Independent mutations in Ad2ts111 cause degradation of cellular DNA and defective viral DNA replication

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145

The adenovirus mutant Ad2tslll has been previously shown to contain a mutation in the early region 2A gene encoding the single-stranded-DNA-binding protein that results in thermolabile replication of virus DNA and a mutation in early region 1 that causes degradation of intracellular DNA. A recombinant virus, Ad2cytlO6, has been constructed which contains the Adltslll early region 1 mutation and the wild-type early region 2A gene from adenovirus 5. This virus, like its parent Ad2tslll, has two temperature-independent phenotypes; first, it has the ability to cause an enhanced and unusual cytopathic effect on the host cell (cytocidal [cyt] phenotype) and second, it induces degradation of cell DNA (DNA degradation [deg] phenotype). The mutation responsible for these phenotypes is a single point mutation in the gene encoding the adenovirus early region 1B (ElB) 19,000-molecular-weight (19K) tumor antigen. This mutation causes a change from a serine to an asparagine in the 20th amino acid from the amino terminus of the protein. Three other mutants that affect the E1B 19K protein function have been examined. The mutants Ad21p5 and Ad5dl337 have both the cytocidal and DNA degradation phenotypes (cyt deg), whereas Ad21p3 has only the cytocidal phenotype and does not induce degradation of cell DNA (cyt deg'). Thus, the DNA degradation is not caused by the altered cell morphology. Furthermore, the mutant Ad5dl337 does not make any detectable E1B 19K protein product, suggesting that the absence of E1B 19K protein function is responsible for the mutant phenotypes. A fully functional E1B 19K protein is not absolutely required for lytic growth of adenovirus 2 in HeLa cells, and its involvement in transformation of nonpermissive cells to morphological variants is discussed.

Section: Discussionmentioning

confidence: 81%

Section: Personal Communication) (3)mentioning

confidence: 99%

Section: Personal Communication) (3)mentioning

confidence: 99%

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Mutations in the gene encoding the adenovirus early region 1B 19,000-molecular-weight tumor antigen cause the degradation of chromosomal DNA

White¹,

Grodzicker²,

Stillman³

1984

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145

“…Although little or nothing is known about the mechanism(s) by which this protein acts in the transforming process, the Ad5 55K species is known to interact with the protein encoded by the p53 tumor suppressor gene (47), and this could represent a step in the progression to complete transformation. Likewise, many mutants with alterations in the 19K coding sequence have been shown to be defective for transformation of rat cells in vitro, leading to the general consensus that the E1B 19K protein is also required for the complete transformation of such cells (1,3,5,9,43,52,54). The mechanism by which the 19K protein might act in this process has not yet been made clear, but it is known that in productively infected human cells the main function of the protein is likely to be the protection of the input DNA from nuclease digestion (32).…”

mentioning

confidence: 99%

The E1B 19-kilodalton protein is not essential for transformation of rodent cells in vitro by adenovirus type 5

Telling

Williams

1993

The newly constructed adenovirus type 5 mutant inl carries a single AT base pair insertion immediately after nucleotide position 1715 in the E1B gene sequence which destroys the proximal AUG normally present in E1B messages and prevents production of intact E1B 19-kDa protein in infected cells. We have used inl, variants of inl containing mutant alleles of viral genes known to enhance transformation frequency, and adenovirus type 5 mutant d1337 (S.

“…The mutations in aa 280 and 282 are in a region of the DBP that closely resembles the consensus sequence of a zinc finger (47, 48), a structure thought to be important for protein binding to double-stranded DNA (2). These two mutants, Ad2tslll (aa 280) and Ad2+ NDlts23 (aa 282), both produce DBPs that do not support DNA replication in vitro at the nonpermissive temperature (34,40). The ts23 DBP, however, supports initiation in vitro at the nonpermissive temperature at a level almost equal to that of the wild-type DBP.…”

mentioning

confidence: 99%

Conserved region 3 of the adenovirus type 5 DNA-binding protein is important for interaction with single-stranded DNA

Neale

Kitchingman

1990

The adenovirus-encoded single-stranded DNA-binding protein (DBP) functions in viral DNA replication and several aspects of RNA metabolism. Previous studies (G. A. M. Neale and G. R. Kitchingman, J. Biol. Chem. 264:3153-3159, 1989) have defined three highly conserved regions in the carboxy-terminal domain of the protein (amino acids 178 to 186, 322 to 330, and 464 to 475) that may be involved in the binding of the protein to single-stranded DNA. We examined the role of conserved region 3 (464 to 475) by constructing nine classes of point mutants with from one to four amino acid changes. The point mutants were tested for their ability to assist adeno-associated virus DNA replication. All nine differed from wild-type DBP; seven were essentially nonfunctional, whereas two had 55 and 145%, respectively, of the wild-type DBP helper activity. Three of the mutants were found to be temperature sensitive, with significantly greater helper activity at 33°C than at 37°C. All nine mutants produced essentially wild-type levels of protein. One monoclonal antibody against the DBP, termed 2/4, did not immunoprecipitate the mutant DBPs as well as wild-type DBP, indicating either that the antibody recognized sequences around CR3 or that the conformation of the protein around the epitope recognized by 2/4 had changed. Two of the three temperature-sensitive DBP mutants bound to single-stranded DNA-cellulose with the same affinity as wild-type DBP at 4°C; the remaining mutants all showed reduced affinity. These results demonstrated that many of the residues within conserved region 3 of the DBP are important for interaction of the protein with nucleic acid.