Graphical Abstract Highlights d A method for comprehensive analysis of HTLV-1 proviruses in infected individuals d The method provides viral sequence, integration site, and degree of cell expansion d Defective proviruses are present in asymptomatic carriers and HAM/TSP, as well as ATL d Infected cells with defective proviruses proliferate more than those with intact ones Correspondence y-satou@kumamoto-u.ac.jp In Brief Katsuya et al. demonstrate that HTLV-1 DNA-capture-seq provides comprehensive information, including the entire viral sequence, integration site, and clonal abundance of infected cells.Infected clones with defective-type proviruses are present in disease states and in asymptomatic carriers, and they proliferate more than full-length proviruses. SUMMARYThe retrovirus human T-cell leukemia virus type 1 (HTLV-1) integrates into the host DNA, achieves persistent infection, and induces human diseases.Here, we demonstrate that viral DNA-capture sequencing (DNA-capture-seq) is useful to characterize HTLV-1 proviruses in naturally virus-infected individuals, providing comprehensive information about the proviral structure and the viral integration site. We analyzed peripheral blood from 98 naturally HTLV-1-infected individuals and found that defective proviruses were present not only in patients with leukemia, but also in those with other clinical entities. We further demonstrated that clones with defective-type proviruses exhibited a higher degree of clonal abundance than those with full-length proviruses. The frequency of defective-type proviruses in HTLV-1-infected humanized mice was lower than that in infected individuals, indicating that defective proviruses were rare at the initial phase of infection but preferentially selected during persistent infection. These results demonstrate the robustness of viral DNA-captureseq for HTLV-1 infection and suggest potential applications for other virus-associated cancers in humans.

Regardless of recent advances in the development of anti-retroviral drugs, it is still extremely difficult to eradicate HIV-1 from infected individuals. The characterization of the HIV-1 provirus, a type of viral reservoir, with a high resolution is key to HIV-1 cure research. Here, we demonstrate that DNA-capture-seq is a powerful tool to obtain comprehensive information on the HIV-1 provirus. We use biotinylated DNA probes targeting the entire HIV-1 sequence to capture fragments containing HIV-1 sequences from DNA-seq libraries prepared for high throughput sequencing. We demonstrate that the protocol provided the entire proviral sequence from the beginning of the 5′ LTR to the end of the 3′ LTR. Since HIV-1 DNA-probes can hybridize not only viral fragments but also virus-host chimeric ones, the viral integration site information can also be obtained. We verify the efficiency of the protocol by using latently infected cell lines, such as ACH-2 and J1.1, and newly generated ones. The results reveal that the 2 new clones that we analyse harbour one copy of replication-competent provirus, suggesting that latency is not caused by genetic mutations or deletions of the provirus. In conclusion, HIV-1 DNA-capture-seq is a powerful tool to characterize the HIV-1 provirus at a single nucleotide resolution and therefore might be useful for various experiments aiming for an HIV-1 cure.

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Human T-cell leukemia virus type 1 (HTLV-1) is a retrovirus that causes adult T-cell leukemia/lymphoma (ATL), a cancer of infected CD4+ T-cells. There is both sense and antisense transcription from the integrated provirus. Sense transcription tends to be suppressed, but antisense transcription is constitutively active. Various efforts have been made to elucidate the regulatory mechanism of HTLV-1 provirus for several decades; however, it remains unknown how HTLV-1 antisense transcription is maintained. Here, using proviral DNA-capture sequencing, we found a previously unidentified viral enhancer in the middle of the HTLV-1 provirus. The transcription factors, SRF and ELK-1, play a pivotal role in the activity of this enhancer. Aberrant transcription of genes in the proximity of integration sites was observed in freshly isolated ATL cells. This finding resolves certain long-standing questions concerning HTLV-1 persistence and pathogenesis. We anticipate that the DNA-capture-seq approach can be applied to analyze the regulatory mechanisms of other oncogenic viruses integrated into the host cellular genome.

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