RT Journal Article SR Electronic T1 Kinetics of HTLV-1 reactivation from latency quantified by single-molecule RNA FISH and stochastic modelling JF bioRxiv FD Cold Spring Harbor Laboratory SP 631697 DO 10.1101/631697 A1 Michi Miura A1 Supravat Dey A1 Abhyudai Singh A1 David Rueda A1 Charles R. M. Bangham YR 2019 UL http://biorxiv.org/content/early/2019/05/08/631697.abstract AB 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 rapid transcriptional burst. 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. The model indicated that the average duration of an HTLV-1 sense-strand transcriptional burst is less than one hour. HTLV-1 antisense transcription was stable during reactivation of the sense-strand. The antisense transcript HBZ was constantly produced at ∼0.1 molecules per hour per HTLV-1+ cell; however, 20% to 70% of HTLV-1-infected cells were HBZ-negative at a given time. 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.Author summary 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 visualised 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 burst of rapid transcription. Computer simulation, based on the experimental results, indicated that HTLV-1 terminates the transcriptional bursting in less than an hour. The HTLV-1 antisense transcript HBZ was constantly produced at a low level, and 20% to 70% 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.