.
Aim 1 seeks to determine how late viral translation is suppressed in G1-infected cells by comparing viral mRNA synthesis/ed in G1- and S phase-infected cells and by measuring the rates of key translational steps in synchronized and infected cells. This will test the hypothesis that ribosome shunting is decreased in the G1-infected cell.
Aim 2 will elucidate the role of E4orfl and E4orf2 in attenuating late viral translation and restricting virus growth in G1. Viruses defective in either E4orlT or E4orf2 and the E1B-55K genes will be prepared and evaluated with respect to the cell cycle-restriction. Translation directed by ribosome scanning and ribosome shunting will he compared in cells infected with cell cycle-restricted and non-restricted mutant viruses to test the hypothesis that E4orf 1 attenuates translation at the level of ribosome shunting and that this is the basis for the G1-restriction.
Aim 3 will identify the RUNX1 isoform important for E1B-55K function and determine if its activity during an infection is cell cycle-dependent. Candidate isoforms will be over expressed to determine if they compensate for the loss of E1B-55K function and RNA interference will be used to ablate RUNX1 expression to determine the importance of RUNX1 to adenovirus infection. ? ? ?
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