Quantification of HTLV-1 expression at the single-cell level in naturally-infected T-lymphocytes

• Main Author: Billman, Martin Rafael
• Other Authors: Bangham, Charles ; Rueda, David
• Published: Imperial College London 2017
• Subjects: 610
• Online Access: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.745254

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. By using single-molecule RNA-FISH, I was able to quantify the expression of HTLV-1 plus-strand and minus-strand transcripts at the single-cell level in naturally-infected T-cell clones isolated from peripheral blood. While hbz is the lone transcript located on the HTLV-1 minus-strand, it is not possible to definitively separate tax transcripts from its neighbours on the plus-strand by means of single-molecule RNA-FISH. Detected plus-strand transcripts are referred to as tax transcripts throughout the thesis, but it is important to remember that the probe which targets the tax sequence will also detect the other plus-strand products, even if they are not thought to be expressed in the same volume as tax. Measuring viral transcription at the single-cell level revealed strong within-clone and between-clone heterogeneity in the expression of tax and hbz. Both tax and hbz are transcribed in bursts; tax expression is enhanced in the absence of hbz, while hbz expression increased in cells with high tax expression. Unexpectedly, hbz expression was found to be strongly associated with the G2/M phase of the cell cycle, independent of tax expression. There is also a link between a cell’s level of hbz expression and its volume. These results offer an explanation for the paradoxical correlations observed between the host immune response and HTLV-1 transcription. While hbz is the lone transcript located on the HTLV-1 minus-strand, it is not possible to definitively separate tax transcripts from its neighbours on the plus-strand by means of single-molecule RNA-FISH. Detected plus-strand transcripts are referred to as tax transcripts throughout the thesis, but it is important to remember that the probe which target the tax sequence will also detect the other plus-strand products, even if they are not thought to be expressed in the same volume as tax. Measuring viral transcription at the single-cell level revealed strong within-clone and between-clone heterogeneity in the expression of tax and hbz. Both tax and hbz are transcribed in bursts; tax expression is enhanced in the absence of hbz, while hbz expression increased in cells with high tax expression. Unexpectedly, hbz expression was found to be strongly associated with the G2/M phase of the cell cycle, independent of tax expression. There is also a link between a cell’s level of hbz expression and its volume. These results offer an explanation for the paradoxical correlations observed between the host immune response and HTLV-1 transcription.