Date of Graduation
1997
Document Type
Dissertation/Thesis
Abstract
The low processivity of retroviral reverse transcriptase and the lack of exonucleolytic proofreading by retroviral reverse trascriptase contribute to the emergence of virus variants in infected hosts. A result of the low processivity of reverse transcriptase is the high frequency deletion of direct repeats in retroviral genomes and vectors. We determined that direct repeats of 383, 788, and 1333 base pairs delete at rates of 28-40, 85, and 93%, respectively, in one round of retroviral replication. Also, we demonstrated that the propensity of reverse transcriptase to switch templates could be used to reconstitute the neomycin resistance gene and delete the retroviral-packaging signal, E, during reverse transcription. These E-minus proviruses are safer vectors for gene therapy since replication competent virus does not efficiently mobilize them. The other experiments relate to the error-prone DNA synthesis catalyzed by retroviral reverse transcriptases. This process generates high levels of variation in retroviral populations. The large numbers of variants allow for the rapid selection of variants that are drug resistant, possess altered cellular tropism, or have the ability to evade the immune response. We demonstrated that the anti-retrovirus drug 3{dollar}\\sp\\prime{dollar}-deoxy, 3{dollar}\\sp\\prime{dollar}-azido thymidine (AZT) increases the in vivo retrovirus mutation rate. SNV- and MLV-based retroviral vectors encoding the lacZ gene as a mutational target and a drug resistance marker for selection of infected cells were utilized. The rate of inactivation of the lacZ gene was measured following one round of retroviral replication. The results indicated that AZT-treatment of target cells increased the mutation rate 3- to 10-fold. DNA sequence analysis of proviral plasmids encoding the {dollar}{lcub}\\it lacZ\\/{rcub}\\alpha{dollar} gene as a mutational target indicated that AZT increased the rates of deletion mutations and substitution mutations in vivo. Also, the effects of hydroxyurea or thymidine induced dNTP pool imbalances on in vivo retrovirus mutation rates were examined. The results indicated that HU-induced dNTP pool imbalances increased the mutation rates 2-fold and 3-fold for SNV and MLV, respectively. Thymidine-treatment increased the mutation rates 5-fold and 4-fold for SNV and MLV, respectively. The results indicated that dNTP pools are a cellular factor that is a determinate of the fidelity of reverse transcriptase.
Recommended Citation
Julias, John George, "Retrovirus variation and RT template switching during retroviral reverse transcription: Development of novel retroviral vectors for gene therapy." (1997). Graduate Theses, Dissertations, and Problem Reports. 9133.
https://researchrepository.wvu.edu/etd/9133