The human T-cell leukaemia virus HTLV-1: transcriptional heterogeneity at the single-cell level

The human T-cell leukaemia virus HTLV-1, a distant cousin of HIV, causes an aggressive type of leukaemia in about 5% of people it in infects, and an inflammatory disease of the nervous system that culminates in paralysis of the legs in a further 1 to 5 %. Like HIV, this virus can remain silent in the infected cells, and so can escape elimination by antiviral drugs and the immune response. The virus can then be reactivated, to spread both within that infected person and to be transmitted to another person. We now aim to discover what controls the balance between the latency of the virus and its reactivation. We have developed a range of techniques to study the dynamics and the molecular mechanisms of HTLV-1 reactivation in single infected cells. The results of these experiments will lead to a fundamental advance in the understanding of this virus and the serious diseases that it causes, and may indicate new ways to treat or prevent these diseases.

The human T-cell leukaemia virus HTLV-1 causes an aggressive leukaemia or inflammatory diseases in ~10% of people it infects. There is no satisfactory treatment available for either condition; the average survival time of the malignancy is less than one year, despite treatment. HTLV-1-infected people typically make a strong immune response to the virus, but the impact on the virus of this response is limited because, as we and others have recently shown, HTLV-1 is transcribed only in intermittent bursts. A central question arises: how does HTLV-1 regulate the balance between transcription and latency, so as to maintain the proviral load while at the same time avoiding destruction by the host's immune response? The aim of this project is twofold: first, to quantify the dynamics and the heterogeneity of HTLV-1 reactivation at the single-cell level, and second, to identify the molecular mechanisms that regulate this reactivation.

We will use recent advances in single-cell analytic techniques to elucidate the regulation of transcriptional latency of HTLV-1. In the collaborating laboratories in Japan and UK, there are strong existing research programmes on HTLV-1 molecular virology. The Co-PI in Japan is doing single cell transcriptome analysis, using peripheral blood mononuclear cells. The Co-PI in UK is also investigating HTLV-1 infection at the single-cell level, using real-time fluorescent microscopy, single-molecule RNA-FISH (smFISH), transcriptomics, and mathematical techniques. By combining these techniques and exchanging materials in the two laboratories, we will accelerate the progress of the research and provide training for doctoral students and postdoctoral workers in the two groups. The results of this work will identify both key mechanisms of viral persistence and disease development, molecular markers to predict disease development, and new potential molecular targets for the treatment of leukaemia.

Lasting collaboration

The Co-PI in Japan, Yorifumi Satou, worked with Charles Bangham for two years from 2011 to 2013. After Yorifumi Satou obtained a PI position in Japan, we started international collaboration and have since generated significant scientific achievements and published co-authored original data papers. We now propose to enhance and consolidate the international collaboration between UK and Japan. We attach special importance to the training and career development of young researchers in developing this lasting international collaboration.

Scientific impact:
The collaboration will deliver novel molecular techniques and approaches whose application and significance go beyond the field of HTLV-1 infection to other retroviral infections (notably HIV-1), transposable genetic elements, and mammalian genome analysis.

Bangham has previously established key techniques for the analysis of HTLV-1 propagation and persistence:
1. High-throughput HTLV-1 integration site analysis (Blood 2011, PLoS Path 2013, Blood 2015);
2. RNA-FISH to monitor proviral transcription (Wellcome Open Res 2017, bioRxiv 2019).

Satou has also established key techniques to characterize the HTLV-1 provirus:
1. HTLV-1 DNA-capture-seq (Sci Rep 2016, Cell Reports 2019).
2. Droplet single cell RNA-seq and bioinformatics analysis of the single cell data (CellRanger, Seurat, Monocle, ect).

Medical impact:
Because there is no satisfactory treatment available for either the malignant or the inflammatory diseases caused by HTLV-1, there is an urgent need for better understanding of the persistence and pathogenesis of the virus and for new therapeutic leads. The proposed collaborative programme will identify new mechanisms of the regulation of latency and persistence that will indicate novel potential therapeutic strategies.

Japan is an endemic area of HTLV-1 infection, with around 800,000 people infected. Kumamoto is located at the centre of the HTLV-1 endemic area in the south island of Kyushu. The Co-PI in Japan, Yorifumi Satou, has established a network to collect additional clinical samples from the neighbouring prefectures of Saga, Miyazaki, and Kagoshima. In these centres, our collaborators are running clinics specific for HTLV-1 carriers; fresh PBMCs from individuals with HTLV-1 infection are available from these clinics, including longitudinal samples.