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> in vivo. </ns4:p> <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: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: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 in vivo. Our results offer an explanation for the paradoxical correlations observed between the host immune response and HTLV-1 transcription. </ns4:p>