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

Abstract

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.

Keywords: HTLV-; RNA-FISH; gene burst; latency; retrovirus; smFISH; transcription.

Figure 1.. Single-molecule RNA-FISH reveals heterogeneity of plus-strand and hbz expression within and between clonal cell populations.

a. Schematic of the HTLV-1 genome, showing the spliced transcripts of the plus-strand and hbz; the sequences targeted by probes on the plus-strand and the minus are marked by red and yellow flags respectively. b. Composite fluorescent micrograph with representative examples of cells expressing different levels of hbz: i) silent, ii) low (<5 spots) and iii) high (≥5 spots); and plus-strand RNA: iv) low (<100 spots), v) high (≥100 spots). The Roman numerals show the respective cells’ positions in panels c and d. Scale bar is 5 µm. c. Frequency distribution of hbz expression (number of hbz spots/cell) in individual cells, from one uninfected and two HTLV-1-infected clones. d. Frequency distribution of plus-strand expression in individual cells (same clones as in c); bins show the number of cells with 0-1 plus-strand spots (“0”), 2-50 spots (“50”), 51-100 spots (“100”), 101-150 spots (“150”) etc.

Figure 2.. Both plus-strand and hbz are expressed in bursts; plus-strand bursts are much larger than hbz bursts.

a. Representative plus-strand and hbz bursts. Dashed lines show the positions of the intensity profiles in panel b. Scale bar is 5 µm. b. Bursts are defined as specifically stained intranuclear spots that are significantly brighter than the main population of spots. c. Frequency of bursts is significantly reduced by blocking transcription with actinomycin D (n = 12,317 DMSO-treated cells, 7,120 actinomycin-D-treated cells from two replicates each of clones A and B). d. hbz bursts are small and uniform in size, whereas plus-strand bursts are significantly larger and more variable. SEM is shown, with 514 plus-strand and 800 hbz bursts from the pooled plus-strand-competent clones.

Figure 3.. plus-strand RNA is exported from the nucleus, whereas the majority of hbz is retained.

a. Representative images showing i) an optical slice illustrating the difference in cellular localisation of plus-strand RNA and hbz, ii) a typical plus-strand -high cell after 22 hours in DMSO and iii) a cell after 22 hours in actinomycin D. Image a.i is a single slice from a z-stack whereas ii and iii are maximum projections. Scale bar is 5 µm. b. hbz mRNA remains largely intranuclear, whereas plus-strand mRNA is mostly cytoplasmic; this difference is accentuated by blocking transcription with actinomycin D. n = 936 untreated, 390 DMSO-treated and 195 actinomycin D-treated cells, each from two pooled biological replicates of clones A and B. c. Rate of disappearance of total hbz spots following treatment with actinomycin D. The slope of the log-transformed data is the rate of decay. Individual measurements from replicates are shown, along with a linear regression and 95% confidence intervals (dashed lines); one of the four replicates failed at the 4 hour mark. n = 8126 total cells. d. Mean hbz spot count in hbz-positive cells was strongly correlated with nuclear volume.

Figure 4.. Relationship between expression of the plus-strand and hbz in individual cells.

a. Representative images of a i) plus-strand -silent cell, ii) plus-strand-high cell (≥100 spots) with a plus-strand burst, iii) plus-strand-high cell without a plus-strand burst (note the presence of an hbz burst), iv) plus-strand-low cell (<100 spots) and v) plus-strand-silent cell, with low-level hbz. b. plus-strand bursts occur more frequently in hbz-negative cells. c. plus-strand bursts are indicative of very high plus-strand expression, with the proportion of cells which have a burst increasing as the level of plus-strand RNA increases. d. hbz bursts occur more frequently in high-plus-strand cells than in plus-strand-negative or plus-strand-low cells. Numbers above columns denote the number of cells in the corresponding population, from four pooled plus-strand-competent clones.

Figure 5.. Expression of HTLV-1 plus-strand and hbz mRNAs varies with the stage of the cell cycle.

a. Integrated intensity of DAPI in individual cells from replicates of two HTLV-1-infected T-cell clones. b. Cells in G ₂/M are more frequently hbz+ and express higher levels of hbz than do cells in G ₀/G ₁. c. Consistent with this observation, cells with high levels of hbz mRNA and/or an hbz burst are more likely to be found in S or G ₂/M (p < 0.0001, logistic regression analysis). d, e. Similarly, cells with high levels of plus-strand mRNA and/or a plus-strand burst are more likely to be found in S or G ₂/M. Cells are categorized according to the level of expression of mRNAs: – (1 or 0 plus-strand spots, 0 hbz spots), + (2-99 plus-strand spots, 1-4 hbz spots) and ++ (>99 plus-strand spots, >4 hbz spots), as well as by the presence or absence of bursts. “NA” denotes cells whose integrated nuclear intensity was too dim or bright to fit in one of the three cell cycle bins. Number of cells with a given level of viral expression stated above each bar. The four plus-strand -competent clones were pooled for this analysis, of which three had three biological replicate samples each, and one had two biological replicates; total n = 14,745 cells.

Figure 6.. Model of regulation of HTLV-1 transcription and replication in vivo.

Constitutive expression of hbz mRNA maintains clonal longevity by promoting cellular proliferation. Intermittent bursts of plus-strand expression, driven by bursts of tax, have three chief consequences: production of HTLV-1 virions and resulting infectious spread; accelerated cell-cycle progression; and stimulation of hbz, of which the RNA and protein both have proliferative effects. HBZ protein can also inhibit tax transcription, terminating the plus-strand burst. It is not known whether hbz mRNA also counteracts plus-strand transcription, but it is suggested by our results. The factors that regulate the frequency of plus-strand bursts include glucose metabolism and hypoxia; cell stress and cell activation may also trigger plus-strand expression. Aside from Tax, other triggers for hbz expression remain to be discovered. Dashed line represents a link indicated by results in the present study; dotted lines represent hypothetical links.