| Human adenovirus type 4 (Ad4), the sole member of subgroup E, contains an open reading frame (ORF) in the E3 region predicted to encode a unique 30 kDa protein (named Ad4E3-30K). Ad4E3-30K is predicted to be an integral membrane protein containing an N-terminal signal sequence, a lumenal domain, a transmembrane domain near the C-terminus, and a 37 amino acid cytoplasmic tail. To determine if this protein is expressed, rabbit polyclonal antisera were raised against 30K-containing fusion proteins expressed in bacteria.;A 30K protein was detected by immunoprecipitation from cell-free translation products and from Ad4-infected A549 cells radiolabeled in the presence of tunicamycin. The protein was detected at only low levels in infected cells. It was not synthesized by an E3-deletion mutant that includes the Ad4E3-30K gene.;This E3-deletion mutant grows as well as wild-type Ad4 in culture. Features of Ad4E3-30K were characterized in different transient expression systems. The protein underwent glycosylation by addition of approximately six asparagine-linked oligosaccharides. These glycans were sensitive to endoglycosidase H, indicating that they were either high-mannose or hybrid types, but not complex types, and that the protein did not pass through the Golgi apparatus. Immunofluorescence staining of transfected cells revealed that Ad4E3-30K was localized primarily in the endoplasmic reticulum and nuclear envelope.;Mutants of Ad4E3-30K in Ad4 displayed a similar pattern as wild type Ad4 in plaque development assays, viral infection, viral growth, cytopathic effect and cytotoxicity in tissue culture.;To determine if Ad4E3-30K has a function similar to that Ad2 ADP plays in mediating exit of virus from infected cells, an 30K recombinant virus (Ad5DeltaE3/30K +) was constructed for comparison with the similar recombinant virus GZ3 (Ad5DeltaE3/ADP+). The 30K recombinant virus didn't show the enhanced cytolysis and cell-to-cell spread seen with Ad5DeltaE3/ADP + recombinant virus. These experimental results suggest that the Ad4E3-30K does not have an ADP-like function. |
