Single-cell heterogeneity and cell-cycle-related viral gene bursts in the human leukaemia virus HTLV-1

<ns4:p><ns4:bold>Background</ns4:bold>: The human leukaemia virus HTLV-1 expresses essential accessory genes that manipulate the expression, splicing and transport of viral mRNAs. Two of these genes,<ns4:italic>tax</ns4:italic>and<ns4:italic>hbz</ns4:italic>, also promote proliferation of the 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. <ns4:italic>tax,</ns4:italic>on the proviral plus strand, is usually silent in freshly-isolated cells, whereas the minus-strand-encoded<ns4:italic>hbz</ns4:italic>gene is persistently expressed at a low level. However, the persistently activated host immune response to Tax indicates frequent expression of<ns4:italic>tax</ns4:italic><ns4:italic>in vivo</ns4:italic>. </ns4:p><ns4:p><ns4:bold>Methods</ns4:bold>: We used single-molecule RNA-FISH to quantify the expression of 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. </ns4:p><ns4:p><ns4:bold>Results</ns4:bold>: We found strong heterogeneity both within and between clones in the expression of the proviral plus-strand (detected by hybridization to the<ns4:italic>tax</ns4:italic>gene) and the minus-strand (<ns4:italic>hbz</ns4:italic>gene). Both genes are transcribed in bursts;<ns4:italic>tax</ns4:italic>expression is enhanced in the absence of<ns4:italic>hbz</ns4:italic>, while<ns4:italic>hbz</ns4:italic>expression increased in cells with high<ns4:italic>tax</ns4:italic>expression. Surprisingly, we found that<ns4:italic>hbz</ns4:italic>expression is strongly associated with the S and G<ns4:sub>2</ns4:sub>/M phases of the cell cycle, independent of<ns4:italic>tax</ns4:italic>expression. Contrary to current belief,<ns4:italic>hbz</ns4:italic>is not expressed in all cells at all times, even within one clone. In<ns4:italic>hbz</ns4:italic>-positive cells, the abundance of<ns4:italic>hbz</ns4:italic>transcripts showed a very strong positive linear correlation with nuclear volume.</ns4:p><ns4:p><ns4:bold>Conclusions</ns4:bold>: The occurrence of intense, intermittent plus-strand gene bursts in independent primary HTLV-1-infected T-cell clones from unrelated individuals strongly suggests that the HTLV-1 plus-strand is expressed in bursts<ns4:italic>in vivo</ns4:italic>. Our results offer an explanation for the paradoxical correlations observed between the host immune response and HTLV-1 transcription.</ns4:p>