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Analysis of early region 3 mutants of mouse adenovirus type 1.

ABSTRACT: 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.

SUBMITTER: Beard CW

PROVIDER: S-EPMC190604 | biostudies-other | 1996 Sep

REPOSITORIES: biostudies-other

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Analysis of early region 3 mutants of mouse adenovirus type 1.

Beard C W CW Spindler K R KR

Journal of virology 19960901 9

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 dele ...[more]

PMID: 8709206

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Project description:Mouse adenovirus type 1 (MAV-1) infects endothelial cells and disrupts the blood-brain barrier (BBB), causing encephalitis in inbred and outbred mice. Using a virus mutant that does not produce the early region 1A protein E1A, we investigated whether the activity of this known viral transcriptional regulator is needed for BBB disruption and other phenotypes associated with encephalitis. The wild-type (wt) virus and E1A mutant virus caused similar levels of permeability of sodium fluorescein in brains of infected mice. In an in vitro assay of BBB integrity, wt and mutant virus caused similar decreases in transendothelial electrical resistance in primary mouse brain endothelial cell monolayers. These results indicate that E1A protein does not contribute to disruption of BBB integrity in animals or cultured cells. Both wt and E1A mutant virus infection of mice led to similar increases in the activity of two matrix metalloproteinases known to correlate with BBB disruption, MMP2 and MMP9, while causing no increase in the steady-state expression of MMP2 or MMP9 mRNA. In contrast, the amount of MMP3 transcripts increased upon infection by both viruses and to a higher level in infections by the mutant virus lacking E1A protein production. There was no difference in the levels of steady-state expression of mRNA for tight junction proteins among mock virus, wt virus, and mutant virus infections. Thus, the MAV-1 E1A protein does not measurably affect BBB integrity in the parameters assayed, although it reduces the amount of MMP3 mRNA steady-state expression induced in brains upon infection. IMPORTANCE Encephalitis can be caused by viruses, and it is potentially life-threatening because of the vital nature of the brain and the lack of treatment options. MAV-1 produces viral encephalitis in its natural host, providing a model for investigating factors involved in development of encephalitis. MAV-1 infection disrupts the BBB and increases activity of matrix metalloproteinases in brains of infected mice. We investigated whether the major transcriptional regulator of adenoviruses, E1A protein, is responsible for any of the specific phenotypes that result from MAV-1 infection. For some of the functions assayed, an E1A mutant virus behaved like wild-type virus. However, expression of mRNA for one matrix metalloproteinase was higher in the virus lacking E1A protein production. This highlights the complex nature of encephalitis and suggests that E1A may have transcriptional effects on host genes important for the development of encephalitis.

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Analysis of early region 3 mutants of mouse adenovirus type 1.

Publications

Analysis of early region 3 mutants of mouse adenovirus type 1.

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