ORCID

https://orcid.org/0000-0003-2343-8817

Date of Award

Winter 12-15-2015

Author's School

Graduate School of Arts and Sciences

Author's Department

Biology & Biomedical Sciences (Immunology)

Degree Name

Doctor of Philosophy (PhD)

Degree Type

Dissertation

Abstract

Human Immunodeficiency Virus Type 1 (HIV-1) envelope protein is the sole determinant for viral entry and tropism. The ability of HIV-1 to infect susceptible host cells depends on the ability of its envelope protein to engage host cell receptor CD4 and coreceptor C-C Chemokine Receptor Type 5 (CCR5) and/or C-X-C Chemokine Receptor Type 4 (CXCR4). Most naturally occurring infections start with a single CCR5-tropic virion. In approximately 50% of HIV-1 Clade B infected patients, the viral population spontaneous develops the ability to utilize CXCR4 at a late disease stage, and this coreceptor shift corresponds to a poor prognosis for the patients. Clinical application of a CCR5 antagonist drug accelerates this coreceptor shift process. Despite the important implications of HIV-1 tropism on disease pathogenesis, prognosis, and treatment, molecular mechanisms for coreceptor shift and the contributing envelope determinants have not yet been clearly defined due to the dynamic, multimeric, multi-step nature of envelope-coreceptor interaction. In order to better understand the regions on envelope that are important for coreceptor specificity, we studied a panel of HIV-1 envelope amplicon samples from patients who experienced coreceptor shift and treatment failure in a CCR5 antagonist clinical trial. In order to further understand the structure-function relationship of HIV-1 envelope, we utilized both in-depth single clone analysis to fully characterize the functional attributes of individual envelope isolates, and high-throughput deep sequencing analysis to investigate the global envelope variant landscape before and after treatment.

In the single clone analysis, we applied both tissue-culture based phenotypic tropism characterization, as well as sequence-based genotypic analysis towards 97 single envelope clones isolated from four samples of two study subjects. Unique quasispecies composition were found post coreceptor shift in two individuals who had different courses of disease. In addition, one subject showed drastic sequence variation between isolates from before and after treatment, and a highly homogeneous viral population post treatment. This suggested a rapid shift to CXCR4-using variants that accounted for failure to respond to CCR5 antagonist treatment.

In the deep sequencing analysis, we developed a novel comprehensive approach to sequence a large number of phenotypic validated variants on two next-generation sequencing platforms. This approach offered an unprecedented view of the viral quasispecies landscape in vivo, as well as on the dynamic population change in response to drug selection. Regions of interest that strongly associated with the usage of CXCR4 coreceptor were identified, including previously reported coreceptor specificity sites within and outside V3, the CD4 binding site, and gp41-gp120 interaction site within the same protomer and between neighboring protomers in the envelope trimer spike. These findings have potentially implications in rational design of better coreceptor inhibitors, and development of more accurate predictive algorithms for HIV-1 envelope tropism.

Language

English (en)

Chair and Committee

Lee Ratner

Committee Members

Robyn Klein, Thomas Baranski, Daved Fremont, Marco Colonna, Makedonka Mitreva, Christina Stallings

Comments

Permanent URL: https://doi.org/10.7936/K7MG7MS2

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