Interaction of mouse adenovirus type 1 early region 1A protein with cellular proteins pRb and p107

We demonstrated functional associations between mouse adenovirus type 1 (MAV-1) early region 1A (E1A) protein and both the mouse retinoblastoma protein (pRb) and the mouse pRb-related protein, p107. Interactions between MAV-1 E1A and mouse pRb or mouse p107 proteins were examined in infected cell lysates using a mouse embryonic fibroblast cell line infected with wild-type and mutant MAV-1 viruses. Using a polyclonal antibody to MAV-1 E1A, exogenously added mouse pRb or mouse p107 was coimmunoprecipitated from wild-type-, dIE105 (CR1 Delta)-, and dIE106 (CR3 Delta)-infected cell lysates. No coimmunoprecipitation was seen with cell lysates from dIE102 (CR2 Delta) or pmE109, a mutant virus that produces no detectable E1A protein due to an ATG to TTG point mutation in the initiator methionine. Introduction of mouse pRb into SAGS-2 cells resulted in a flat and enlarged cell phenotype, whereas cotransfection of mouse pRb and MAV-1 E1A resulted in a significant reduction of flat cells, presumably due to El A binding pRb, CR1 Delta and CR2 Delta E1A proteins were less effective at reducing the number of flat, enlarged cells induced by pRb expression than were the CR3a or wild-type E1A proteins. The reduced ability of these mutants to inactivate pRb relative to wild-type E1A correlated with their reduced ability to bind pRb in the in vitro coimmunoprecipitation experiments. As a measure of p107/MAV-1 E1A complex formation in MAV-1-infected cells, we used mobility shift assays to examine cell extracts for the presence of p107-containing E2F protein-DNA complexes, Mock-, dIE102-, and pmE109-iniected cell extracts formed a p107-containing complex, whereas wild-type-infected cell extracts did not. Thus the formation of a p107-E2F complex in wild-type- or these mutant-infected extracts inversely correlated with the presence of E1A-p107 complexes identified in the in vitro coimmunoprecipitation experiments. This is consistent with E1A-p107 complexes forming in wild-type MAV-1-infected cells. (C) 1996 academic Press, Inc.