Abstract
BackgroundHIV-infected cell lines are widely used to study latent HIV infection, which is considered the main barrier to HIV cure. We hypothesized that these cell lines differ from each other and from cells from HIV-infected individuals in the mechanisms underlying latency.ResultsTo quantify the degree to which HIV expression is inhibited by blocks at different stages of HIV transcription, we employed a recently-described panel of RT-ddPCR assays to measure levels of 7 HIV transcripts (“read-through,” initiated, 5′ elongated, mid-transcribed/unspliced [Pol], distal-transcribed [Nef], polyadenylated, and multiply-sliced [Tat-Rev]) in bulk populations of latently-infected (U1, ACH-2, J-Lat) and productively-infected (8E5, activated J-Lat) cell lines. To assess single-cell variation and investigate cellular genes associated with HIV transcriptional blocks, we developed a novel multiplex qPCR panel and quantified single cell levels of 7 HIV targets and 89 cellular transcripts in latently- and productively-infected cell lines. The bulk cell HIV transcription profile differed dramatically between cell lines and cells from ART-suppressed individuals. Compared to cells from ART-suppressed individuals, latent cell lines showed lower levels of HIV transcriptional initiation and higher levels of polyadenylation and splicing. ACH-2 and J-Lat cells showed different forms of transcriptional interference, while U1 cells showed a block to elongation. Single-cell studies revealed marked variation between/within cell lines in expression of HIV transcripts, T cell phenotypic markers, antiviral factors, and genes implicated in latency. Expression of multiply-spliced HIV Tat-Rev was associated with expression of cellular genes involved in activation, tissue retention, T cell transcription, and apoptosis/survival.ConclusionsHIV-infected cell lines differ from each other and from cells from ART-treated individuals in the mechanisms governing latent HIV infection. These differences in viral and cellular gene expression must be considered when gauging the suitability of a given cell line for future research on HIV. At the same time, some features were shared across cell lines, such as low expression of antiviral defense genes and a relationship between productive infection and genes involved in survival. These features may contribute to HIV latency or persistence in vivo, and deserve further study using novel single cell assays such as those described in this manuscript.
Highlights
HIV-infected cell lines are widely used to study latent HIV infection, which is considered the main bar‐ rier to HIV cure
HIV DNA levels differ between cell lines As a surrogate measure of HIV infection frequency in each cell line, we quantified HIV DNA levels using ddPCR assays for 5 different proviral regions and normalized them to cell numbers by DNA mass and by measured copies of the human gene Telomere Reverse Transcriptase (TERT) (Additional file 1: Fig. S1A, B)
Single cell multiplexed qPCR is sensitive and reproducible for detection of cellular and HIV transcripts To determine whether there is cell-to-cell variability within cell lines, and to investigate cellular genes that may be associated with blocks to HIV expression, we developed a novel panel of assays to quantify multiple HIV targets and cellular transcripts at the single cell level using the Fluidigm C1 and Biomark HD platforms
Summary
HIV-infected cell lines are widely used to study latent HIV infection, which is considered the main bar‐ rier to HIV cure. We hypothesized that these cell lines differ from each other and from cells from HIV-infected individu‐ als in the mechanisms underlying latency. Latently-infected CD4+ T cells are exceedingly rare in HIV-infected ART-suppressed individuals (about 1 per million cells [6]), and no methods currently exist to isolate or characterize these cells without ex vivo activation, which reverses latency. It is extremely challenging to investigate these questions using cells from ART-suppressed individuals because no methods exist to distinguish latently-infected cells from uninfected cells or cells infected with defective or non-inducible proviruses
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