Retroviruses replicate through a DNA intermediate which covalently associates with host chromosomal DNA. Many steps in the synthesis, structure, integration and expression of viral DNA are not completely understood. By using recombinant DNA techniques we wish to study some of these events in avian sarcoma virus-infected cells. The direction and temporal relationship of (-) and (+) strands will be examined by using cloned viral DNA fragments as probes. The structure of various circular DNA intermediates and the role of host cell in the formation of circles will be assessed. Specific nucleotide sequences in the viral DNA that are involved in the integrative recombination will be determined. This will be approached by creating deletions in the inverted repeats of the LTRs. The wild type and mutated DNA will be introduced into permissive cells by gene transfer techniques, virus produced by the cells will be used to re-infect chicken QT6 cells and viral DNA synthesis and integration will be monitored. Whether integration of viral DNA is mandatory for gene expression will also be studied by maintaining viral DNA in an episomal state using bovine papilloma DNA vector. The requirement of a unique sequence in the viral genome, generally referred to as NT (nontranslatable), in virus replication will be studied by constructing deletions and by analyzing for new transcripts specific for this region. The role of TATA box and the upstream regulatory sequences in the LTR on the efficiency of transcription initiation will be determined. These studies will be approached by site directed mutagenesis and by introducing specific deletions using recombinant DNA techniques. More specifically, oligonucleotide primers with altered bases will be synthesized and the restriction fragments containing these base changes will be put back into pATV-8 and the effect of these altered nucleotides on gene expression will be determined. The significance of the U3 sequence downstream from the CAAT box (-170) and upstream from TATA box (-30) will be studied by replacing this sequence with a nonspecific sequence from pBR DNA. Finally, the mechanism by which Pr180 is translated from 35S mRNA will be studied. We would like to determine whether a splice, to remove the amber codon between gag and pol genes, is involved in the genesis of Pr180 or whether other mechanisms such as ribosome stuttering at the gag-pol junction is responsible for the synthesis of low levels of Pr180.
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