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Mutations in CR1 of E1A 12S yield dominant negative suppressors of immortalization and the lytic cycle

A Malladi1, M P Quinlan

  • 1Department of Microbiology and Immunology, University of Tennessee, Memphis 38163, USA.

Virology
|June 23, 1997
PubMed

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.

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