HIV-1 Fusion Monitored in Real Time

Date of Award

Spring 1-1-2025

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Microbiology

First Advisor

Mothes, Walther

Abstract

Human immunodeficiency virus 1 (HIV-1) is a retrovirus that is responsible for the ongoing AIDS pandemic. Understanding HIV-1 biology is critical for the development of future treatments and a cure for HIV/AIDS. HIV-1 fusion is the first step in the viral life cycle, and this process is mediated by the HIV-1 envelope glycoprotein (Env). Despite extensive study, questions remain about HIV-1 fusion. While current methods have provided data on many aspects of HIV-1 entry, these methods could not assess the earliest time points of HIV-1 fusion for a population of virions. Here, I present a new method utilizing split NanoLuc to measure HIV-1 fusion with a time resolution of 2 minutes. I then use this method to investigate further questions about HIV-1 fusion. I show that different strains of Env show different fusion kinetics, which is consistent with existing hypotheses that HIV-1 Env strains require different numbers of trimers for fusion. I then show that HIV-1 fusion is dependent on maturation, specifically MA maturation, and that defects in immature fusion can be rescued by truncating the C-terminal tail (C-tail) of Env. Finally, I present another method using two types of virus particles that is compatible with cryogenic electron tomography (cryo-ET). I show that this method can be used to image membrane-membrane interfaces, and thus to obtain structures of Env-CD4 binding in the context of native membranes. I show that by using this method, we can observe CD4-induced clustering of Env into small, large, and ring structures as membranes approach one another. Taken together, the data obtained using these two methods suggest that clustering of Env at fusion sites is an important step in HIV-1 fusion, that this clustering is dependent on MA maturation, and that Env strain-dependent differences in HIV-1 fusion kinetics, particularly delays in the start of fusion, may be explained by different numbers of Env trimers required to cluster at fusion sites before fusion can be initiated. Though the data I present here suggest these conclusions, more work is needed to elucidate the mechanisms of Env clustering and its effect on HIV-1 fusion. While understanding HIV-1 entry is necessary for developing treatments for HIV/AIDS, understanding HIV-1 also paves the way to understanding other viruses such as SARS-CoV-2 and influenza. Further, the tools developed to study HIV-1 can be used to study these and other viruses, including emerging viruses that may cause the next pandemic.

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