Shuffling adenovirus promoters: a viral recombinant with early region 1A under late transcriptional control.

Solnick, David

doi:10.1002/j.1460-2075.1983.tb01512.x

Cited by 8 publications

(4 citation statements)

References 46 publications

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“…Similar conclusions have been drawn by others, studying genes encoding ETa and SV40 Tag (5,6). This observation suggested that translation of late mRNAs in vivo might require sequences from the second and third leaders.…”

Section: Introductionsupporting

confidence: 90%

Effect of the tripartite leader on synthesis of a non-viral protein in an adenovirus 5 recombinant

Berkner

Sharp

1985

Nucl Acids Res

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The EIa region of an Adenovirus 5 recombinant has been substituted by a modular gene encoding dihydrofolate reductase (DHFR). In this recombinant, the mouse DHFR cDNA was positioned behind sequences of the major late promoter and the complete tripartite leader. The leader sequences end in the normal 5' splice site (SS) of the third leader, so that RNA splicing joins the tripartite leader to a 3' splice site immediately upstream of the DHFR cDNA. At late stages of infection, high levels of DHFR mRNAs were synthesized. At early times in the late stage, this mRNA was efficiently translated; however, at later times translation of DHFR decreased probably due to poor competition with other late mRNAs. Synthesis of DHFR protein from an analogous Adenovirus 5 recombinant containing only the first late leader was studied in parallel. Equivalent levels of DHFR mRNA were expressed after infection with this recombinant virus; however, the efficiency of DHFR translation was at least 20 fold lower than that of the DHFR mRNA containing the tripartite leader. This suggests that the tripartite leader sequence is important for translation in the late stage of infection. As reported previously, the Ad5 recombinant containing only the first leader vastly overexpresses polypeptide IX from a novel mRNA, formed by the splicing of the first leader in the modular DHFR gene to the 3' splice site in the EIb region. Cells infected with this recombinant synthesize very little normal mRNA from the EIb region. Here, we demonstrated that coinfection of 293 cells with this recombinant and wild type Adenovirus 5 also results in decreased EIb mRNA synthesis. We propose that the overproduction of polypeptide IX suppresses mRNA expression from the EIb and IX promoter sites, probably by an autoregulation loop active during lytic growth.

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Section: Introductionsupporting

confidence: 90%

Effect of the tripartite leader on synthesis of a non-viral protein in an adenovirus 5 recombinant

Berkner

Sharp

1985

Nucl Acids Res

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“…Most late adenovirus mRNAs have the tripartite leader segments spliced to their 5' termini, and this common element might contain the signals for preferential translation. We, and others, have shown that synthesis of high levels of mRNAs without the tripartite leader in late-infected cells results in synthesis of only low levels of the corresponding polypeptide (7, 38,43). For example, mRNAs with only the first leader, or the first and second leader, have been shown to be inefficiently translated at late-infection times.…”

Section: Discussionmentioning

confidence: 95%

“…Most of the late mRNAs possess a common tripartite leader at their 5' termini which may contain sequences important for efficient translation at late times (4,12). We, and others, have described recombinants in which foreign genes were inserted downstream of the adenovirus type 2 (Ad2) MLP (7,8,17,21,23,38,43). Many of these constructs included the tripartite leader, which apparently increased the efficiency of translation.…”

mentioning

confidence: 99%

Abundant expression of polyomavirus middle T antigen and dihydrofolate reductase in an adenovirus recombinant

Berkner¹,

Schaffhausen²,

Roberts³

et al. 1987

J Virol

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A modular gene with a cDNA encoding the polyomavirus middle T antigen positioned behind the adenovirus type 2 major late promoter and tripartite leader was substituted for the Ela region in an adenovirus vector. Permissive human cells infected with this recombinant produce middle T protein at levels as high as those of the most abundant late adenoviral proteins, e.g., hexon or fiber. This level represents at least a 40-fold increase over that observed in a polyomavirus lytic infection of murine cells. Partial proteolytic mapping showed that this protein has the same primary structure as middle T protein produced in polyomavirus-infected murine cels. The adenovirus recombinant-generated middle T protein exhibited in vitro kinase activity, although at an approximately 10-fold-lower specific activity than that of middle T protein from polyomavirus-infected murine cells. Comparison of the expression levels of this middle T antigen-containing adenovirus vector with a similar construction encoding dihydrofolate reductase suggested that the translation efficiency of the inserted gene was dependent upon the proximity of its initiation codon to the tripartite leader. We tested this possibility by comparing three dihydrofolate reductase recombinants among which the spacing between the initiation codon and tripartite leader varied from 188 to 36 nucleotides. The efficiency of expression of dihydrofolate reductase protein dramatically increased as this spacing was reduced. MATERIALS AND METHODS Cells and viruses. Wild-type py NG59RA used for controls was derived from strain NG59 by marker rescue (3, 16).

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“…Adenovirus recombinants. Adenovirus-SV40 recombinants containing the gene for T antigen (mutant or wild type) were generated by cotransfection of KpnI-linearized pSKAT and XbaI fragment A of adenovirus type 5 d1309 (21) (map units 4 to 100) in adenovirus-transformed 293 cells as described previously (3,25,42,45). Transfections were carried out by using a CaPO4 precipitation technique (6).…”

Section: Methodsmentioning

confidence: 99%

Four major sequence elements of simian virus 40 large T antigen coordinate its specific and nonspecific DNA binding

Simmons

Loeber

Tegtmeyer

1990

J Virol

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By mutational analysis, we have identified a motif critical to the proper recognition and binding of simian virus 40 large tumor antigen (T antigen) to virus DNA sequences at the origin of DNA replication. This motif is tripartite and consists of two elements (termed A1 and B2) that are necessary for sequence-specific binding of the origin and a central element (B1) which is required for nonspecific DNA-binding activity. Certain amino acids in elements A1 (residues 152 to 155) and B2 (203 to 207) may make direct contact with the GAGGC pentanucleotide sequences in binding sites I and II on the DNA. Alternatively, these two elements could determine the proper structure of the DNA-binding domain, although for a number of reasons we favor the first possibility. In contrast, element B1 (183 to 187) is most likely important for recognizing a general structural feature of DNA. Elements A1 and B2 are nearly identical in all known papovavirus T antigens, whereas B1 is identical only in the closely related papovaviruses simian virus 40, BK virus, and JC virus. In addition to these three elements, a fourth (B3; residues 215 to 219) is necessary for the binding of T antigen to site II but not to site I. We propose that additional contact sites on T antigen are involved in the interaction with site II to initiate the replication of the viral DNA.

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Shuffling adenovirus promoters: a viral recombinant with early region 1A under late transcriptional control.

Cited by 8 publications

References 46 publications

Effect of the tripartite leader on synthesis of a non-viral protein in an adenovirus 5 recombinant

Effect of the tripartite leader on synthesis of a non-viral protein in an adenovirus 5 recombinant

Abundant expression of polyomavirus middle T antigen and dihydrofolate reductase in an adenovirus recombinant

Four major sequence elements of simian virus 40 large T antigen coordinate its specific and nonspecific DNA binding

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