CRISPR-Cas9-based Genome Engineering to Generate Jurkat Reporter Models for HIV-1 Infection with Selected Proviral Integration Sites

Abstract Introduction Transplantation among HIV positive patients may be a valuable therapeutic intervention. This study involves an HIV D+/R+ kidney–liver transplantation, where PBMC-associated HIV quasispecies were analyzed in donor and transplant recipients (TR) prior to transplantation and thereafter, together with standard viral monitoring. Methods The donor was a 54 year of age HIV infected woman: kidney and liver recipients were two HIV infected men, aged 49 and 61. HIV quasispecies in PBMC was analyzed by ultra-deep sequencing of V3 env region. During TR follow-up, plasma HIV-1 RNA, HIV-1 DNA in PBMC, analysis of proviral integration sites and drug-resistance genotyping were performed. Other virological and immunological monitoring included CMV and EBV DNA quantification in blood and CD4 T cell counts. Results Donor and TR were all ART-HIV suppressed at transplantation. Thereafter, TR maintained a nearly suppressed HIV-1 viremia, but HIV-1 RNA blips and the increase of proviral integration sites in PBMC attested some residual HIV replication. A transient peak in HIV-1 DNA occurred in the liver recipient. No major changes of drug-resistance genotype were detected after transplantation. CMV and EBV transient reactivations were observed only in the kidney recipient, but did not require specific treatment. CD4 counts remained stable. No intermixed quasispecies between donor and TR was observed at transplantation or thereafter. Despite signs of viral evolution in TR, HIV genetic heterogeneity did not increase over the course of the months of follow up. Conclusions No evidence of HIV superinfection was observed in the donor nor in the recipients. The immunosuppressive treatment administrated to TR did not result in clinical relevant viral reactivations.

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Pinpointing recurrent proviral integration sites in new models for latent HIV-1 infection

Virus Research ◽

10.1016/j.virusres.2018.03.007 ◽

2018 ◽

Vol 249 ◽

pp. 69-75 ◽

Cited By ~ 2

Author(s):

Ulrike C. Lange ◽

Julia K. Bialek ◽

Thomas Walther ◽

Joachim Hauber

Keyword(s):

Proviral Integration ◽

New Models ◽

Integration Sites ◽

Latent Hiv ◽

Hiv 1

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Isolation and Analysis of Retroviral Integration Targets by Solo Long Terminal Repeat Inverse PCR

Journal of Virology ◽

10.1128/jvi.76.11.5540-5547.2002 ◽

2002 ◽

Vol 76 (11) ◽

pp. 5540-5547 ◽

Cited By ~ 8

Author(s):

Yi Feng Jin ◽

Toshio Ishibashi ◽

Akio Nomoto ◽

Michiaki Masuda

Keyword(s):

Long Terminal Repeat ◽

Dna Sequences ◽

Target Selection ◽

Terminal Repeat ◽

Inverse Pcr ◽

Proviral Integration ◽

Host Chromosome ◽

Retroviral Integration ◽

Integration Sites ◽

Slip Method

ABSTRACT Upon retroviral infection, the genomic RNA is reverse transcribed to make proviral DNA, which is then integrated into the host chromosome. Although the viral elements required for successful integration have been extensively characterized, little is known about the host DNA structure constituting preferred targets for proviral integration. In order to elucidate the mechanism for the target selection, comparison of host DNA sequences at proviral integration sites may be useful. To achieve simultaneous analysis of the upstream and downstream host DNA sequences flanking each proviral integration site, a Moloney murine leukemia virus-based retroviral vector was designed so that its integrated provirus could be removed by Cre-loxP homologous recombination, leaving a solo long terminal repeat (LTR). Taking advantage of the solo LTR, inverse PCR was carried out to amplify both the upstream and downstream cellular flanking DNA. The method called solo LTR inverse PCR, or SLIP, proved useful for simultaneously cloning the upstream and downstream flanking sequences of individual proviral integration sites from the polyclonal population of cells harboring provirus at different chromosomal sites. By the SLIP method, nucleotide sequences corresponding to 38 independent proviral integration targets were determined and, interestingly, atypical virus-host DNA junction structures were found in more than 20% of the cases. Characterization of retroviral integration sites using the SLIP method may provide useful insights into the mechanism for proviral integration and its target selection.

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An Improved Sequencing-Based Bioinformatics Pipeline to Track the Distribution and Clonal Architecture of Proviral Integration Sites

Frontiers in Microbiology ◽

10.3389/fmicb.2020.587306 ◽

2020 ◽

Vol 11 ◽

Author(s):

Nicolas Rosewick ◽

Vincent Hahaut ◽

Keith Durkin ◽

Maria Artesi ◽

Snehal Karpe ◽

...

Keyword(s):

Proviral Integration ◽

Bioinformatics Pipeline ◽

Clonal Architecture ◽

Integration Sites

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HIV-1 Proviral Integration Is Blocked by Agonists to the VpPAC2 Neuroendocrine Receptor.

Blood ◽

10.1182/blood.v106.11.1427.1427 ◽

2005 ◽

Vol 106 (11) ◽

pp. 1427-1427

Author(s):

Donald R. Branch ◽

Payman Baradar Bokaei ◽

Darinka Sakac ◽

Xue-Zhong Ma

Keyword(s):

Mononuclear Cells ◽

Receptor Expression ◽

Productive Infection ◽

Proviral Integration ◽

Peripheral Blood Mononuclear ◽

Low Concentrations ◽

Circle Formation ◽

G Protein Coupled ◽

Hiv 1 ◽

Receptor Family

Abstract VPAC1 is a 7-transmembrane G-protein-coupled neuroendocrine receptor previously shown to transduce a facilitation signal for HIV-1 infection (Branch DR, et al., AIDS.2002;16:309–319). VPAC2, a related receptor, has been reported to have opposing function when compared to VPAC1 (Xia M, et al., J Immunol.1996;157:1132–1138; Tsutsumi M, et al., Diabetes.2002;51:1453–1460). We therefore examined whether stimulation of VPAC2, in contrast to VPAC1, may act to inhibit HIV-1 infection. Using three different and specific agonists of VPAC2, helodermin, RO 25-1553, and R3P55, daily treatment with low concentrations (10−9M) resulted in ~75% to 95% inhibition of either X4 or R5 HIV-1 productive infection in cell lines or primary peripheral blood mononuclear cells. The agonists VIP, PACAP, and secretin, that stimulate the two other VPAC receptor family members VPAC1 and PAC1, did not inhibit HIV-1 infection. Also, Hut78 cells, that lack VPAC2 and are infected by HIV-1, show no effect of VPAC2 agonists on HIV-1 infection. However, Hut78 cells transfected with human VPAC2 cDNA to overexpress this receptor become resistant to HIV-1 infection when treated with VPAC2 agonists. VPAC2-mediated inhibition of productive HIV-1 infection was not due to effects on CD4 or chemokine co-receptor expression, cell growth, or apoptosis. Treatment with VPAC2 agonists also did not inhibit HIV-1 entry into the host cell. However, compared to untreated or cells treated with VPAC1-specific agonists, VPAC2 stimulation profoundly inhibits HIV-1 proviral DNA integration into the host genome. The block in integration was found to be due to the ability of agonists to VPAC2 to inhibit formation of 2-LTR circles, required for HIV-1 integration within the nucleus. We conclude that VPAC2-specific agonists are strongly inhibitory for productive HIV-1 infection. Furthermore, this inhibition is mediated by suppression of 2-LTR circle formation preventing proviral integration. Agonists of VPAC2 appear to be excellent candidates for future development as possible drugs for the amelioration of treatments aimed at the prevention of HIV/AIDS.

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Resting CD4+ T Cells from Human Immunodeficiency Virus Type 1 (HIV-1)-Infected Individuals Carry Integrated HIV-1 Genomes within Actively Transcribed Host Genes

Journal of Virology ◽

10.1128/jvi.78.12.6122-6133.2004 ◽

2004 ◽

Vol 78 (12) ◽

pp. 6122-6133 ◽

Cited By ~ 224

Author(s):

Yefei Han ◽

Kara Lassen ◽

Daphne Monie ◽

Ahmad R. Sedaghat ◽

Shino Shimoji ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

T Cells ◽

Cell Populations ◽

Rt Pcr ◽

Immunodeficiency Virus ◽

Integration Sites ◽

Hiv 1 ◽

Host Genes

ABSTRACT Resting CD4+ T-cell populations from human immunodeficiency virus type 1 (HIV-1)-infected individuals include cells with integrated HIV-1 DNA. In individuals showing suppression of viremia during highly active antiretroviral therapy (HAART), resting CD4+ T-cell populations do not produce virus without cellular activation. To determine whether the nonproductive nature of the infection in resting CD4+ T cells is due to retroviral integration into chromosomal regions that are repressive for transcription, we used inverse PCR to characterize the HIV-1 integration sites in vivo in resting CD4+ T cells from patients on HAART. Of 74 integration sites from 16 patients, 93% resided within transcription units, usually within introns. Integration was random with respect to transcriptional orientation relative to the host gene and with respect to position within the host gene. Of integration sites within well-characterized genes, 91% (51 of 56) were in genes that were actively expressed in resting CD4+ T cells, as directly demonstrated by reverse transcriptase PCR (RT-PCR). These results predict that HIV-1 sequences may be included in the primary transcripts of host genes as part of rapidly degraded introns. RT-PCR experiments confirmed the presence of HIV-1 sequences within transcripts initiating upstream of the HIV-1 transcription start site. Taken together, these results demonstrate that HIV-1 genomes reside within actively transcribed host genes in resting CD4+ T cells in vivo.

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HIV-1 Latency Due to the Site of Proviral Integration

Virology ◽

10.1006/viro.1993.1545 ◽

1993 ◽

Vol 196 (2) ◽

pp. 849-854 ◽

Cited By ~ 45

Author(s):

Barbara J. Winslow ◽

Roger J. Pomerantz ◽

Omar Bagasra ◽

Didier Trono

Keyword(s):

Proviral Integration ◽

Hiv 1

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

mBio ◽

10.1128/mbio.00568-21 ◽

2021 ◽

Vol 12 (2) ◽

Author(s):

Michael J. Bale ◽

Mary Grace Katusiime ◽

Daria Wells ◽

Xiaolin Wu ◽

Jonathan Spindler ◽

...

Keyword(s):

T Cell ◽

Infected Cell ◽

Host Cells ◽

T Cell Clones ◽

Immune Systems ◽

Cell Clones ◽

Integration Sites ◽

Selection For ◽

Hiv 1

ABSTRACT Little is known about the emergence and persistence of human immunodeficiency virus (HIV)-infected T-cell clones in perinatally infected children. We analyzed peripheral blood mononuclear cells (PBMCs) for clonal expansion in 11 children who initiated antiretroviral therapy (ART) between 1.8 and 17.4 months of age and with viremia suppressed for 6 to 9 years. We obtained 8,662 HIV type 1 (HIV-1) integration sites from pre-ART samples and 1,861 sites from on-ART samples. 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 to 9 years on ART. A comparison of integration sites in the samples obtained on ART with healthy donor PBMCs 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. IMPORTANCE HIV-1 integrates its genome into the DNA of host cells. Consequently, HIV-1 genomes are copied with the host cell DNA during cellular division. Pediatric immune systems differ significantly from adults, consisting primarily of naive T cells, which have low expression of the HIV-1 coreceptor CCR5. This difference may result in variances in the number or size of infected cell clones that persist in children on ART. Here, we provide the most extensive analysis of the integration landscape of HIV-1 in children. We found that, despite the largely naive cell populations in neonatal immune systems, patterns of HIV-1 integration and the size of infected cell clones are as large and widespread as those in adults. Furthermore, selection for integration events in proto-oncogenes were observed in children despite early ART. If such cell clones persist for the life span of these individuals, there may be long-term consequences that have yet to be realized.

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HIV-1 replication complexes accumulate in nuclear speckles and integrate into speckle-associated genomic domains

Nature Communications ◽

10.1038/s41467-020-17256-8 ◽

2020 ◽

Vol 11 (1) ◽

Cited By ~ 8

Author(s):

Ashwanth C. Francis ◽

Mariana Marin ◽

Parmit K. Singh ◽

Vasudevan Achuthan ◽

Mathew J. Prellberg ◽

...

Keyword(s):

Viral Replication ◽

Nuclear Import ◽

Integration Site ◽

Nuclear Speckles ◽

Nuclear Entry ◽

Primary Monocyte ◽

Integration Sites ◽

Cleavage And Polyadenylation ◽

Specificity Factor ◽

Hiv 1

AbstractThe early steps of HIV-1 infection, such as uncoating, reverse transcription, nuclear import, and transport to integration sites are incompletely understood. Here, we imaged nuclear entry and transport of HIV-1 replication complexes in cell lines, primary monocyte-derived macrophages (MDMs) and CD4+ T cells. We show that viral replication complexes traffic to and accumulate within nuclear speckles and that these steps precede the completion of viral DNA synthesis. HIV-1 transport to nuclear speckles is dependent on the interaction of the capsid proteins with host cleavage and polyadenylation specificity factor 6 (CPSF6), which is also required to stabilize the association of the viral replication complexes with nuclear speckles. Importantly, integration site analyses reveal a strong preference for HIV-1 to integrate into speckle-associated genomic domains. Collectively, our results demonstrate that nuclear speckles provide an architectural basis for nuclear homing of HIV-1 replication complexes and subsequent integration into associated genomic loci.

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