Kinetics of HTLV-1 reactivation from latency quantified by single-molecule RNA FISH and stochastic modelling

Abstract

20The human T cell leukemia virus HTLV-1 establishes a persistent infection in vivo in which the viral sense-21 strand transcription is usually silent at a given time in each cell. However, cellular stress responses 22 trigger the reactivation of HTLV-1, enabling the virus to transmit to a new host cell. Using single-23 molecule RNA FISH, we measured the kinetics of the HTLV-1 transcriptional reactivation in peripheral 24 blood mononuclear cells (PBMCs) isolated from HTLV-1 + individuals. The abundance of the HTLV-1 sense 25 and antisense transcripts was quantified hourly during incubation of the HTLV-1-infected PBMCs ex vivo. 26We found that, in each cell, the sense-strand transcription occurs in two distinct phases: the initial low-27 rate transcription is followed by a phase of rapid transcription. The onset of transcription peaked 28 between 1 and 3 hours after the start of in vitro incubation. The variance in the transcription intensity 29 was similar in polyclonal HTLV-1 + PBMCs (with tens of thousands of distinct provirus insertion sites), and 30 in samples with a single dominant HTLV-1 + clone. A stochastic simulation model was developed to 31 estimate the parameters of HTLV-1 proviral transcription kinetics. In PBMCs from a leukemic subject 32 with one dominant T-cell clone, the model indicated that the average duration of HTLV-1 sense-strand 33 activation by Tax (i.e. the rapid transcription) was less than one hour. HTLV-1 antisense transcription 34 was stable during reactivation of the sense-strand. The antisense transcript HBZ was produced at an 35 average rate of ~0.1 molecules per hour per HTLV-1 + cell; however, between 20% and 70% of HTLV-1-36 infected cells were HBZ-negative at a given time, the percentage depending on the individual subject. 37 HTLV-1-infected cells are exposed to a range of stresses when they are drawn from the host, which 38 initiate the viral reactivation. We conclude that whereas antisense-strand transcription is stable 39 throughout the stress response, the HTLV-1 sense-strand reactivation is highly heterogeneous and 40 occurs in short, self-terminating bursts. 41 3 Author summary 42 Human retroviruses such as HIV-1 and HTLV-1 (human T cell leukemia virus) can establish a latent 43 infection in the host cell. However, these viruses need to be able to produce viral genome to propagate 44 in a new host. HTLV-1-infected cells are transmitted through breastfeeding, blood transfusion and sexual 45 contact, and HTLV-1 restores transcription once the infected cells are drawn from infected individuals. 46We measured the kinetics of the HTLV-1 transcriptional reactivation in blood cells isolated from HTLV-1 + 47 individuals by single-molecule RNA FISH. Viral transcripts were visualised as diffraction-limited spots and 48 their abundance was quantified at one-hour intervals. The onset of the virus transcription peaked after 49 one to three hours of incubation. In each cell, a short period of slow HTLV-1 transcription was followed 50 by a phase of rapid transcription. Computer simu...

Real-time single-cell characterization of the eukaryotic transcription cycle reveals correlations between RNA initiation, elongation, and cleavage

“…Measurements of cell-to-cell variability in transcription initiation rate have uncovered, for example, the existence of transcriptional bursting and mechanisms underlying the establishment of precise developmental boundaries [ 39 , 40 , 45 , 47 , 48 , 55 , 57 ]. Yet, to date, these studies have mostly employed techniques such as single-molecule FISH to count the number of nascent transcripts on a gene or the number of cytoplasmic mRNA molecules [ 39 – 41 , 43 , 45 , 48 – 52 , 56 – 58 , 77 – 84 ]. In principle, these techniques do not report on the variability in transcription initiation alone; they convolve this measurement with variability in other steps of the transcription cycle [ 76 , 81 ].…”

“…Additionally, while fixed-tissue technologies such as single-molecule RNA-FISH provide superior spatial and molecular resolution to current live imaging technologies [ 45 , 57 ], the fixation process necessarily prevents temporal analysis of the same single cell to study these dynamic transcriptional processes. Thus, live imaging approaches offer a complementary approach to widespread RNA-FISH studies of transcriptional dynamics [ 39 – 41 , 43 , 45 , 48 – 52 , 56 – 58 , 77 – 84 ].…”

HDAC inhibitors Panobinostat and Romidepsin enhance tax transcription in HTLV-1-infected cell lines and freshly isolated patients’ T-cells

“…HBZ served as a surrogate for the minus-strand transcription of HTLV-1. In contrast to Tax, which is only expressed in infrequent bursts, HBZ is continuously produced, albeit at a very low level (59,60). The HBZ gene exists as unspliced (usHBZ) and spliced version (sHBZ), whereas the sHBZ protein inhibits Tax-mediated transcriptional activation of the sense strand more strongly than usHBZ (38).Thus, we decided to focus on sHBZ.…”