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|Title:||Construction and characterization of E1B mutants of adenovirus type 12|
Branton, Philip E.
|Abstract:||<p>Early region 1 (E1) of the adenovirus (Ad) genome contains two overlapping transcription units, E1A and E1B, which encode proteins essential for productive viral infection of permissive human cells and for transformation of nonpermissive rodent cells. E1B of Ad12 produces two major proteins of 482 and 163 amino acid residues. termed 482R and 163R (495R and 175R in Ad2, or 496R and 176R in Ad5). Studies with a variety of mutants affecting the 175R, 176R or 163R proteins have indicated that this product is multifunctional and involved in the protection from degradation of viral and cellular DNA and in the determination of the cytopathic phenotype of infected cells. In addition it is required for complete and efficient transformation of cells. However, a recent report in which the mutants with no initiation codon for 163R or with a downstream stop codon in the 163R coding region were found to be capable of transforming primary baby rat kidney cells has seriously questioned the transformation function of 163R. The functions of the E1B 482R protein in Ad12 have also been characterized. This protein is involved in cellular transformation and is required for accumulation of viral late mRNA and proteins while blocking the same processes for cellular mRNAs and proteins. It is also required for tumour induction and plays some role in viral DNA synthesis. To examine the role of the Ad12 E1B 163R protein more thoroughly, in particular to determine the transforming function of the protein, deletion and point mutations were introduced at various positions in the coding sequence of 163R by oligonucleotide-directed mutagenesis, including a mutant that lacks the 163R AUG initiation codon. The results indicated that many of these mutants yielded unstable 163R-related products induced DNA degradation and enhanced cytopathic effect (cyt/deg phenotype) in infected KB cells, and transformed primary rodent cells at efficiencies significantly lower than wild-type Ad12. Deletion of the final 16 residues at the carboxy terminus did not affect the phenotypes. Alteration of residue 105 reduced transforming efficiency significantly, suggesting that this region of 163R is functionally import. Removal of the initiation codon at nucleotide 1541 blocked production of 163R but resulted in a large increase in the synthesis of the E1B 482R protein and a transforming efficiency similar to that of wild-type Ad12. These results suggested that 163R plays a role in transformation but that normal transforming efficiencies can be obtained in its absence if a sufficient quantity of 482R is produced. To define the functional role of Ad12 E1B 163R and 482R in viral gene expression, viral mRNA accumulation from infected cells was assessed by Northern blot analysis. The results demonstrated that the E1B 163R protein does not play a crucial role in modulating viral gene expression. However, a 482R mutant lacking residues 114-155 produced greatly reduced levels of both early (E2B and E4) mRNA and late protein and was defective for viral DNA replication. These results indicated that the 482R protein plays a role in viral early mRNA metabolism resulting in secondary effects on viral DNA replication and viral late protein synthesis.</p>|
|Appears in Collections:||Open Access Dissertations and Theses|
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