The Nature of the HTLV-1 Provirus in Naturally Infected Individuals Analyzed by the Viral DNA-Capture-Seq Approach

Abstract

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.

Provirus Mutations of Human T-Lymphotropic Virus 1 and 2 (HTLV-1 and HTLV-2) in HIV-1-Coinfected Individuals

“…Another important detail that emerged from this study was the high rate of defective provirus particles (around 30%) detected in HIV/HTLV-1-and HIV/HTLV-2coinfected individuals, which is similar to the overall rates described in ATL individuals infected with HTLV-1 (20)(21)(22). However, the percentages differ depending on the type of defective provirus (type 1 or type 2).…”

“…However, the percentages differ depending on the type of defective provirus (type 1 or type 2). While in HTLV-1-infected individuals the majority of cases are defective provirus type 2 instead of type 1 (26.7% versus 2.2% in ATL, 14.5% versus 2.9% in asymptomatic carriers, and 13.1% versus 2.0% in HAM/TSP, respectively) (22), in the present study we detected more defective type 1 particles in HIV/HTLV-1 coinfection (20.4% versus 11.4%) and the same rates of type 1 and type 2 in HIV/HTLV-2-coinfected individuals (16.0%).…”

“…The examination of defective proviruses was conducted according to the lack of LTR, env, and tax segments after several amplifications and sequencing events, including testing different amounts of DNA inputs and primer pairs. The criterion to be considered an HTLV-1 defective type 1 provirus was the lack of the env and/or tax segment, and that for type 2 was the lack of a 5=LTR region (20)(21)(22). The same criterion used for HTLV-1 was employed for HTLV-2, since there is no publication or classification of defective proviruses of HTLV-2 in the literature.…”

“…Two types of defective HTLV-1 provirus were described in the 1990s: type 1, which retains both LTRs and lacks internal sequences, and type 2, which lacks the 5=LTR region (20,21). Interestingly, infected cells with HTLV-1-defective proviruses proliferate more than those with intact ones (22) and were detected at high frequencies in ATL cells and in aggressive forms of ATL (20,21,23). These defective sequences were also identified in asymptomatic carriers and HAM/TSP patients, albeit at low frequencies (22,24).…”

An Improved Sequencing-Based Bioinformatics Pipeline to Track the Distribution and Clonal Architecture of Proviral Integration Sites

“…While NGS techniques based on capture probes have generated a genome-wide picture of HTLV-1 and HIV-1 integration sites and their associated proviral genomes (Iwase et al, 2019;Katsuya et al, 2019), capture-dependent approaches are limited in their throughput and are not well suited to exploring early nonmalignant stages of infection where only a small fraction of the cells carries the integrated provirus. We and others have used more high-throughput NGS integration site profiling approaches consisting of LAM-PCR followed by NGS (Paruzynski et al, 2010;Gillet et al, 2011;Firouzi et al, 2014;Sunshine et al, 2016;Percher et al, 2017;Rosewick et al, 2017).…”