The role of Ad E1 proteins in determining host range differences exemplified by abortive infection will be addressed in this proposal. Interactions between Ad12 E1 proteins, viral DNA and cellular proteins will be characterized in hamster cells in which Ad12 infection is usually blocked.
Specific Aim 1 will determine whether the concentration of E1 proteins is critical for allowing productive Ad12 infection of hamster cells. This goal will be accomplished by cloning the rat metallothionein or other inducible promoter upstream of the Ad12 E1A and E1B genes; BHK cells will be transformed with this gene construct and a stable cell line isolated. This cell line will then be induced to express E1 proteins and infected with Ad12 virus to test the hypothesis. If increased E1 protein does result in productive infection, then in Specific Aim 2, promoter deletions or hybrid Ad12/Ad5 E1 promoters will be used to increase the level of E1 protein and the relative functions of wild type and mutated promoters will be assessed using a CAT reporter gene transfected into KB and BHK cells. If the concentration of Ad12 E1 proteins does not overcome the block to virus replication in Specific Aim 1, then chemical mutagenesis mutated genes will be recombined back into Ad12 DNA, to identify mutations that might allow productive Ad12 infection of hamster cells.
