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        Construction of Mutations in the Adenovirus Early Region 3 (E3) Transcription Units

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        954
        The E3 transcription unit of the well-studied subgroup C adenoviruses (Ad) (prototypic serotypes 2 [Ad2] and 5 [Ad5]) is located between map units 76–86 ( see Fig. 1 ). The E3 region is surrounded by the genes for virion protein VIII and fiber, and is transcribed off the r-strand. The E3-region transcription unit is complex Extensive splicing controls expression of seven identified E3 proteins, four of which have functions that modify the host immune response to the viral infection (reviewed in ref. 1 ), Table 1 )
        Fig 1  Methods used to insert a mutated E3 region into the viral genome ( A ) Schematic illustrating some of the adenovirus transcription units expressed during early stages of infection ( B ) Construction of E3 mutations Ligation method Cleave cloned Ad2 Eco RI-D fragment containing a mutation with Eco RI, then ligate to Eco RI-cleaved Ad5 TP-DNA complex. Transfect ligation mixture into A549 cells and allow plaques to form. Overlap recombination method. Cotransfect cloned Ad2 Kpn I-A fragment containing a mutation in the E3 region with Eco RI-cleaved Ad5 TP-DNA complex into A549 cells and plaques were allowed to form

        Table 1  Location of the E3 Genes Within the Ad5 and Ad2 Genomic Sequence

        Coding sequence

        Ad5 (2)

        Ad2 (3)

        E3 protein

        start

        stop

        start

        stop

        Function

        12.5 K

        27,858

        28,179

        27,919

        28,220

        Unknown

        67K

        28,547

        28,736

        28,630

        28,819

        Unknown

        gp 19 K

        28,735

        29,215

        28,812

        29,289

        Inhibits Ad-specific cytotoxic T-lymphocyte recognition and killing of the virally infected cell by binding MHC class I molecules and inhibiting their transport to the cell surface

        11.6K(ADP) a

        29,491

        29,770

        29,468

        29,771

        Required for efficient cell lysis and release of adenovirus from infected cells (4,5)

        10.4 K (RID α) b

        29,784

        30,057

        29,781

        30,054

        10.4 K and 14.5 K proteins form a complex that. Prevents TNF cytolysis Clears epidermal growth factor and insulin receptors from the infected cell surface Clears Fas antigen from the infected cell surface (6)

        14.5 K (RIDβ) b

        30,062

        30,458

        30,059

        30,449

         

        14.7 K

        30,453

        30,837

        30,444

        30,828

        Inhibits TNF-mduced apoptosis GTPase binding protein (7)

        a ADP, adenovirus death protein
        b RID, receptor internalization and degradation complex, made up of 10.4-K (RID α ) and 14 5-K (RID β ) proteins
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