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 retinoblast oma protein (pRb) and the mouse pRb-related protein, p107. Interaction s 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 c oimmunoprecipitated from wild-type-, dIE105 (CR1 Delta)-, and dIE106 ( CR3 Delta)-infected cell lysates. No coimmunoprecipitation was seen wi th cell lysates from dIE102 (CR2 Delta) or pmE109, a mutant virus that produces no detectable E1A protein due to an ATG to TTG point mutatio n in the initiator methionine. Introduction of mouse pRb into SAGS-2 c ells resulted in a flat and enlarged cell phenotype, whereas cotransfe ction of mouse pRb and MAV-1 E1A resulted in a significant reduction o f flat cells, presumably due to El A binding pRb, CR1 Delta and CR2 De lta E1A proteins were less effective at reducing the number of flat, e nlarged cells induced by pRb expression than were the CR3a or wild-typ e E1A proteins. The reduced ability of these mutants to inactivate pRb relative to wild-type E1A correlated with their reduced ability to bi nd 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 p10 7-containing E2F protein-DNA complexes, Mock-, dIE102-, and pmE109-ini ected cell extracts formed a p107-containing complex, whereas wild-typ e-infected cell extracts did not. Thus the formation of a p107-E2F com plex in wild-type- or these mutant-infected extracts inversely correla ted with the presence of E1A-p107 complexes identified in the in vitro coimmunoprecipitation experiments. This is consistent with E1A-p107 c omplexes forming in wild-type MAV-1-infected cells. (C) 1996 academic Press, Inc.