Date of Award


Document Type


Degree Name


Organizational Unit

College of Natural Science and Mathematics, Biological Sciences

First Advisor

Schuyler van Engelenburg


HIV, AIDS, Cellular biology


Human immunodeficiency virus type-1 (HIV-1) infects and destroys immune cells (e.g. CD4+ T cells), leading to acquired immunodeficiency syndrome (AIDS). Antiretroviral therapy (ART) can suppress HIV-1 replication, but there is still no vaccine or cure. Here, we have addressed open questions about two key steps of the HIV-1 life cycle which are not targeted by current ART and which rely crucially on host cell factors, presenting potential avenues for new treatment strategies.

First, to release newly formed virus particles from the plasma membrane (PM) of an infected cell, HIV-1 hijacks the cellular ESCRT membrane scission machinery by binding ESCRT-I. In order to probe the organization of ESCRT-I in this process, we used CRISPR/Cas9-mediated genome editing in human cell lines to tag endogenously expressed ESCRT-I with GFP. These knock-in cell lines enable fluorescent imaging of endogenous ESCRT-I, avoiding artifacts of overexpression, and offer a valuable tool not only for HIV-1 research but also for research on the many cellular functions of the ESCRT machinery. We then applied this tool to measure HIV-1 particle incorporation of ESCRT-I. We showed that ESCRT-I is in released HIV-1 particles, indicating that ESCRT-I mediates HIV release from inside the virion, and we quantified the number of molecules of ESCRT-I per virion, giving insight into the mechanism by which ESCRT-mediated abscission may be initiated.

Second, the HIV-1 Envelope (Env) spike must be incorporated into HIV-1 particles during viral assembly at the PM for the particles to be infectious, but non-virus-incorporated Env is rapidly endocytosed to avoid detection by the host immune system. The fate of this internalized Env has been unclear. We used pulse-labeling with a monovalent anti-Env Fab, together with CRISPR/Cas9-mediated endogenous tagging of host cell Rab GTPases, to investigate the post-endocytic trafficking of Env in physiologically relevant CD4+ T cell lines. We found that endocytosed Env traffics to Rab14+ late endosomes and lysosomes, where it is retained mostly without being degraded, and we showed the first direct evidence that Env can return to the PM after endocytosis to be incorporated in HIV-1 particles.

Publication Statement

Copyright is held by the author. This work may only be accessed by members of the University of Denver community. The work is provided by permission of the author for individual research purposes only and may not be further copied or distributed. User is responsible for all copyright compliance.

Rights Holder

Huxley Kaminsky Hoffman


Received from author

File Format




File Size

146 pgs


Molecular biology, Biophysics