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. 1998 Aug;72(8):6325-31.
doi: 10.1128/JVI.72.8.6325-6331.1998.

Mouse adenovirus type 1 early region 1A is dispensable for growth in cultured fibroblasts

B Ying  1 , K Smith, K R Spindler
Affiliations

Mouse adenovirus type 1 early region 1A is dispensable for growth in cultured fibroblasts

B Ying et al. J Virol. 1998 Aug.

Abstract

Mouse adenovirus type 1 (MAV-1) mutants with deletions of conserved regions of early region 1A (E1A) or with point mutations that eliminate translation of E1A were used to determine the role of E1A in MAV-1 replication. MAV-1 E1A mutants expressing no E1A protein grew to titers comparable to wild-type MAV-1 titers on mouse fibroblasts (3T6 fibroblasts and fibroblasts derived from Rb+/+, Rb+/-, and Rb-/- transgenic embryos). To test the hypothesis that E1A could induce a quiescent cell to reenter the cell cycle, fibroblasts were serum starved to stop DNA replication and cellular replication and then infected with the E1A mutant and wild-type viruses. All grew to equivalent titers. Steady-state levels of MAV-1 early mRNAs (E1A, E1B, E2, E3, and E4) from 3T6 cells infected with wild-type or E1A mutant virus were examined by Northern analysis. Steady-state levels of mRNAs from the mutant-infected cells were comparable to or greater than the levels found in wild-type virus infections for most of the early regions and for two late genes. The E2 mRNA levels were slightly reduced in all mutant infections relative to wild-type infections. E1A mRNA was not detected from infections with the MAV-1 E1A null mutant, pmE109, or from infections with similar MAV-1 E1A null mutants, pmE112 and pmE113. The implications for the lack of a requirement of E1A in cell culture are discussed.

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Figures

FIG. 1
FIG. 1
Expression of E1A protein in wt- and mutant-infected mouse 3T6 cells. Mouse 3T6 cells were infected with either wt virus (pmE301) or the indicated mutant virus at an MOI of 5, or they were mock infected. [35S]methionine- and [35S]cysteine-labeled infected cell lysates were harvested at 22 h (early [E]) and 40 h (late [L]) p.i. and immunoprecipitated with an anti-MAV-1 E1A antibody (E1A) or preimmune serum (pre). Immunoprecipitated products were electrophoresed on an SDS–12% polyacrylamide gel followed by phosphorimager analysis. Marker designates 14C-labeled protein standards, whose sizes are indicated on the left.
FIG. 2
FIG. 2
Growth curve of wt and mutant viruses on mouse fibroblasts. Mouse 3T6 or 37.1 cells were infected with the indicated viruses at an MOI of 5. Virus growth was monitored through 96 h p.i. by determining titers on 37.1 cells. Two assays of the titers at the 96-h time point were done, and the standard error bars determined from multiple plaque assay plates are indicated. The standard errors for the stocks grown on 3T6 cells are not visible because they are so small, ranging from 2.0 × 106 to 1.4 × 107.
FIG. 3
FIG. 3
Virus titers on serum-starved mouse cell lines. Monolayers of mouse 3T6 cells or secondary cells derived from embryos of Rb+/+, Rb+/−, or Rb−/− knockout mice were serum starved for 3 days prior to infection. Virus titers were determined on day 3 p.i. for wt and mutant MAV-1. The cell type is given in the upper left corner of each panel. Two independent Rb+/− cell strains and two independent Rb−/− cell strains were tested.
FIG. 4
FIG. 4
Northern analysis of MAV-1 early mRNAs from wt- or mutant-infected 3T6 cells. Total cytoplasmic RNA was isolated at 20 h p.i. from mouse 3T6 cells infected with wt virus and each of the indicated E1A mutant viruses at an MOI of 5. Steady-state levels of E1A, E1B, E2, E3, and E4 mRNAs were determined by using gene-specific probes and were normalized to 1 for wt levels. The results represent the means of three independent experiments.
FIG. 5
FIG. 5
Viral and host protein production at early (22 h p.i.) and late (40 h p.i.) times of infection of 3T6 cells with wt or mutant virus. [35S]methionine- and [35S]cysteine-labeled cellular extracts were harvested from mock-, wt-, or mutant-infected cells at 22 or 40 h p.i. as indicated and analyzed by SDS-PAGE (10% polyacrylamide gel). The migration of protein size markers is indicated on the right. Several virus-infected cell-specific proteins are indicated by the arrows at the left.
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