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J. Virol., Sep 1996, 5867-5874, Vol 70, No. 9
Copyright © 1996, American Society for Microbiology

Analysis of early region 3 mutants of mouse adenovirus type 1

CW Beard and KR Spindler
Department of Genetics, University of Georgia, Athens 30602, USA.

Early region 3 (E3) of mouse adenovirus type 1 has the potential to produce three proteins which have identical amino termini but unique carboxy-terminal sequences. Three recombinant deletion viruses were constructed so that each could produce only one of the three E3 proteins. A fourth mutant that should produce no E3 proteins was also constructed. These recombinants were able to grow in mouse 3T6 cells and produced wild-type levels of viral mRNAs and proteins except for those specifically deleted by the mutations. Early mRNA production from the mutant viruses was analyzed by reverse transcriptase PCR and confirmed that each deletion mutant would be able to produce only one of the three E3 proteins. Late mRNA production was analyzed by Northern (RNA) blotting and found to be similar in wild-type and mutant viruses. Capsid morphology was unaltered in the mutant viruses as seen by electron microscopy. Immunoprecipitation of E3 proteins from infections of mouse 3T6 cells using an antiserum specific for all three E3 proteins was used to examine the effect of the introduced mutations on protein expression. Two mutants produced only one class of E3 protein as predicted from their specific mutations and mRNA expression profiles. One mutant virus failed to produce any detectable E3 proteins. The predicted E3-null mutant was found to be leaky and could produce low levels of E3 proteins. Outbred Swiss mice were infected with the E3 mutant viruses to determine if the E3 proteins have an effect on the pathogenicity of the virus in mice. All of the mutants showed decreased pathogenicity as determined by increased 50% lethal doses, indicating that the proteins of the E3 region are important determinants of the pathogenesis of mouse adenovirus in its natural host.

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