Gary L. Buchschacher, Jr 1987-1995
Assistant Clinical Professor, Department of Medicine, Division of Hematology/Oncology
University of California-San Diego
University of Wisconsin - Madison 1987
Molecular Biology PhD 1993
Antonito Panganiban
The development of human immunodeficiency virus vectors and dominant
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One possible way to inhibit the spread of HIV is to express, in infected cells, a foreign gene that interferes with viral replication. The experiments described in the following chapters were designed to investigate the feasibility of this idea. Human immunodeficiency virus type 1 (HIV-1) vectors were developed for delivery of foreign genes and for the evaluation of potential anti-viral genes. Also, a mutant HIV-1 envelope glycoprotein that interferes with viral infectivity was identified and the ability of this mutant to interfere with virus spread was characterized.
A relatively complex HIV-1 vector containing a defective envelope gene and constitutively expressing a marker gene from an internal promoter was constructed and, along with two other vectors, tested for its ability to be propagated when envelope glycoprotein was provided in trans. However, analysis of gene expression demonstrated that complex HIV vectors containing an internal SV40 early promoter did not express genes from the HIV LTR at detectable levels.
Other HIV vectors that had a relatively simple structure and that expressed a marker gene from the HIV LTR also were constructed. The results indicated that sequences extending into the gag open reading frame are necessary in cis for efficient vector propagation. This vector system was used to create a cell line that stably contained a marker gene whose expression from the HIV promoter could be induced by the viral protein Tat. Activation of marker gene expression by infecting virus was determined to be an efficient process. A potential alternate delivery method for simple HIV vectors was investigated. Results indicated that frequent deletion of the internal construct limited the usefulness of this approach for delivery of HIV vectors.
A HIV-1 envelope glycoprotein mutant was tested and found to interfere with the function of wild-type envelope glycoprotein. To determine if wild-type virus replication could be slowed, the 41.2 mutant was used to make a CD4-positive cell line that might inhibit the spread of wild-type virus. The 41.2 envelope gene expressed from the HIV promoter was introduced into HeLa T4 cells. These cells produced mutant envelope glycoprotein and were resistant to the cytopathic effects caused by wild-type envelope. The titer of infectious vector virus produced from these cells also was decreased. When these cells were challenged with wild-type virus, viral spread through the cultures was inhibited. (Abstract shortened by UMI.) |
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| Thesis Publications |
- Buchschacher GL Jr, Freed EO, Panganiban AT. Effects of second-site mutations on dominant interference by a human immunodeficiency virus type 1 envelope glycoprotein mutant. J Virol 69:1344-1348, 1995.
- Buchschacher GL Jr. Molecular targets of gene transfer therapy for HIV infection. JAMA, 269:2880-2886, 1993.
- Cunningham DB, Buchschacher GL Jr, Burland TG, Dove WF, Kessler D, Paul EC. Cloning and characterization of the altA alpha-tubulin gene of Physarum. J Gen Microbiol 139:137-151, 1993.
- Delwart EL, Buchschacher GL Jr, Freed EO, Panganiban AT. Analysis of HIV-1 envelope mutants and pseudotyping of replication-defective HIV-1 vectors by genetic complementation. AIDS Res Hum Retroviruses 8:1669-1677, 1992.
- Buchschacher GL Jr, Freed EO, Panganiban AT. Cells induced to express a human immunodeficiency virus type 1 envelope gene mutant inhibit the spread of wild-type virus. Hum Gene Ther 3:391-397, 1992.
- Buchschacher GL Jr, Panganiban AT. Human immunodeficiency virus vectors for inducible expression of foreign genes. J Virol 66:2731-2739, 1992.
- Freed EO, Delwart EL, Buchschacher GL Jr, Panganiban AT. A mutation in the human immunodeficiency virus type 1 transmembrane glycoprotein gp41 dominantly interferes with fusion and infectivity. Proc Natl Acad Sci USA 89:70-74, 1992.
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