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

Abstract

Clonal expansion of HIV-infected cells contributes to the long-term persistence of the HIV reservoir in ART-suppressed individuals. However, the contribution from cell clones that harbor inducible proviruses to plasma viremia 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 show 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 reveal that cell clones with predicted pathogen-specificity can harbor inducible proviruses integrated into cancer-related genes. Furthermore, we find several matches between proviruses retrieved with STIP-Seq and plasma viruses obtained during ART and upon treatment interruption, suggesting that STIP-Seq can capture clones that are responsible for low-level viremia or viral rebound.

Chemistry and Bioinformatics Considerations in Using Next-Generation Sequencing Technologies to Inferring HIV Proviral DNA Genome-Intactness

“…FLIP-seq and similar technologies have been applied to cross-sectionally examine viral reservoir landscapes in various CD4 + T-cell subsets [ 7 , 8 ] and to longitudinally examine the evolution of the viral DNA genome populations over time [ 20 , 50 ]. MIP-seq has been applied to compare viral integration sites of intact versus defective genomes [ 32 ], reveal unique patterns of genome-intact viral integration sites in HIV elite controllers [ 24 ], and has been further developed by another group of researchers to include co-capturing of T-cell receptor sequences for antigen specificity inferences of the infected cells [ 73 ]. Application of these sequencing technologies to various cohorts have resulted in a rich collection of HIV-DNA genome sequences archived in public repositories, such as the HIV Proviral Sequence Databases [ 73 ] and the Los Alamos HIV Sequence Database [ 74 ], which are used in part to guide the design of relatively low-cost ddPCR-based assays, such as IPDA [ 27 ] and a multiplex assay by Levy et al [ 29 ] for the quantification of intact versus defective HIV-DNA genomes.…”

“…MIP-seq has been applied to compare viral integration sites of intact versus defective genomes [ 32 ], reveal unique patterns of genome-intact viral integration sites in HIV elite controllers [ 24 ], and has been further developed by another group of researchers to include co-capturing of T-cell receptor sequences for antigen specificity inferences of the infected cells [ 73 ]. Application of these sequencing technologies to various cohorts have resulted in a rich collection of HIV-DNA genome sequences archived in public repositories, such as the HIV Proviral Sequence Databases [ 73 ] and the Los Alamos HIV Sequence Database [ 74 ], which are used in part to guide the design of relatively low-cost ddPCR-based assays, such as IPDA [ 27 ] and a multiplex assay by Levy et al [ 29 ] for the quantification of intact versus defective HIV-DNA genomes. In summary, this commentary highlights that deep sequencing like FLIP-seq and MIP-seq offers advantages, such as high-resolution data quality enabling post-hoc quality control for true single-genome amplification; but in order to take full advantage of these technologies, one has to be mindful to take necessary quality control steps to monitor data quality.…”

Clonal Expansion of Infected CD4+ T Cells in People Living with HIV

“…Techniques that make it possible to obtain both the structure of the provirus and its integration site have been developed. One method uses multiple displacement amplification of endpoint-diluted DNA to create multiple copies of genomic DNA containing a single integrated provirus, which can then be used for both integration site analysis and proviral sequencing [32,43,44]. At present, these methods are labor intensive, and the number of cases in which both the structure of a provirus and its cognate integration site are known is limited.…”

The Clonal Expansion Dynamics of the HIV-1 Reservoir: Mechanisms of Integration Site-Dependent Proliferation and HIV-1 Persistence

“…Blood sampling before and after analytical treatment interruptions (ATIs) in therapeutic intervention clinical trials may provide more clues regarding the origin of viral rebound. The Chomont group developed the simultaneous T cell receptor sequence (TCR), integration site, and provirus sequencing (STIP-Seq) method that captures HIV-1 clonal expansion dynamics [ 40 ]. Briefly, STIP-seq involves sorting HIV-1 p24 + single-cells for genomic DNA extraction and phi29-mediated whole-genome amplification.…”

“…The amplified DNA is then split into separate aliquots for TCR sequencing, integration site sequencing, and HIV-1 near full-length proviral genome sequencing. Some HIV-1 proviruses in clonally expanded CD4 + T cells captured during viral suppression are identical to plasma viruses captured during ATIs, suggesting that clonally expanded HIV-1-infected CD4 + T cells can contribute to viral rebound [ 40 ] ( Figure 1 a).…”