Abstract
Background: The human retrovirus HTLV-1 inserts the viral complementary DNA of 9 kb into the host genome. Both plus- and minus-strands of the provirus are transcribed, respectively from the 5' and 3' long terminal repeats (LTR). Plus-strand expression is rapid and intense once activated, whereas the minus-strand is transcribed at a lower, more constant level. To identify how HTLV-1 transcription is regulated, we investigated the epigenetic modifications associated with the onset of spontaneous plus-strand expression and the potential impact of the host factor CTCF. Methods: Patient-derived peripheral blood mononuclear cells (PBMCs) and in vitro HTLV-1-infected T cell clones were examined. Cells were stained for the plus-strand-encoded viral protein Tax, and sorted into Tax + and Tax - populations. Chromatin immunoprecipitation and methylated DNA immunoprecipitation were performed to identify epigenetic modifications in the provirus. Bisulfite-treated DNA fragments from the HTLV-1 LTRs were sequenced. Single-molecule RNA-FISH was performed, targeting HTLV-1 transcripts, for the estimation of transcription kinetics. The CRISPR/Cas9 technique was applied to alter the CTCF-binding site in the provirus, to test the impact of CTCF on the epigenetic modifications. Results: Changes in the histone modifications H3K4me3, H3K9Ac and H3K27Ac were strongly correlated with plus-strand expression. DNA in the body of the provirus was largely methylated except for the pX and 3' LTR regions, regardless of Tax expression. The plus-strand promoter was hypomethylated when Tax was expressed. Removal of CTCF had no discernible impact on the viral transcription or epigenetic modifications. Conclusions: The histone modifications H3K4me3, H3K9Ac and H3K27Ac are highly dynamic in the HTLV-1 provirus: they show rapid change with the onset of Tax expression, and are reversible. The HTLV-1 provirus has an intrinsic pattern of epigenetic modifications that is independent of both the provirus insertion site and the chromatin architectural protein CTCF which binds to the HTLV-1 provirus.
Highlights
Human T cell leukemia virus type 1 (HTLV-1) was the first pathogenic exogenous retrovirus identified in humans
· Results section: A phrase was inserted - “The DNA methylation pattern was much less variable in the HTLV-1-infected T cell clones than in the peripheral blood mononuclear cells (PBMCs): each clone is derived from a single cell, so every cell in that clone carries the HTLV-1 provirus in the same genomic site.”
To identify the epigenetic modifications associated with transcriptional activity in the provirus, we sorted the cells based on Tax protein expression and performed ChIP and DNA methylation analyses for each fraction (Figure 2) unless stated otherwise
Summary
Human T cell leukemia virus type 1 (HTLV-1) was the first pathogenic exogenous retrovirus identified in humans. The main routes of infection are breast feeding, sexual contact and blood transfusion, each of which transmits cells carrying HTLV-1 and capable of infecting other cells in a new host. The majority of infected individuals remain asymptomatic throughout life. Some 5% develop adult T cell leukemia (ATL), and up to another 5% develop HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP)[1,2]. HTLV-1 reverse-transcribes its 9 kb genomic RNA into complementary double-stranded DNA which is inserted into the host cellular DNA upon infection. Thereafter the virus remains as a chromatinized provirus and is replicated as a part of the host genome. Each infected cell carries a single copy of the HTLV-1 provirus in a given location in the host genome[3,4]
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