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,taxandhbz, 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-encodedhbzgene is persistently expressed at a low level. However, the persistently activated host immune response to Tax indicates frequent expression oftaxin 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 thetaxgene) and the minus-strand (hbzgene). Both genes are transcribed in bursts;taxexpression is enhanced in the absence ofhbz, whilehbzexpression increased in cells with hightaxexpression. Surprisingly, we found thathbzexpression is strongly associated with the S and G2/M phases of the cell cycle, independent oftaxexpression. Contrary to current belief,hbzis not expressed in all cells at all times, even within one clone. Inhbz-positive cells, the abundance ofhbztranscripts 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.