Dynamics and consequences of the HTLV-1 proviral plus-strand burst

Abstract Expression of the transcriptional transactivator protein Tax, encoded on the proviral plus-strand of human T-cell leukaemia virus type 1 (HTLV-1), is crucial for the replication of the virus, but Tax-expressing cells are rarely detected in fresh blood ex vivo . The dynamics and consequences of the proviral plus-strand transcriptional burst remain insufficiently characterised. We combined time-lapse live-cell imaging, single-cell tracking and mathematical modelling to study the dynamics of Tax expression at single-cell resolution in two naturally-infected T-cell clones transduced with a short-lived enhanced green fluorescent protein (d2EGFP) Tax reporter system. Five different patterns of Tax expression were observed during the 30-hour observation period; the distribution of these patterns differed between the two clones. The mean duration of Tax expression in the two clones was 94 and 417 hours respectively, estimated from mathematical modelling of the experimental data. Tax expression was associated with decreased proliferation, increased apoptosis, enhanced activation of the DNA damage response pathways, and slower cell-cycle progression. Longer-term follow-up (14 days) revealed an increase in the proportion of proliferating cells and a decrease in the fraction of apoptotic cells as the cells ceased Tax expression, resulting in a greater net expansion of the initially Tax-positive population. Time-lapse live-cell imaging showed enhanced cell-to-cell adhesion among Tax-expressing cells, and decreased cell motility of Tax-expressing cells at the single-cell level. The results demonstrate the within-clone and between-clone heterogeneity in the dynamics and patterns of HTLV-1 plus-strand transcriptional bursts and the balance of positive and negative consequences of the burst for the host cell. Author Summary Human T-cell leukaemia virus type 1 (HTLV-1) causes disabling or fatal diseases in up to 10% of the infected individuals. The expression of viral protein Tax is essential to cause new infections and contributes to HTLV-1-associated diseases. The proviral plus-strand, which encodes Tax, is expressed in intense intermittent bursts. However, the kinetics of Tax expression and its short and longer-term impact on the infected cell are not well understood. We combined live-cell imaging and mathematical modelling to study Tax expression kinetics in two naturally-infected T-cell clones. Single-cell analysis showed five patterns of Tax expression, with most Tax-positive cells expressing continuously during the 30-hour imaging. The average duration of Tax expression in the two clones was 94 and 417 hours respectively, by mathematical modelling. Tax expression correlated with decreased proliferation, increased apoptosis, enhanced activation of DNA damage response pathways and delayed progression through the cell-cycle. Extended observation showed an increase in the proportion of proliferating cells and a decrease in the percentage of apoptotic cells as cells ceased Tax expression, resulting in a greater net growth of the originally Tax-positive population. Tax-expressing cells also formed cell clumps and showed reduced cell movement. This study highlights prolonged Tax expression and its short and longer-term impact on naturally-infected cells.