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

Abstract

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. ), Table 1) Open image in new window 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 EcoRI-D fragment containing a mutation with EcoRI, then ligate to EcoRI-cleaved Ad5 TP-DNA complex. Transfect ligation mixture into A549 cells and allow plaques to form. Overlap recombination method. Cotransfect cloned Ad2 KpnI-A fragment containing a mutation in the E3 region with EcoRI-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 (RIDα) and 14 5-K (RIDβ) proteins