Kinetics of HIV-1 Latency Reversal Quantified on the Single-Cell Level Using a Novel Flow-Based Technique

Abstract

O ne major obstacle toward an effective human immunodeficiency virus type 1 (HIV-1) cure is the establishment of a pool of long-lived latently infected cells early after infection (1). Using the rhesus macaque model of simian immunodeficiency virus (SIV) infection, it was recently shown that latent reservoirs could be seeded as early as 3 days after SIV exposure and before the detection of viremia in blood (2). The vast majority of cells infected with HIV-1 will die as a consequence of the infection or will be eliminated by the immune system. A minority of infected cells, however, turns into latently infected resting cells. These cells can either be reactivated and release de novo HIV-1 particles (3) or persist and homeostatically proliferate as long-lived memory T cells (4, 5). While current antiretroviral treatments (ARTs) can efficiently suppress HIV-1 replication and have dramatically improved the life expectancy and life quality of infected individuals, ART cannot eradicate the latent viral reservoir.Several different biological processes have been described to maintain latency in HIV-1-infected cells. Host transcription factors (TFs) such as nuclear factor kappa light-chain enhancer of activated B cells (NF-B) have multiple binding sites in the 5= long terminal repeat (LTR) of the HIV-1 genome, and their binding has been demonstrated to be necessary to initiate HIV-1 transcription (6). Sequestration of these TFs in the cytoplasm is one of the mechanisms enabling viral latency (7). Another described HIV-1 latency mechanism involves histone deacetylase (HDAC)-mediated epigenetic silencing (8). During latency establishment, HDAC molecules are recruited toward the 5= LTR of HIV-1 (9, 10) and therefore maintain the LTR in a repressed state (11). Several HDAC inhibitors (HDACis) targeting HDAC molecules have been tested for their ability to reactivate latently HIV-1-infected cells, including vorinostat, panobinostat, entinostat, and romidepsin (RMD). These HDACis proved to efficiently induce HIV-1 expression in latently infected resting CD4 ϩ T cells from HIV-1-infected individuals (8,12,13). RMD, a drug that has been used for the treatment of peripheral T-cell lymphoma,

Single-Cell Analysis of Quiescent HIV Infection Reveals Host Transcriptional Profiles that Regulate Proviral Latency

“…Flow cytometry using probes for viral transcripts has also yielded insights into diversity of virus and host gene expression in individual cells during infection (Baxter et al, 2016. ; Bolton et al, 2017; Martrus et al, 2016). Two recent publications have also examined HIV latency using scRNA-seq of a primary cell model (Golumbeanu et al, 2018) and from sorted patient samples (Cohn et al, 2018), yielding important insights.…”

Cancer‐related mRNA expression analysis using a novel flow cytometry‐based assay

“…To the best of our knowledge, this is the first report describing this technique in a cancer-related setting, and only a handful reports have addressed its application worldwide (20)(21)(22)(23)(24)(25). Subsequently, guidelines are lacking on how to interpret the mRNA amplification signal.…”

“…Only a handful of reports have documented its application and, to the best of our knowledge, cancer-related research has not yet been addressed (20)(21)(22)(23)(24)(25). Herein, the single-cell resolution of FCM is combined with a hybridization-based branched DNA (bDNA) technology, allowing intracellular RNA detection followed by exponential signal amplification.…”

“…Herein, the single-cell resolution of FCM is combined with a hybridization-based branched DNA (bDNA) technology, allowing intracellular RNA detection followed by exponential signal amplification. Only a handful of reports have documented its application and, to the best of our knowledge, cancer-related research has not yet been addressed (20)(21)(22)(23)(24)(25). Here, we evaluated the feasibility of PrimeFlow TM RNA assay in detecting key target mRNAs in NB and AML, using cell lines and patient samples, respectively.…”

The Alphabet Soup of HIV Reservoir Markers

“…Simultaneous use of PrimeFlow and p24 protein staining via flow cytometry was successfully reported in cell lines and in vitro infected cells [53]. The PrimeFlow assay is reported to detect different classes of HIV RNA transcripts with a limit of detection (LOD) of 10–100 infected cells within 10 6 total cells [54].…”