Transformation-deficient adenovirus mutant defective in expression of region 1A but not region 1B

Abstract

A adenovirus type 5 host range mutant (hr440) has been isolated which is defective in a splicing event required to generate the middle-sized mRNA from early region 1A. This defect has been ascribed to two adjacent nucleotide changes which lie five and six nucleotides from the 5' splice site for this mRNA (Solnick, Nature 291:508-510, 1981). One of these changes introduces an amber codon into the reading frame of the largest region 1A mRNA, resulting in the production of a truncated polypeptide. Like other region 1A mutants, hr440 is defective in the production of mRNA from early regions 2 and 3, but hr440 is unusual in that transcription from regions 1B and 4 is normal. Furthermore, although region 1B expression is unaffected, hr440 does not transform baby rat kidney cells. Therefore, expression of early region 1B is insufficient for transformation, eliminating the possibility that region 1A is required only to induce such expression.

E1A 12S and 13S of the transformation-defective adenovirus type 12 strain CS-1 inactivate proteins of the RB family, permitting transactivation of the E2F-dependent promoter

“…These proteins are synthesized immediately after viral infection and are predominant in transformed cells. In primary rodent cells, stable E1A protein expression leads to immortalization (18,25) and, in cooperation with the Ad early region 1B (E1B) gene product (53,57) or a cellular oncoprotein, such as Ras (8), to complete oncogenic transformation. Mutational analysis of the sequences of E1A proteins from different serotypes revealed two highly conserved regions, 1 and 2 (CR1 and CR2), which are common in both E1A proteins and the amino terminus responsible for the transforming activity (27,29,54,60).…”

The first exon of region E1a genes of adenoviruses 5 and 12 encodes a separate functional protein domain.

“…This was further supported by the observation that an Adl2 Ela mutant, which carries a frameshift mutation at the 3' end of the first exon of region Ela, affecting only the 13S mRNA product, is transformation deficient, although it activates the Elb promoter at a normal level . The same conclusion followed from experiments with the Ad5 mutant hr440, which also carries a mutation in the 13S Ela transcript and is transformation defective but normally expresses the Elb region (Solnick and Anderson, 1982 which also contains a frameshift mutation close to the 3' end of the first exon, can transform BRK cells (Graham et al, 1978), and, at a slightly elevated temperature, also other primary rat cells (Ho et al, 1982). Despite their structural similarity, the Ela regions of Ad5 and Adl2 differ in certain biological properties, in particular in transformation.…”

Production of a monospecific antiserum against the early region 1A proteins of adenovirus 12 and adenovirus 5 by an adenovirus 12 early region 1A-beta-galactosidase fusion protein antigen expressed in bacteria

“…Not all mutations in the ElA gene of Ad5 exhibit the same pleiotropic effect. For example, the defects contained in the frameshift mutants H5hrl and H5in500 and the nonsense mutant hr440 each prevent full-length synthesis of the larger ElA acidic protein (9,34,40). However, the H5hrl mutant does not synthesize an E1B, E2, E3, or E4 transcript (4), whereas the hr440 mutant expresses E1B and E4 transcripts (40), and the H5in500 mutant produces wild-type levels of E1B and E3 transcripts but low levels of E2 and E4 transcripts (9).…”

“…For example, the defects contained in the frameshift mutants H5hrl and H5in500 and the nonsense mutant hr440 each prevent full-length synthesis of the larger ElA acidic protein (9,34,40). However, the H5hrl mutant does not synthesize an E1B, E2, E3, or E4 transcript (4), whereas the hr440 mutant expresses E1B and E4 transcripts (40), and the H5in500 mutant produces wild-type levels of E1B and E3 transcripts but low levels of E2 and E4 transcripts (9). Taken altogether, these findings indicate that the protein product of the larger ElA transcript, in fact, does modulate expression of other early genes, but that, depending upon the location and nature of the mutation in the ElA DNA, manifestation of this effect will vary.…”

“…In rodent cells, integration of both ElA and E1B genes (0 to 11 map units [mu]) is necessary for complete transformation (18,20,44), but direct linkage of ElA and E1B genes is not necessary (43). An argument for an independent transforming function of ElA can be made from the analysis of the ElA hr mutants hr440 and H5in500, which are capable of transcribing E1B yet are incapable of transformation (9,40).…”