Droplet microfluidic sequencing of HIV genomes and integration sites

AbstractSequencing individual HIV-proviruses and their adjacent cellular junctions can elucidate mechanisms of infected cell persistence in vivo. Here, we present a high throughput microfluidic method to sequence entire proviruses in their native integration site context. We used the method to analyze infected cells from people with HIV on suppressive antiretroviral therapy, demonstrating >90% capture and sequencing of paired proviral genomes and integration sites. This method should enable comprehensive genetic analysis of persistent HIV-infected cell reservoirs, providing important insights into the barriers to HIV cure.

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Stable integrant-specific differences in bimodal HIV-1 expression patterns revealed by high-throughput analysis

10.1101/666941 ◽

2019 ◽

Author(s):

David F. Read ◽

Edmond Atindaana ◽

Kalyani Pyaram ◽

Feng Yang ◽

Sarah Emery ◽

...

Keyword(s):

Gene Expression ◽

Infected Cell ◽

High Throughput ◽

Expression Patterns ◽

Clonal Expansion ◽

High Throughput Analysis ◽

Integration Sites ◽

Infected Cells ◽

Hiv 1 ◽

Over Time

AbstractHIV-1 gene expression is regulated by host and viral factors that interact with viral motifs and is influenced by proviral integration sites. Here, expression variation among integrants was followed for hundreds of individual proviral clones within polyclonal populations throughout successive rounds of virus and cultured cell replication. Initial findings in immortalized cells were validated using CD4+ cells from donor blood. Tracking clonal behavior by proviral “zip codes” indicated that mutational inactivation during reverse transcription was rare, while clonal expansion and proviral expression states varied widely. By sorting for provirus expression using a GFP reporter in thenefopen reading frame, distinct clone-specific variation in on/off proportions were observed that spanned three orders of magnitude. Tracking GFP phenotypes over time revealed that as cells divided, their progeny alternated between HIV transcriptional activity and non-activity. Despite these phenotypic oscillations, the overall GFP+ population within each clone was remarkably stable, with clones maintaining clone-specific equilibrium mixtures of GFP+ and GFP-cells. Integration sites were analyzed for correlations between genomic features and the epigenetic phenomena described here. Integrants inserted in genes’ sense orientation were more frequently found to be GFP negative than those in the antisense orientation, and clones with high GFP+ proportions were more distal to repressive H3K9me3 peaks than low GFP+ clones. Clones with low frequencies of GFP positivity appeared to expand more rapidly than clones for which most cells were GFP+, even though the tested proviruses were Vpr-. Thus, much of the increase in the GFP-population in these polyclonal pools over time reflected differential clonal expansion. Together, these results underscore the temporal and quantitative variability in HIV-1 gene expression among proviral clones that are conferred in the absence of metabolic or cell-type dependent variability, and shed light on cell-intrinsic layers of regulation that affect HIV-1 population dynamics.SummaryVery few HIV-1 infected cells persist in patients for more than a couple days, but those that do pose life-long health risks. Strategies designed to eliminate these cells have been based on assumptions about what viral properties allow infected cell survival. However, such approaches for HIV-1 eradication have not yet shown therapeutic promise, possibly because much of the research underlying assumptions about virus persistence has been focused on a limited number of infected cell types, the averaged behavior of cells in diverse populations, or snapshot views. Here, we developed a high-throughput approach to study hundreds of distinct HIV-1 infected cells and their progeny over time in an unbiased way. This revealed that each virus established its own pattern of gene expression that, upon infected cell division, was stably transmitted to all progeny cells. Expression patterns consisted of alternating waves of activity and inactivity, with the extent of activity differing among infected cell families over a 1000-fold range. The dynamics and variability among infected cells and within complex populations that the work here revealed has not previously been evident, and may help establish more accurate correlates of persistent HIV-1 infection.

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Comprehensive Integration Site Analysis of Human Immunodeficiency Virus during In Vivo Infections Reveals Genomic Regions of Enrichment and Clonal Expansion

Blood ◽

10.1182/blood.v128.22.2518.2518 ◽

2016 ◽

Vol 128 (22) ◽

pp. 2518-2518

Author(s):

Kevin G Haworth ◽

Lauren E Schefter ◽

Zachary K. Norgaard ◽

Jennifer E Adair ◽

Hans-Peter Kiem

Keyword(s):

Integration Site ◽

Site Analysis ◽

Genome Wide ◽

Integration Site Analysis ◽

Integration Sites ◽

Infected Cells ◽

Genomic Regions ◽

Hiv Integration

Abstract BACKGROUND A key event in the lifecycle of Human Immunodeficiency Virus (HIV) is permanent integration into the infected cells genome. In addition to allowing long-term persistence of the virus, this results in a trackable mark carried in all infected cells. Active HIV replication represses cellular pathways, preventing further cell division. This would imply that any specific integration site (IS) which is clonally expanded either during active or repressed viral infection arises from either a dormant/inactive virus, or is perturbing local gene expression, leading to increased cell proliferation. Alternatively, a cell carrying HIV provirus could proliferate due to T-cell specific antigen stimulation. By analyzing the patterns of integration sites detected in cell cultures and tissue samples from animal models of HIV infection, we can better understand the basic virology of integration site selection and determine what may potentially drive infected cells to persist despite effective treatment regimens. METHODS Jurkat reporter cell lines or primary human CD4+ cells were cultured and infected with various strains of HIV including both CCR5 and CXCR4 tropic viruses. Infected cells were cultured up to 21 days post infection, then analyzed for HIV proviral integration sites by next-generation sequencing. For in vivo studies, NSG mice were infused with human CD34+ hematopoietic stem/progenitor cells, resulting in a reconstituted human immune system including high levels of CD4+ T cells capable of sustaining HIV infection. After 16 weeks post-challenge, tissues were collected and subjected to integration site analysis for HIV proviral DNA. Identified integration sites were mapped and compared across multiple parameters to identify chromosomal regions and associated genes enriched for integration events, as well as clonally expanded cells in vivo. RESULTS Genome-wide analysis of HIV integration sites reveals a remarkably similar chromosomal landscape both in tissue culture infection of Jurkat cells and in vivo infection data (Figure 1), as well as across multiple HIV strains. As previously observed, the majority of integrations occur near or within gene coding regions thought to be actively transcribed at time of infection. However, certain areas of the genome, and even unique genes, are enriched for IS in individual samples. In addition to these genomic regions of enrichment, we also observe specific clonal outgrowth of unique integration events in genes previously unidentified in the literature. Three genes in particular exhibit a significant increase of integration events during acute infection which are 3x higher than predicted by random chance alone. We also observe integration events in genes that have been documented by other labs in HIV+ clinical patient samples, however in our active infection models, we do not see those specific genes enriched or expanded. This could indicate that these genes play a role in persistence that is only present during anti-retroviral therapy which suppresses active replication. CONCLUSIONS We have cataloged the most extensive HIV IS library to date in both relevant tissue culture models and in vivo infection studies, including over 245,000 unique integration events and three different HIV strains commonly used in research. Genome-wide correlation studies reveal regions significantly enriched for HIV integrations and genes which repeatedly exhibit clonal outgrowth in multiple animals. These types of studies are now being applied to human patient samples to determine if latency and persistence of infection can be mapped to unique integration events or genes of interest. Such information may indicate when and how the latent HIV reservoir is seeded and what types of therapy or treatments are most effective at targeting and eliminating these populations. Circos plot comparing HIV integrations sites (IS) identified either during in vitro cell culture infections (black bars), or in vivo infection studies using humanized mice (red bars). The outer ring is composed of human chromosomes each of which are divided into 25kB fragment bins. Total number of unique integration sites identified in each bin is represented by the height of the histogram bars. The in vitro IS concentric ring scale represents increments of 25 outwards up to 250 while the in vivo IS scales inwards in increments of 2 up to 16. Figure 1 Comparison of in vitro vs in vivo HIV Integration Sites. Figure 1. Comparison of in vitro vs in vivo HIV Integration Sites. Disclosures Adair: Rocket Pharmaceuticals: Consultancy, Equity Ownership.

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Integration in or Near Oncogenes Plays Only a Minor Role in Determining the in Vivo Distribution of HIV Integration Sites Before or During Suppressive Antiretroviral Therapy

10.1101/2020.11.25.397653 ◽

2020 ◽

Author(s):

John M. Coffin ◽

Michael J. Bale ◽

Daria Wells ◽

Shuang Guo ◽

Brian Luke ◽

...

Keyword(s):

Antiretroviral Therapy ◽

Minor Component ◽

Minor Role ◽

Growth And Survival ◽

Integration Sites ◽

Highly Expressed Genes ◽

Infected Cells ◽

A Minor

AbstractHIV persists during antiretroviral therapy (ART) as integrated proviruses in cells descended from a small fraction of the CD4+ T cells infected prior to the initiation of ART. To better understand what controls HIV persistence and the distribution of integration sites (IS), we compared about 16,000 and 54,000 IS from individuals pre-ART and on ART, respectively, with approximately 385,000 IS from PBMC infected in vitro. The distribution of IS in vivo is quite similar to the distribution in PBMC, modified by selection against proviruses in expressed genes, by selection for proviruses integrated into one of 6 specific genes, and by clonal expansion. The clones in which a provirus integrated in an oncogene contributed to the survival of the clone comprise only a small fraction of the clones that persist in HIV-infected individuals on ART. Mechanisms that do not involve the provirus, or its location in the host genome, are more important in determining which clones expand and persist.Author SummaryIn HIV-infected individuals, a small fraction of the infected cells persist and divide. This reservoir persists on ART and can rekindle the infection if ART is discontinued. Because the number of possible sites of HIV DNA integration is very large, each infected cell, and all of its descendants, can be identified by the site where the provirus is integrated (IS). To understand the selective forces that determine the fates of infected cells in vivo, we compared the distribution of HIV IS in freshly-infected cells to cells from HIV-infected donors sampled both before and during ART. We found that, as has been previously reported, integration favors highly-expressed genes. However, over time the fraction of cells with proviruses integrated in highly-expressed genes decreases, implying that they grow less well. There are exceptions to this broad negative selection. When a provirus is integrated in a specific region in one of six genes, the proviruses affect the expression of the target gene, promoting growth and/or survival of the cell. Although this effect is striking, it is only a minor component of the forces that promote the growth and survival of the population of infected cells during ART.

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DNA Palindromes with a Modest Arm Length of ≳20 Base Pairs Are a Significant Target for Recombinant Adeno-Associated Virus Vector Integration in the Liver, Muscles, and Heart in Mice

Journal of Virology ◽

10.1128/jvi.00963-07 ◽

2007 ◽

Vol 81 (20) ◽

pp. 11290-11303 ◽

Cited By ~ 41

Author(s):

Katsuya Inagaki ◽

Susanna M. Lewis ◽

Xiaolin Wu ◽

Congrong Ma ◽

David J. Munroe ◽

...

Keyword(s):

Mouse Liver ◽

Integration Site ◽

Marker Gene ◽

Cpg Islands ◽

Previous Method ◽

Adeno Associated Virus ◽

Transcription Start Sites ◽

Vector Integration ◽

Integration Sites

ABSTRACT Our previous study has shown that recombinant adeno-associated virus (rAAV) vector integrates preferentially in genes, near transcription start sites and CpG islands in mouse liver (H. Nakai, X. Wu, S. Fuess, T. A. Storm, D. Munroe, E. Montini, S. M. Burgess, M. Grompe, and M. A. Kay, J. Virol. 79:3606-3614, 2005). However, the previous method relied on in vivo selection of rAAV integrants and could be employed for the liver but not for other tissues. Here, we describe a novel method for high-throughput rAAV integration site analysis that does not rely on marker gene expression, selection, or cell division, and therefore it can identify rAAV integration sites in nondividing cells without cell manipulations. Using this new method, we identified and characterized a total of 997 rAAV integration sites in mouse liver, skeletal muscle, and heart, transduced with rAAV2 or rAAV8 vector. The results support our previous observations, but notably they have revealed that DNA palindromes with an arm length of ≳20 bp (total length, ≳40 bp) are a significant target for rAAV integration. Up to ∼30% of total integration events occurred in the vicinity of DNA palindromes with an arm length of ≳20 bp. Considering that DNA palindromes may constitute fragile genomic sites, our results support the notion that rAAV integrates at chromosomal sites susceptible to breakage or preexisting breakage sites. The use of rAAV to label fragile genomic sites may provide an important new tool for probing the intrinsic source of ongoing genomic instability in various tissues in animals, studying DNA palindrome metabolism in vivo, and understanding their possible contributions to carcinogenesis and aging.

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HIV Cure Strategies

10.1093/med/9780190493097.003.0006 ◽

2017 ◽

Author(s):

Boris Juelg ◽

Rajesh Gandhi

Keyword(s):

Gene Therapy ◽

Antiretroviral Therapy ◽

Immune Responses ◽

Immune Recognition ◽

Hiv Cure ◽

Active Viral Replication ◽

Infected Cells ◽

Or Gene ◽

Virus Expression ◽

Hiv 1

Although current antiretroviral therapy (ART) is highly effective at controlling HIV-1 replication, it does not eradicate or cure the infection. HIV-1 persists quiescently in cellular reservoirs, not detected by the immune system due to the lack of active viral replication; these reservoirs represent the major obstacle for cure approaches. Reversal of HIV-1 latency and induction of virus expression by a variety of interventions may render infected cells susceptible to immune recognition and active clearance. Strategies to boost immune responses via vaccination, immunomodulation, or gene therapy are being evaluated with the aim of achieving HIV-1 control without antiretroviral therapy, if not viral eradication.

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In-depth single-cell analysis of translation-competent HIV-1 reservoirs identifies cellular sources of plasma viremia

10.1101/2021.02.15.431218 ◽

2021 ◽

Author(s):

Basiel Cole ◽

Laurens Lambrechts ◽

Pierre Gantner ◽

Ytse Noppe ◽

Noah Bonine ◽

...

Keyword(s):

Single Cell ◽

Single Cell Analysis ◽

Integration Site ◽

Donor Site ◽

Treatment Interruption ◽

Splice Donor Site ◽

Plasma Viremia ◽

Cell Clones ◽

Integration Sites ◽

Infected Cells

AbstractClonal expansion of HIV-infected cells contributes to the long-term persistence of the HIV reservoir in ART-suppressed individuals. However, the contribution to plasma viremia from cell clones that harbor inducible proviruses is poorly understood. Here, we describe a single-cell approach to simultaneously sequence the TCR, integration sites and proviral genomes from translation-competent reservoir cells, called STIP-Seq. By applying this approach to blood samples from eight participants, we showed that the translation-competent reservoir mainly consists of proviruses with short deletions at the 5’-end of the genome, often involving the major splice donor site. TCR and integration site sequencing revealed that antigen-responsive cells can harbor inducible proviruses integrated into cancer-related genes. Furthermore, we found several matches between proviruses retrieved with STIP-Seq and plasma viruses obtained during ART and upon treatment interruption, showing that STIP-Seq can capture clones that are responsible for low-level viremia or viral rebound.

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Early Emergence and Long-Term Persistence of HIV-Infected T Cell Clones in Children

10.1101/2020.09.15.298406 ◽

2020 ◽

Author(s):

Michael J. Bale ◽

Mary Grace Katusiime ◽

Daria Wells ◽

Xiaolin Wu ◽

Jonathan Spindler ◽

...

Keyword(s):

T Cell ◽

Infected Cell ◽

Mononuclear Cells ◽

Ex Vivo ◽

Peripheral Blood Mononuclear ◽

T Cell Clones ◽

Cell Clones ◽

Integration Sites ◽

Infected Cells

AbstractLittle is known about the emergence and persistence of HIV-infected T cell clones in perinatally-infected children. We analyzed peripheral blood mononuclear cells for clonal expansion in 11 children who initiated antiretroviral therapy (ART) between 1.8-17.4 months of age and with viremia suppressed for 6-9 years. We obtained 8,662 HIV-1 integration sites from pre-ART and 1,861 sites on ART. Expanded clones of infected cells were detected pre-ART in 10/11 children. In 8 children, infected cell clones detected pre-ART persisted for 6-9 years on ART. A comparison of integration sites in the samples obtained on ART with healthy donor PBMC infected ex-vivo showed selection for cells with proviruses integrated in BACH2 and STAT5B. Our analyses indicate that, despite marked differences in T cell composition and dynamics between children and adults, HIV-infected cell clones are established early in children, persist for up to 9 years on ART, and can be driven by proviral integration in proto-oncogenes.

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HIV Replication and Latency in a Humanized NSG Mouse Model during Suppressive Oral Combinational Antiretroviral Therapy

Journal of Virology ◽

10.1128/jvi.02118-17 ◽

2018 ◽

Vol 92 (7) ◽

pp. e02118-17 ◽

Cited By ~ 13

Author(s):

Sangeetha Satheesan ◽

Haitang Li ◽

John C. Burnett ◽

Mayumi Takahashi ◽

Shasha Li ◽

...

Keyword(s):

Bone Marrow ◽

Antiretroviral Therapy ◽

Hiv Latency ◽

Hiv Replication ◽

Hematopoietic Stem ◽

Latently Infected ◽

Infected Cells ◽

Surgical Manipulation ◽

Nsg Mouse

ABSTRACTAlthough current combinatorial antiretroviral therapy (cART) is therapeutically effective in the majority of HIV patients, interruption of therapy can cause a rapid rebound in viremia, demonstrating the existence of a stable reservoir of latently infected cells. HIV latency is therefore considered a primary barrier to HIV eradication. Identifying, quantifying, and purging the HIV reservoir is crucial to effectively curing patients and relieving them from the lifelong requirement for therapy. Latently infected transformed cell models have been used to investigate HIV latency; however, these models cannot accurately represent the quiescent cellular environment of primary latently infected cellsin vivo. For this reason,in vivohumanized murine models have been developed for screening antiviral agents, identifying latently infected T cells, and establishing treatment approaches for HIV research. Such models include humanized bone marrow/liver/thymus mice and SCID-hu-thy/liv mice, which are repopulated with human immune cells and implanted human tissues through laborious surgical manipulation. However, no one has utilized the human hematopoietic stem cell-engrafted NOD/SCID/IL2rγnull(NSG) model (hu-NSG) for this purpose. Therefore, in the present study, we used the HIV-infected hu-NSG mouse to recapitulate the key aspects of HIV infection and pathogenesisin vivo. Moreover, we evaluated the ability of HIV-infected human cells isolated from HIV-infected hu-NSG mice on suppressive cART to act as a latent HIV reservoir. Our results demonstrate that the hu-NSG model is an effective surgery-freein vivosystem in which to efficiently evaluate HIV replication, antiretroviral therapy, latency and persistence, and eradication interventions.IMPORTANCEHIV can establish a stably integrated, nonproductive state of infection at the level of individual cells, known as HIV latency, which is considered a primary barrier to curing HIV. A complete understanding of the establishment and role of HIV latencyin vivowould greatly enhance attempts to develop novel HIV purging strategies. An ideal animal model for this purpose should be easy to work with, should have a shortened disease course so that efficacy testing can be completed in a reasonable time, and should have immune correlates that are easily translatable to humans. We therefore describe a novel application of the hematopoietic stem cell-transplanted humanized NSG model for dynamically testing antiretroviral treatment, supporting HIV infection, establishing HIV latencyin vivo. The hu-NSG model could be a facile alternative to humanized bone marrow/liver/thymus or SCID-hu-thy/liv mice in which laborious surgical manipulation and time-consuming human cell reconstitution is required.

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A High-Throughput Method for Cloning and Sequencing Human Immunodeficiency Virus Type 1 Integration Sites

Journal of Virology ◽

10.1128/jvi.01737-06 ◽

2006 ◽

Vol 80 (22) ◽

pp. 11313-11321 ◽

Cited By ~ 10

Author(s):

Sanggu Kim ◽

Yein Kim ◽

Teresa Liang ◽

Janet S. Sinsheimer ◽

Samson A. Chow

Keyword(s):

High Throughput ◽

Integration Site ◽

Target Site ◽

Viral Dna ◽

Target Site Selection ◽

Immunodeficiency Virus ◽

High Throughput Method ◽

Integration Sites ◽

Hiv 1

ABSTRACT Integration of retroviral DNA is nonspecific and can occur at many sites throughout chromosomes. However, the process is not uniformly distributed, and both hot and cold spots for integration exist. The mechanism that determines target site specificity is not well understood. Because of the nonspecific and widespread nature of integration, studies analyzing the mechanism and factors that control target site selection require the collection and analysis of a large library of human immunodeficiency virus type 1 (HIV-1) proviral clones. Such analyses are time-consuming and labor-intensive using conventional means. We have developed an efficient and high-throughput method of sequencing and mapping a large number of independent integration sites in the absence of any selection or bias. The new assay involves the use of a modified HIV-1 (NL-Mme) containing a type IIS restriction site, MmeI, at the right end of viral DNA. Digestion of genomic DNA from NL-Mme-infected cells generated viral DNA-containing fragments of a discrete size. Subsequent ligation-mediated PCR yielded short integration site fragments termed Int-tags, which were concatemerized for determining multiple integration sites in a single sequencing reaction. Analysis of chromosomal features and sequence preference associated with integration events confirmed the validity of the new high-throughput assay. The assay will aid the effort in understanding the mechanisms of target site selection during HIV-1 DNA integration, and the described methodology can be adapted easily to integration site studies involving other retroviruses and transposons.

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Common Viral Integration Sites Identified in Avian Leukosis Virus-Induced B-Cell Lymphomas

mBio ◽

10.1128/mbio.01863-15 ◽

2015 ◽

Vol 6 (6) ◽

Cited By ~ 14

Author(s):

James F. Justice ◽

Robin W. Morgan ◽

Karen L. Beemon

Keyword(s):

B Cell ◽

High Throughput ◽

High Throughput Sequencing ◽

Consensus Sequence ◽

B Cell Lymphoma ◽

Avian Leukosis Virus ◽

B Cell Lymphomas ◽

Genome Wide ◽

Integration Sites

ABSTRACTAvian leukosis virus (ALV) induces B-cell lymphoma and other neoplasms in chickens by integrating within or near cancer genes and perturbing their expression. Four genes—MYC,MYB,Mir-155, andTERT—have previously been identified as common integration sites in these virus-induced lymphomas and are thought to play a causal role in tumorigenesis. In this study, we employ high-throughput sequencing to identify additional genes driving tumorigenesis in ALV-induced B-cell lymphomas. In addition to the four genes implicated previously, we identify other genes as common integration sites, includingTNFRSF1A,MEF2C,CTDSPL,TAB2,RUNX1,MLL5,CXorf57, andBACH2. We also analyze the genome-wide ALV integration landscapein vivoand find increased frequency of ALV integration near transcriptional start sites and within transcripts. Previous work has shown ALV prefers a weak consensus sequence for integration in cultured human cells. We confirm this consensus sequence for ALV integrationin vivoin the chicken genome.IMPORTANCEAvian leukosis virus induces B-cell lymphomas in chickens. Earlier studies showed that ALV can induce tumors through insertional mutagenesis, and several genes have been implicated in the development of these tumors. In this study, we use high-throughput sequencing to reveal the genome-wide ALV integration landscape in ALV-induced B-cell lymphomas. We find elevated levels of ALV integration near transcription start sites and use common integration site analysis to greatly expand the number of genes implicated in the development of these tumors. Interestingly, we identify several genes targeted by viral insertions that have not been previously shown to be involved in cancer.

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