Functional characterisation of the HIV-1 preintegration complex
File(s)
Author(s)
Hope, Joshua Alexander
Type
Thesis
Abstract
Covalent insertion of retroviral DNA into the host chromatin is an obligatory step in the retroviral life cycle, termed integration. On completion of reverse transcription, the viral enzyme, integrase (IN), associates with both ends of double stranded viral DNA to form the intasome, which is competent to initiate integration upon encounter with host DNA. While the intasome is responsible for the catalysis of integration, it is part of a much larger nucleoprotein complex, termed the preintegration complex (PIC), which is poorly defined. We aimed to characterise these elusive viral nucleoprotein complexes to gain insights that could not be gathered using purified recombinant IN alone.
In this study, I optimised the extraction and purification of HIV-1 PICs from acutely infected cells by size exclusion chromatography and with a range of affinity reagents. The development of a highly sensitive real-time quantitative PCR assay to detect PIC integration allowed high-throughput experiments to track our manipulation of PICs. To demonstrate feasibility of PIC detection for imaging approaches, I tested the limits of single-molecule fluorescence microscopy techniques to detect extremely dilute nucleoprotein complexes and experimentally found that the detection limit for dilute macromolecules is comparable to the levels of HIV-1 PICs present in extracts of infected cells.
The host factor, LEDGF/p75, is the key candidate to direct integration of HIV-1 PICs into the host chromatin, as it strongly interacts with both HIV-1 IN and histone 3 Lys-36Me3 methylated (H3K36Me3) nucleosomes. I used strand-transfer activity assays to study the interaction of poly-nucleosomal arrays with both PICs and intasome models. I found that LEDGF/p75 is both essential and minimally sufficient to increase the integration activity into H3K36Me3 nucleosomes for PICs and intasome models. Finally, I determined the extent to which LEDGF/p75 redistributes HIV-1 intasome integration sites in different chromatinised templates using a novel long-read Nanopore sequencing assay.
In this study, I optimised the extraction and purification of HIV-1 PICs from acutely infected cells by size exclusion chromatography and with a range of affinity reagents. The development of a highly sensitive real-time quantitative PCR assay to detect PIC integration allowed high-throughput experiments to track our manipulation of PICs. To demonstrate feasibility of PIC detection for imaging approaches, I tested the limits of single-molecule fluorescence microscopy techniques to detect extremely dilute nucleoprotein complexes and experimentally found that the detection limit for dilute macromolecules is comparable to the levels of HIV-1 PICs present in extracts of infected cells.
The host factor, LEDGF/p75, is the key candidate to direct integration of HIV-1 PICs into the host chromatin, as it strongly interacts with both HIV-1 IN and histone 3 Lys-36Me3 methylated (H3K36Me3) nucleosomes. I used strand-transfer activity assays to study the interaction of poly-nucleosomal arrays with both PICs and intasome models. I found that LEDGF/p75 is both essential and minimally sufficient to increase the integration activity into H3K36Me3 nucleosomes for PICs and intasome models. Finally, I determined the extent to which LEDGF/p75 redistributes HIV-1 intasome integration sites in different chromatinised templates using a novel long-read Nanopore sequencing assay.
Version
Open Access
Date Issued
2023-06-20
Date Awarded
2024-03-01
Copyright Statement
Creative Commons Attribution NonCommercial Licence
License URL
Advisor
Cherepanov, Peter
Sponsor
National Institutes of Health
The Francis Crick Institute
Grant Number
US NIH grant P50 AI150481
Publisher Department
Department of Chemistry
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)