HIV-1 uncoating occurs in the nucleus through small lattice defect formation culminating in terminal loss of the capsid lattice Open Access
Gifford, Levi (Summer 2024)
Abstract
HIV-1 is an enveloped positive sense retrovirus that is results in lifelong infection that over 39 million individuals are afflicted with. The HIV-1 capsid, a large viral structure comprised of roughly 250 capsid (CA) hexamer and 12 pentamers that protects the viral genome from the innate immune surveillance, acts as a scaffolding platform for host factor interactions, and serves as a nanoscale reactor for efficient reverse transcription of the viral genome. During the HIV-1 early infection, the capsid needs to disassemble through a process referred to as uncoating to release the reverse transcribed viral DNA for integration into host genome. The process of uncoating is poorly characterized with conflicting models being proposed. To delineate the complex process of uncoating, we utilized the amber codon suppression system to label the HIV-1 capsid protein at the isoleucine-91 position within the cyclophilin A binding loop to produce fluorescently labeled pseudoviruses (CA*). CA* pseudoviruses were colabeled with a fluid-phase marker to monitor both capsid integrity loss and terminal uncoating on the single particle level. With this system, we found that HIV-1 capsid uncoating occurs through two distinct steps in vitro and in cells including (1) loss of capsid integrity (release of a fluid-phase marker) and (2) terminal loss of the capsid lattice (loss of CA* signal). Importantly, our results suggest that capsid integrity is retained during nuclear import and that loss of integrity is a key intermediate step for nuclear capsid uncoating, preceding capsid uncoating by roughly 30 minutes. These results further our understanding of the early stages of HIV-1 replication and provide information for the design and targeting of HIV-1 with antiviral therapies.
Table of Contents
Distribution agreement …………………………………………………………………………..i
Approval page …………………………………………………………………………………...ii
Abstract cover page ……………..…………………..………………………………….……...iii
Abstract ……………………..…………………………………...……………………….……..iv
Acknowledgments …………………………..………………………………………………….vi
Table of contents ……………………………………..……………………………………….viii
List of figures ……………………………………..…………………………………………….xii
List of supplementary figures …………………………………………………………………xv
Publications and conferences ……………………………………..…………………………xvi
List of Acronyms and abbreviations …………………………………..…………………….xviii
Chapter 1. Introduction ……………………..………….……………………………………….1
1.1. HIV-1: A human pathogen ………………………………………..……………1
1.2. The HIV-1 genome ……………………………………………………………...4
1.3. The early stages of HIV-1 life cycle …………………..…………………..……9
1.3.1. Virus-cell membrane fusion and entry …………..…………………..10
1.3.2. Cytoplasmic HIV-1 events ………..…………………………………..11
1.3.3. HIV- Nuclear import …………….……………………………………..15
1.3.4. Nuclear events of HIV-1 …………..…………………………………..18
1.3.5. Nuclear capsid uncoating ….………………………………………….21
1.3.6. Integration of viral DNA into host chromatin …………………………24
1.4. Biochemical techniques to investigate the HIV-1 capsid….………………. 26
1.4.1. Approaches to fluorescently label the HIV-1 capsid ……………….31
1.5. The capsid as an antiviral target ……………………………………………..38
1.6. Genetic code expansion …………………………………………..………….42
1.6.1. Utilizing amber codon suppression in mammalian cells………….. 43
1.7. Amber suppression for HIV labeling ...….……………………………………46
1.8. Goals of thesis …………………………………………………………………48
Chapter 2. HIV-1 uncoating in the nucleus progresses through a localized loss of capsid integrity preceding terminal loss of the capsid lattice ………………………………………50
2.1. Selection of amber suppression sites in CA ……………… ………………...50
2.2. Characterization and validation of direct HIV-1 capsid protein labeling with unnatural amino acids …………………………………………………………………………51
2.3. Co-labeling of HIV-1 capsid and core-entrapped fluid phase marker reveals 2-step uncoating of cell-free viruses ………………………………………………..……….58
2.4. HIV-1 uncoating in the cytosol and at the nuclear pore occurs in two steps …………………………………………………………………………………………………...63
2.5. No detectable loss of CA* or iYFP occurs upon HIV-1 nuclear import ….….66
2.6. Terminal CA* loss from nuclear HIV-1 capsids is preceded by a loss of core-trapped iYFP …………………………………………………………………………………...68
2.7. Reverse transcription does not majorly impact the lag between two uncoating steps in the nucleus ……………………………………………………………………………74
2.8. HIV-1 undergoes two-step uncoating in different cell types …………………75
2.9. HIV-1 core uncoating in the nucleus correlates with productive infection….78
Chapter 3. Defining the nuclear compartments in which uncoating occurs and visualization of vDNA ………………………………………………………………………….82
3.1. HIV-1 resides within nuclear speckles ……………………………………….82
3.2. Visualization of two-step uncoating in nuclear speckles ……………………..83
3.3. HIV-1 vDNA visualization in living cells with the ANCHOR system …………86
3.4. HIV-1 PICs vDNA are is accessible within the nuclear speckle and are trafficked to SPAD domains …………………………………………………………………..87
Chapter 4. Discussion ..……………………………………………………………………….92
4.1. Advantages and limitation of CA amber suppression labeling ……………….93
4.2. Discordant models of cytosolic and NPC capsid uncoating ………………….94
4.3. Nuclear uncoating of HIV-1 capsids ……………………………………………96
4.4. Release of the vDNA after capsid uncoating …………………………………100
Chapter 5. Future directions ………………………………………………………………...105
5.1. Remaining questions on capsid uncoating ……………………………………105
5.2. Future directions of uncoating within nuclear speckles ……………………..106
5.3. Future directions with the ANCHOR system ………………………………….109
Chapter 6. Conclusions …………………………………………………………………...…110
Chapter 7. Materials and Methods ………………………………………………………….111
7.1. Methods for Gifford et al. 2024 …………………………………………………111
7.2. SRRM2-TagBFP and ANCHOR system materials and methods …………..125
Chapter 8. Refences cited ………..…………………………………………………………131
Chapter 9. Supplemental appendix ………………………………………………………...144
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