Date of Award

Fall 2011

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Cell & Molecular Biology

First Advisor

Robert W. Doms

Abstract

For HIV to enter cells, the viral surface protein Envelope (Env) must sequentially bind the host protein CD4 and one of two coreceptors, either CCR5 or CXCR4. This triggers conformational changes in Env that result in fusion of the host and viral membranes. Our understanding of this process has led to the development of successful anti-viral drugs and provided insights into viral pathogenesis. One critical question is whether we can further exploit our knowledge of the HIV entry process to develop an effective therapeutic vaccine. To do this, we need to better understand HIV-1 transmission, which may reveal viral properties that could be targeted in future vaccine efforts. In chapter two, we conduct a phenotypic comparison of clade B transmitted/founder (T/F) and chronic HIV-1 Envs in an attempt to reveal viral properties associated with successful transmission. We demonstrate that T/F Envs are more sensitive to neutralization by CD4 binding site antibodies and that this correlates with antibody binding suggesting T/F Envs have subtle but potentially important structural differences that may have implications for HIV-1 transmission and vaccine design. A second critical question is how can we provide long-term viral control in the absence of anti-retroviral therapy. Recently, an HIV-1 infected individual was ‘cured’ after receiving a bone marrow transplant from an uninfected donor who had a naturally occurring mutation in CCR5. This suggests genetic disruption of the HIV coreceptors may provide clinical benefit. Previously ccr5-specific zinc finger nucleases (R5-ZFNs) were developed to disrupt ccr5 and engineer HIV-resistant cells. ZFNs are DNA binding proteins that specifically bind and cleave a specific 24 base pair DNA target. After cleavage, error-prone host DNA repair pathways often introduce mutations resulting in a non-functional gene product. Since 50% of late-stage HIV-infected people harbor virus that can use CXCR4, we developed cxcr4-specific ZFNs (X4-ZFNs) that safely and efficiently disrupt cxcr4 conferring resistance to X4 HIV both in vitro and in humanized mice in vivo. Genome editing with ZFNs results in HIV-resistant cells that can be re-infused into a patients own body and hopefully confer therapeutic benefit.

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