Single-cell heterogeneity and cell-cycle-related viral gene bursts

The human leukaemia virus HTLV-1 expresses essential Background accessory genes that manipulate the expression, splicing and transport of viral mRNAs. Two of these genes, and , also promote proliferation of the tax hbz infected cell, and both genes are thought to contribute to oncogenesis in adult T-cell leukaemia/lymphoma. The regulation of HTLV-1 proviral latency is not understood. on the proviral plus strand, is usually silent in freshly-isolated tax, cells, whereas the minus-strand-encoded gene is persistently expressed at hbz a low level. However, the persistently activated host immune response to Tax indicates frequent expression of in vivo. tax : We used single-molecule RNA-FISH to quantify the expression of Methods HTLV-1 transcripts at the single-cell level in a total of >19,000 cells from five T-cell clones, naturally infected with HTLV-1, isolated by limiting dilution from peripheral blood of HTLV-1-infected subjects. : We found strong heterogeneity both within and between clones in the Results expression of the proviral plus-strand (detected by hybridization to the tax gene) and the minus-strand ( gene). Both genes are transcribed in bursts; hbz expression is enhanced in the absence of , while expression tax hbz hbz increased in cells with high expression. Surprisingly, we found that tax hbz expression is strongly associated with the S and G /M phases of the cell cycle, independent of expression. Contrary to current belief, is not expressed tax hbz in all cells at all times, even within one clone. In -positive cells, the hbz abundance of transcripts showed a very strong positive linear correlation hbz with nuclear volume. : The occurrence of intense, intermittent plus-strand gene bursts Conclusions in independent primary HTLV-1-infected T-cell clones from unrelated individuals strongly suggests that the HTLV-1 plus-strand is expressed in bursts in vivo. Our results offer an explanation for the paradoxical correlations observed between the host immune response and HTLV-1 transcription. 1 2