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Interaction of mouse adenovirus type 1 early region 1A protein with cellular proteins pRb and p107
Abstract:
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 SAOS-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 E1A 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 CR3 delta 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-infected 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 vitro coimmunoprecipitation experiments. This is consistent with E1A-p107 complexes forming in wild-type MAV-1-infected cells.
Insights
Mouse adenovirus type 1 (MAV-1) early region 1A (E1A) protein functionally associates with mouse retinoblastoma protein (pRb) and p107. E1A binding to pRb and p107 is crucial for MAV-1
Area of Science:
- Virology
- Molecular Biology
- Cell Biology
Background:
- Mouse adenovirus type 1 (MAV-1) is a significant viral pathogen.
- The early region 1A (E1A) protein of adenoviruses plays a critical role in viral replication and cellular transformation.
- Retinoblastoma protein (pRb) and pRb-related proteins (like p107) are key regulators of the cell cycle.
Purpose of the Study:
- To investigate the functional interactions between MAV-1 E1A protein and mouse pRb and p107.
- To determine the regions of E1A responsible for these interactions.
- To elucidate the impact of these interactions on cellular processes.
Main Methods:
- Coimmunoprecipitation assays using MAV-1 infected cell lysates and specific antibodies.
- Analysis of wild-type and mutant MAV-1 viruses (dIE105, dIE106, dIE102, pmE109).
- Cellular phenotype analysis in SAOS-2 cells upon transfection with mouse pRb and MAV-1 E1A.
- Mobility shift assays to detect E2F protein-DNA complexes.
Main Results:
- MAV-1 E1A coimmunoprecipitated with mouse pRb and p107 in infected cells.
- Mutant viruses with deletions in CR1 and CR3 regions showed reduced binding to pRb.
- MAV-1 E1A expression reduced the pRb-induced flat cell phenotype, indicating pRb inactivation.
- E1A-p107 complex formation in infected cells correlated with the absence of p107-E2F complexes.
Conclusions:
- MAV-1 E1A protein directly interacts with mouse pRb and p107.
- The CR1 and CR3 regions of E1A are important for pRb binding and inactivation.
- E1A-p107 complex formation in infected cells disrupts p107-E2F complexes, suggesting a role in cell cycle regulation.