The Impact of Cellular Proliferation on the HIV-1 Reservoir
Abstract
:1. Introduction
2. Cell Type, Proliferation Potential, and Growth Stimulus
2.1. Introduction to HIV Infection in Patients
2.2. Hematopoietic Potential
2.3. T Cell Reservoirs
3. HIV Sequence Integrity
3.1. Clonality and HIV Sequence Integrity in T Cell Subsets and HSPCs
3.2. Using HIV Sequences to Determine Clonality and Decay
4. Methods of Viral Spread
4.1. Cell-to-Cell Viral Spread
4.2. Viral Evolution and Insufficient ART
4.3. Expression from Unintegrated Virus
5. Insertion Site
5.1. Integration Site and Genome Capture Techniques
5.2. Integration Site Preferences
5.3. Informing Cure Strategies from Integration Sites
6. Techniques
7. Conclusions
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
Abbreviations
PBMC | peripheral blood mononuclear cell |
BMMC | bone marrow mononuclear cell |
HSPC | hematopoietic stem and progenitor cell |
HDI | histone deacetylase inhibitor |
TCR | T cell antigen receptor |
RT | reverse transcriptase |
pMHCII | peptide-bound major histocompatibility complex II |
APC | antigen presenting cell |
ORF | open reading frame |
ELISA | enzyme-linked immunosorbent assay |
rCD4 | resting CD4 |
MOLT-4/CCR5 | cells from the CCR5-expressing T cell line MOLT-4 |
FACS | Fluorescence Activated Cell Sorting |
TH | T helper cell |
TEM | T effector memory |
TCM | T central memory |
TTM | T transitional memory |
TSCM | T stem cell memory |
ART | antiretroviral therapy |
PV | peripheral virus |
MSD | major splice donor site |
LTR | long terminal repeat |
NFL | near full length. |
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Technique | Description | Advantages | Limitations |
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Modified TC-Seq [7,122] | Sonicate genomic DNA into small fragments. End-repair DNA fragments. Add dA-linker to 3’ ends. Attach linkers to 3’ ends using dT. Perform Nested PCR on fragments. Attach Illumina sequencing adaptors. Paired-end Illumina sequencing across integration sites. |
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ISLA (Integration site loop amplification) (Figure 5) [76] | Linear extension PCR products from the 3’end of HIV into chromosome are amplified with a random decamer complementary to the host genome with a U5-priming sequence-tail. The U5 primer is used as a reverse primer to convert the ssPCR into a dsPCR of 3’HIV-chromosome product. Linearized products produce a genetic barbell with LTR sequence on either side of human sequence which can be PCR amplified and prepared for NextGen sequencing. |
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Circular Template [11,132] | PCR amplification and sequencing across the insertion site starting from genomic DNA from HIV infected cells. A restriction enzyme (PstI) creates small provirus-host DNA fragments that can be ligated together into circular units and amplified using HIV-specific primers oriented in opposing directions across the integration site, then sequenced by Sanger sequencing. |
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Technique | Description | Advantages | Limitations |
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FLIPS (Full-length individual proviral sequencing) [15] | An assay used to capture nearly full-length genomes by two non-overlapping, nested-PCR reactions amplifying from the U5 region in the 5’ and 3’ LTRs. PCR products are sequenced by paired-end Illumina sequencing using the Nextera library preparation kit (Illumina). Reads are mapped by de novo genome assembly. |
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SCS Assay (single-cell sequencing) [108] | CD4+ T cells are FACS sorted from HIV-infected patients into a 96-well plate such that there is no more than one cell in any one well. Once the cell in each well is lysed, the DNA is distributed from one well into 10. PCR is used to amplify HIV DNA between gag-pol, identified through gel-electrophoresis, and sequenced. Sequenced products can then be aligned to a reference HIV genome. |
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SPS/SGS (Single-genome (provirus) sequencing and single genome amplification) [67,133,134]. | Virions collected from the peripheral blood or cultured media containing HIV virion-producing cells are collected, lysed, and converted to cDNA. The cDNA is serially diluted and used for qPCR with primer probe directed at pol (SGS) or env (SGA). Once HIV-containing samples are identified, the cDNA is then subjected to PCR amplification between gag-pol or env and sequenced. |
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Technique | Description | Advantages | Limitations |
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Matched integration site and proviral sequencing (MIP-Seq) [115] | Genomic DNA is isolated from CD4+ T cells, quantified using ddPCR for viral gag, and diluted to single proviral genomes based on ddPCR and Poisson distribution. This is followed by multiple displacement amplification (MDA) and whole genome amplification (WGA), generating 1,000-10,000 copies of gDNA. Amplified gDNA is divided such that some is used for NFL sequencing [14] and some for integration site analysis (ISLA; Table 1; Figure 5) or other methods. |
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Multiple-displacement amplification with single-genome sequencing (MDA-SGS) [131] | Genomic DNA is extracted from PBMCs or other primary cells and diluted across a 96-well plate. Whole-gDNA is amplified in-well using MDA. MDA wells are screened for proviruses of interest using SGS (subgenomic fragments) from P6 through part of RT. Then integration sites are determined using modified TC-Seq (Table 1) followed by NFL amplification using Sanger sequencing or PacBio sequencing. |
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Technique | Description | Advantages | Limitations |
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ddPCR (droplet digital PCR) (reviewed [135]) | Target molecules are emulsified into thousands of nanoliter droplets and amplified by PCR using a primer-probe set. Droplets containing HIV genomic material that fluoresces above a certain threshold will be considered positive. The ratio between the positive and negative droplets is used to calculate the absolute number of starting molecules using a Poisson distribution. |
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IPDA (intact proviral DNA assay) [136] | Uses two amplicons covering the packaging signal (ψ) and env region and ddPCR to designate deleted proviruses as defective. In parallel, multiplex PCR is performed with two unique primer-probe sets targeting ψ and env with unique labeling probes and measured by ddPCR. Primer-probe sets amplify validated, highly conserved regions of the genome. Droplets are scored based on expression of combinations of probe fluorescent patterns. |
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TILDA(Tat/Rev induced limiting dilution assay) [137] | CD4+ T cells are stimulated in vitro (PMA and ionomycin) to maximally produce tat/rev transcripts. Cells are serially diluted as replicates. Real-time PCR with primer-probe pairs are used to quantify inducible viral RNA. The frequency of cells with inducible HIV RNA can then be calculated from the number of positive wells at each dilution by the maximum likelihood method. |
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QVOA (quantitative viral outgrowth assay) [138,139] | Culture method to quantify the replication-competent viral reservoir. HIV(+) donor rCD4+ T cells are cultured with irradiated PBMCs and CD4+ T cells from an HIV(-) donor and stimulated (PHA; IL-2). Replication -competent virus can spread to HIV(-) CD4+ T cells, amplifying the infection, allowing detection and quantification of viral outgrowth. |
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mQVOA (Modified quantitative viral outgrowth assay) [140] | A more sensitive adaptation of the gold-standard assay; CD4+ T cells from patients are serially diluted and stimulated (αCD3/CD28 antibodies). MOLT-4/CCR5 cells are co-cultured with the primary cells. HIV RNA is extracted, and RT-qPCR is performed to amplify pol. The number of wells positive for HIV RNA at each dilution level are used to determine the infection frequency by maximum likelihood estimate. |
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dQVOA (differentiation culture quantitative viral Outgrowth assay) [141] | Measures the impact of TEM differentiation on induction and outgrowth of replication-competent HIV. rCD4+ T cells from patients are activated through culture with a differentiation cytokine cocktails to drive cells towards the TEM terminally differentiated subset. Cells are distributed at limiting dilutions and cultured in differentiation cytokines, then activated. Titer measured by p24 ELISA. |
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Virgilio, M.C.; Collins, K.L. The Impact of Cellular Proliferation on the HIV-1 Reservoir. Viruses 2020, 12, 127. https://doi.org/10.3390/v12020127
Virgilio MC, Collins KL. The Impact of Cellular Proliferation on the HIV-1 Reservoir. Viruses. 2020; 12(2):127. https://doi.org/10.3390/v12020127
Chicago/Turabian StyleVirgilio, Maria C., and Kathleen L. Collins. 2020. "The Impact of Cellular Proliferation on the HIV-1 Reservoir" Viruses 12, no. 2: 127. https://doi.org/10.3390/v12020127
APA StyleVirgilio, M. C., & Collins, K. L. (2020). The Impact of Cellular Proliferation on the HIV-1 Reservoir. Viruses, 12(2), 127. https://doi.org/10.3390/v12020127