Student Theses and Dissertations

Date of Award

1983

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

RU Laboratory

Hanafusa Laboratory

Abstract

Integration of Rous sarcoma virus DNA into its host genome was analyzed under conditions where secondary integration via virus spread was inhibited. This was accomplished by using the noninfectious pol- ,env- alpha variant of the Bryan high titer strain of Rous sarcoma virus (BH-RSV). Twelve independent BHRSV- transformed chicken embryo fibroblast clones were obtained and the provirus-cell junction fragments were mapped by restriction endonuclease cleavage and Southern blotting analyses. We found that expression of the viral genes could occur after proviral integration at many sites on the chicken genome and that there was no apparent preference for specific integration sites. BH-RSV DNA was further analyzed in order to precisely determine the defects in this strain. A 5kb EcoRI fragment which contained the entire pol and src genes was molecularly cloned from integrated proviruses of BH-RSV alpha and its pol+ parent BH-RSV beta. DNA sequencing of the pol-src junction of BH-RSV revealed that the env sequence was almost entirely absent; only 6 bp following the pol stop codon remained. Starting at position 7 (relative to the end of pol), a 91 bp sequence identical to the 91 bp immediately upstream from src in other strains of Rous sarcoma virus (RSV) was found. This was followed by the src coding sequence. It appears remarkable that in all RSV strains, including this defective BH-RSV, as well as in cellular-src DNA, these 91 bp are conserved. The helper virus-related sequence of about 100 bp, which is present as a direct repeat in the 5' and 3' sides of src in other RSVs, was present only on the 3' side of src in BH-RSV. The sequence of about 100 nucleotides immediately following src in BH-, PR-, and SR-RSV seems to be completely unconserved. Restriction enzyme mapping analysis showed the structure of the BH-RSV alpha pol gene to be basically unchanged from that of BH-RSV beta. Using cloned DNAs, we have constructeds molecular clones in which the pol gene of non-defective RSV was replaced with the pol gene of BH-RSV alpha or BH-RSV beta. The BH-RSV alpha pol gene was found to be biologically inactive in a transfection assay. Further in vitro recombination localized the lesion to an 859 bp Xbal-Bglll fragment in the second third of the pol gene. Analysis of the proteins synthesized in BH-RSV alpha infected cells revealed the fact that BH-RSV alpha directed the synthesis of a full-sized Prl80 gag-pol precursor, yet no polymerase related proteins were found in BH-RSV alpha virion particles. The pol lesion in the BH-RSV alpha genome appears to cause a defect in the processing or packaging of reverse transcriptase.

Comments

A thesis submitted to the Faculty of The Rockefeller University in partial fulfillment of the requirements for the degree of Doctor of Philosophy

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