Characterization of HIV-1 antisense RNA and enhanced transcription with interference of LEDGF/p75-directed integration Restricted; Files Only
Mahboubi, Darius (Spring 2023)
Published
Abstract
Viral latency is the biggest barrier to complete clearance of HIV infection; the inability to clear HIV infection means that treatment is lifelong. Latently infected CD4+ T cells contain inactive virus and are not killed by antiretrovirals. The interaction between host factor Lens Epithelium-Derived Growth Factor (LEDGF/p75) and HIV-1 integrase (IN) directs integration into highly spliced, intron dense regions of the genome, and towards the center of genes. Disruption of this interaction leads to more random integration, increase in latent infection and, in our data, accumulation of antisense RNA (asRNA). This phenotype was not observed when Cleavage and Polyadenylation Specificity Factor Subunit 6 (CPSF6), another host factor involved in integration, is depleted, suggesting that loss of canonical integration site selection alone is not the main contributor, rather a result of the specific consequence of loss of IN-LEDGF/p75. The role of antisense HIV-1 transcripts in latency and infection remains unclear. Understanding the mechanism by which HIV-1 transcription is regulated will advance our knowledge of latency.
We recently discovered that Jurkat cells that were either genetically altered to knock-out LEDGF/p75 or treated with allosteric HIV-1 integrase inhibitor (ALLINI) BI-D, displayed an increase in frequency and abundance of HIV-1 asRNA. This effect was accompanied by a reduction in the number of cells expressing the HIV-1 sense RNA and gag protein, indicative of replication suppression. These data sparked our interest to investigate whether asRNA plays a role in the HIV-1 replication cycle and the significance of the loss of IN-LEDGF/p75 in its expression levels. Moreover, the HIV-1 antisense RNA (asRNA) transcripts appeared to reside predominantly in the nucleus of infected cells, implying that asRNA may function as a non-coding RNA to regulate sense RNA levels. To better understand this phenotype, we have initiated characterization of the transcripts by Illumina and nanopore sequencing on infected primary CD4+ T cells treated with DMSO or BI-D. Our data suggest that transcription of the asRNA can also originate outside the provirus, which is enhanced with BI-D treatment, and that its splicing is perturbed following integration in the absence of LEDGF/p75. Loss of canonical Tat activity on transcription elongation also enhanced asRNA levels in all cells tested providing evidence that asRNA transcription can be regulated by viral transcription factors. We hypothesize that accumulation of HIV-1 asRNA is a result of changes to integration site selection from loss of IN-LEDGF/p75, which can potentially lead to suppression of HIV-1 transcription and replication, seen in latently infected cells. Understanding the relationship between LEDGF/p75, HIV-1 asRNA, and latency may help in the discovery of regimens that follow the “shock and kill” or “block and lock” therapeutic strategies.
Table of Contents
ACKNOWLEDGEMENTS
TABLE OF FIGURES
LIST OF ABBREVIATIONS
CHAPTER I: OVERVIEW OF THE HUMAN IMMUNODEFICIENCY VIRUS TYPE 1 (HIV-1)
PANDEMIC, VIRAL INTEGRATION & LATENCY, AND TRANSCRIPTIONAL REGULATION
BY NON-CODING RNA (ncRNA)
The HIV disease pathogenesis: the global pandemic, epidemiology, and AIDS…………1
The HIV-1 Genome……………………………………………………………………..................3
The HIV-1 Genome: gag……………………………………………………...…...............6
The HIV-1 Genome: pol…………………………………………………….............……..7
The HIV-1 Genome: env……………………………………………………...............….10
The HIV-1 Genome: regulatory and accessory proteins……………………......…..12
HIV-1 replication……….……………………………………………….…………….............….15
HIV-1 replication: Virion structure……….....…………………………….........….….15
HIV-1 replication: Binding and fusion...…...……….…..………………......…...…..17
HIV-1 replication: Reverse transcription………….……...….………….......……..…19
HIV-1 replication: Integration………….………………....……………..........…….…19
HIV-1 replication: Transcription/replication…………....….………………......……20
HIV-1 replication: Assembly………….…………....……………………....…..........…21
HIV-1 replication: Budding……….……………....……………………….........…....…21
Cellular and molecular mechanisms required for HIV-1 integration…………………....22
Molecular mechanisms of HIV-1 viral latency………………………………………........….24
Molecular mechanisms of HIV-1 viral latency: chromatin architecture……....…25
Molecular mechanisms of HIV-1 viral latency: DNA methylation……………......26
Molecular mechanisms of HIV-1 viral latency: RNA Pol II…………………….......26
Molecular mechanisms of HIV-1 viral latency: Therapeutic approaches……..….27
Small and long non-coding RNAs……………...………………………….………….........….29
Diseases linked to long non-coding RNAs…………….…..……………………….......…….32
Sense and antisense direction to produce long non-coding RNAs…………..…………..34
HIV-1 antisense RNA and protein……………………………………………………..........….35
HIV-1 antisense RNA and protein: Potential function of ASP………..…….......…36
HIV-1 antisense RNA and protein: Natural antisense transcripts (NATs).….…..38
HIV-1 antisense RNA and protein: Possible role of asRNA………………….......…39
HIV-1 antisense RNA and protein: asRNA and latency………….…………......…..42
Inhibitors targeting the LEDGF/p75 binding pocket of HIV-1 Integrase………………..42
Research goals……………………………………………………………………….................…47
CHAPTER II: CHARACTERIZATION OF HIV-1 ANTISENSE RNA AND ENHANCED
TRANSCRIPTION WITH INTERFERENCE OF LEDGF/p75-DIRECTED INTEGRATION......85
Abstract……..…….………………………………………………………………..............……...85
Background……...……………………………………………………………..............………….87
Results…..……………………………………………………………………………...............…..90
Disruption of IN-LEDGF/p75 interaction leads to increased asRNA expression
and decreased sense RNA levels…………………………...................................…96
Localization of HIV-1 sense and antisense RNA……………………………….........99
Disruption of 5’ LTR- transcription leads to increased asRNA levels…………....101
Characterization of the asRNA transcripts…………………………………..........…103
Discussion…..………………………………………………………………………................…109
Conclusion.....……………………………………..............…………………………………..…113
Materials and methods…………………………………………………………...…...........…..114
Statistical analyses…………………………………………….………….............……………..121
Acknowledgements……….…………………………………………………………..............…122
Abbreviations…….…………..………………………………………………………..............…122
References………………………………………………………………………................………126
CHAPTER III: HIV-1 ANTISENSE RNA DETECTION, CHARACTERIZATION, AND
REGULATION DURING LOSS OF IN-LEDGF/p75……………..……………………..................136
Background…….....………………………………………………………………...............….…136
Role of LEDGF on cell survival during environmental stress………..............………….137
Influence of cellular environment on endogenous LEDGF/p75 levels…....……...........138
Fusion of Inhibitor of growth protein 2 (ING2) to LEDGF/p75 results in a
loss of asRNA levels …………...………………………………………...............………………144
Role of HRP-2 on asRNA……………………………………………........................…………148
Nanopore seq. (ONT), the third generation of sequencing………..............……………..151
Nanopore seq. (ONT), the third generation of sequencing: Advantages……...….152
Nanopore seq. (ONT), the third generation of sequencing: Disadvantages…......152
Nanopore seq. (ONT), the third generation of sequencing: Experiment design...154
Nanopore seq. (ONT), the third generation of seq.: Nanopore seq……………......160
Nanopore seq. (ONT), the third generation of seq.: SSP primer……………….......163
Nanopore seq. (ONT), the third generation of seq.: 5D9 treatment experiment..167
Nanopore seq. (ONT), the third generation of seq.: 5D9 sequencing run #1..……168
Nanopore seq.(ONT), the third generation of seq.: 5D9 sequencing run #2…......174
Materials and methods……………………………………………..............……….……………182
References………………………….…………………………………....................………………187
CHAPTER IV: DISCUSSION…………………………………….................…………………………..194
Final remarks…………………………………………………………....................……………….211
References…………………………………………………………….....................……………….213
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