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

Background : The human leukaemia virus HTLV-1 expresses essential accessory genes that manipulate the expression, splicing and transport of viral mRNAs.  Two of these genes, tax and hbz , 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.  tax, on the proviral plus strand, is usually silent in freshly-isolated cells, whereas the minus-strand-encoded hbz gene is persistently expressed at a low level.  However, the persistently activated host immune response to Tax indicates frequent expression of tax in vivo.  Methods : 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.  Results : We found strong heterogeneity both within and between clones in the expression of the proviral plus-strand (detected by hybridization to the tax gene) and the minus-strand ( hbz gene). Both genes are transcribed in bursts; tax expression is enhanced in the absence of hbz , while hbz expression increased in cells with high tax expression. Surprisingly, we found that hbz expression is strongly associated with the S and G 2 /M phases of the cell cycle, independent of tax expression.  Contrary to current belief, hbz is not expressed in all cells at all times, even within one clone.  In hbz -positive cells, the abundance of hbz transcripts showed a very strong positive linear correlation with nuclear volume. Conclusions : 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.