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

Abstract

The human T cell leukemia virus HTLV-1 establishes a persistent infection in vivo in which the viral sense-strand transcription is usually silent at a given time in each cell. However, cellular stress responses trigger the reactivation of HTLV-1, enabling the virus to transmit to a new host cell. Using single-molecule RNA FISH, we measured the kinetics of the HTLV-1 transcriptional reactivation in peripheral blood mononuclear cells (PBMCs) isolated from HTLV-1⁺ individuals. The abundance of the HTLV-1 sense and antisense transcripts was quantified hourly during incubation of the HTLV-1-infected PBMCs ex vivo. We found that, in each cell, the sense-strand transcription occurs in two distinct phases: the initial low-rate transcription is followed by a phase of rapid transcription. The onset of transcription peaked between 1 and 3 hours after the start of in vitro incubation. The variance in the transcription intensity was similar in polyclonal HTLV-1⁺ PBMCs (with tens of thousands of distinct provirus insertion sites), and in samples with a single dominant HTLV-1⁺ clone. A stochastic simulation model was developed to estimate the parameters of HTLV-1 proviral transcription kinetics. In PBMCs from a leukemic subject with one dominant T-cell clone, the model indicated that the average duration of HTLV-1 sense-strand activation by Tax (i.e. the rapid transcription) was less than one hour. HTLV-1 antisense transcription was stable during reactivation of the sense-strand. The antisense transcript HBZ was produced at an average rate of ~0.1 molecules per hour per HTLV-1⁺ cell; however, between 20% and 70% of HTLV-1-infected cells were HBZ-negative at a given time, the percentage depending on the individual subject. HTLV-1-infected cells are exposed to a range of stresses when they are drawn from the host, which initiate the viral reactivation. We conclude that whereas antisense-strand transcription is stable throughout the stress response, the HTLV-1 sense-strand reactivation is highly heterogeneous and occurs in short, self-terminating bursts. Human retroviruses such as HIV-1 and HTLV-1 (human T cell leukemia virus) can establish a latent infection in the host cell. However, these viruses need to be able to produce viral genome to propagate in a new host. HTLV-1-infected cells are transmitted through breastfeeding, blood transfusion and sexual contact, and HTLV-1 restores transcription once the infected cells are drawn from infected individuals. We measured the kinetics of the HTLV-1 transcriptional reactivation in blood cells isolated from HTLV-1⁺ individuals by single-molecule RNA FISH. Viral transcripts were visualized as diffraction-limited spots and their abundance was quantified at one-hour intervals. The onset of the virus transcription peaked after one to three hours of incubation. In each cell, a short period of slow HTLV-1 transcription was followed by a phase of rapid transcription. Computer simulation, based on experimental data on PBMCs from a leukemic patient with a single dominant HTLV-1-infected T cell clone, indicated that this rapid transcription from the HTLV-1 sense-strand promoter activated by Tax was terminated in less than an hour. The HTLV-1 antisense transcript HBZ was constantly produced at a low level, and 50% ± 20% of HTLV-1⁺ cells were negative for HBZ at a given time. These results demonstrate how rapidly HTLV-1 is reactivated and potentially becomes infectious, once HTLV-1⁺ cells are transmitted into a new host.