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

互联网2014-02-13

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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