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Abstract
Early region 3 (E3) of mouse adenovirus type 1 was analyzed and three major classes of early mRNAs were identified; all were approximately 1 kb long, consisted of three exons, and had the same 5$\sp\prime$ and 3$\sp\prime$ ends. The three classes had alternative splicing at the junction between the second and third exon. These E3 mRNAs have the potential to produce three proteins which have identical amino termini but unique carboxy-terminal sequences. An antiserum specific for one of these proteins detected a 14K protein on a Western blot of infected cell lysates. Immunoprecipitation and endoglycosidase H digestion revealed that the 14K protein was a glycoprotein, and we have designated this protein E3 gp11K. We determined that the predicted signal sequence of gp11K was cleaved when the protein was expressed during an infection. Biochemical and immunofluorescence microscopy data indicated that E3 gp11K was localized to the endoplasmic reticulum of infected cells. Biochemical experiments further indicated that gp11K is a peripheral membrane protein.
Three recombinant deletion viruses were constructed each expected to produce only one of the three E3 proteins. A fourth mutant was constructed that was predicted to produce no E3 proteins. The mutant virus early mRNA production was analyzed by reverse transcriptase (RT)-PCR, which confirmed that each deletion mutant produced a single, unique E3 mRNA. Late mRNA production and capsid morphology were found to be similar in wild-type and mutant viruses. The effect of the mutations on protein expression was examined by immunoprecipitation of E3 proteins from infections of mouse 3T6 cells. Two mutants produced only one class of E3 protein as expected 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 produced low levels of E3 proteins. All of the virus mutants showed decreased pathogenicity in adult outbred Swiss mice as determined by increased 50% lethal doses. This indicates that the proteins of the E3 region are important determinants of the pathogenesis of mouse adenovirus in its natural host.
