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Mutations in CR1 of E1A 12S yield dominant negative suppressors of immortalization and the lytic cycle
1Department of Microbiology and Immunology, University of Tennessee, Memphis 38163, USA.
Abstract:
The Adenovirus 5 E1A 12S gene is responsible for the establishment of immortalization of primary cells by Adenovirus. We have identified two mutants of 12S (30K and NTdl814), which, when coexpressed with wild-type 12S in primary baby rat kidney cells, were capable of suppressing the immortalizing function of the wild-type 12S gene, even when the mutant proteins were expressed at levels lower than wild type. 30K and NTdl814 did not affect the ability of the coexpressed 12S to activate the cell cycle, but have a suppressive effect on 12S-induced DNA synthesis and proliferation at late times in the immortalization pathway. Both the dominant negative mutants have a deletion in conserved region (CR)1 in the first exon of E1A, which encompasses one of the pRb-family binding regions. However, the mutants did not effect the binding of cellular proteins to full-length 12S. A suppressive effect on wild-type 12S was not observed with mutants that have lost any other region or function. In addition, expression of 30K, which is equivalent to the protein encoded by the 10S mRNA of E1A, inhibited E1A function in lytic cycle. Thus, loss of the CR1 seems to be a prerequisite for a mutant to have a dominant negative effect on E1A functions.
Insights
Two Adenovirus 5 E1A 12S gene mutants, 30K and NTdl814, suppress cell immortalization. Loss of conserved region 1 in E1A is crucial for this dominant-negative effect on Adenovirus 5 functions.
Area of Science:
- Molecular Biology
- Virology
- Cell Biology
Background:
- Adenovirus 5 E1A 12S gene drives primary cell immortalization.
- Understanding E1A's role is key to controlling viral oncogenesis.
Purpose of the Study:
- Identify and characterize dominant-negative mutants of Adenovirus 5 E1A 12S.
- Determine the structural requirements for E1A's dominant-negative activity.
Main Methods:
- Co-expression of wild-type 12S with mutant 30K and NTdl814 in primary baby rat kidney cells.
- Analysis of cell cycle activation, DNA synthesis, and proliferation.
- Mapping deletions in conserved regions (CR) of E1A mutants.
- Assessment of pRb-family binding and other cellular protein interactions.
Main Results:
- Mutants 30K and NTdl814 exhibit dominant-negative suppression of wild-type 12S immortalizing function, even at lower expression levels.
- These mutants do not impede cell cycle activation but suppress late-stage DNA synthesis and proliferation.
- Deletions in conserved region 1 (CR1) encompassing a pRb-family binding site are essential for dominant-negative activity.
- Mutants lacking other regions or functions did not show suppressive effects.
- 30K also inhibited E1A function during the lytic cycle.
Conclusions:
- Conserved region 1 deletion is a prerequisite for dominant-negative effects on E1A functions.
- Specific structural alterations in E1A can inhibit Adenovirus-mediated cell immortalization and lytic functions.