The host genomic environment of the provirus determines the abundance of HTLV-1–infected T-cell clones

Abstract

Human T-lymphotropic virus type 1 (HTLV-1) persists by driving clonal proliferation of infected T lymphocytes. A high proviral load predisposes to HTLV-1-associated diseases. Yet the reasons for the variation within and between persons in the abundance of HTLV-1-infected clones remain unknown. We devised a highthroughput protocol to map the genomic location and quantify the abundance of > 91 000 unique insertion sites of the provirus from 61 HTLV-1 ؉ persons and > 2100 sites from in vitro infection. We show that a typical HTLV-1-infected host carries between 500 and 5000 unique insertion sites. We demonstrate that negative selection dominates during chronic infection, favoring establishment of proviruses integrated in transcriptionally silenced DNA: this selection is significantly stronger in asymptomatic carriers. We define a parameter, the oligoclonality index, to quantify clonality. The high proviral load characteristic of HTLV-1- IntroductionHuman T-lymphotropic virus type 1 (HTLV-1) causes adult T-cell leukemia-lymphoma (ATLL), HTLV-1-associated myelopathy/ tropical spastic paraparesis (HAM/TSP), uveitis, and infective dermatitis. It is estimated that 15 to 20 million persons live with HTLV-1 infection worldwide. A small proportion (up to 7%, depending on the area) of HTLV-1-infected persons develop disease, whereas the majority remain asymptomatic carriers (ACs). Infection occurs via breastfeeding, transfusion of infected cellular blood products, or sexual intercourse. Symptoms appear after a long period (years or decades) of clinical latency. 1 The HTLV-1 proviral load (PVL) remains stable within each infected person and correlates with the outcome of infection. However, the PVL varies widely among infected people, even within a particular diagnostic group. [2][3][4] The sequence of HTLV-1 is also stable within a person, 5,6 indicating that the PVL is maintained in vivo mainly by mitosis of infected cells during the chronic phase of the infection. This interpretation is supported by the observation that individual clones of infected cells can persist in patients for several years. 7-9 Thus, it has been hypothesized that infectious transmission of HTLV-1 is important early in infection across the virologic synapse, 10 whereas mitotic replication is responsible for maintaining proviral load once a persistent infection has been established and reached an equilibrium with the immune response. 11 In approximately 5% of infected people, persistent clonal proliferation culminates in malignant transformation in the disease ATLL. 7,8 The leukemic clones carry generally one (complete or defective) provirus per cell. [12][13][14] There has been a longstanding debate on the question of whether HTLV-1 is latent or persistently expressed in vivo. Persistent expression is strongly suggested by the extensive evidence that the strong, chronically activated cytotoxic T lymphocyte (CTL) response to HTLV-1 limits the proviral load and reduces the risk of HAM/TSP. 11 Furthermore, there is both experimental evidence 15 and th...

Reducing the global burden of HTLV-1 infection: An agenda for research and action

“…-genes promoting viral replication and persistence (Tax and HBZ) (Matsuoka and Jeang, 2007); -modes of viral entry and replication (infectious and mitotic cycles) (Ghez et al, 2010;Boxus et al, 2012;Gillet et al, 2011Gillet et al, , 2013Sibon et al, 2006); -genomic and epigenetic mechanisms leading to ATL (oncogenic stress, driver mutations) (Kataoka et al, 2015;Yamagishi et al, 2012); -the role of the immune response in the control of infection (Asquith and Bangham, 2008); -the pathogenic potential of HTLV-2, -3 and -4 (Calattini et al, 2006).…”

Quantitative Shearing Linear Amplification Polymerase Chain Reaction: An Improved Method for Quantifying Lentiviral Vector Insertion Sites in Transplanted Hematopoietic Cell Systems

“…13,17,35 Recently, random DNA shearing by sonication and count of shear site was introduced and appear to have better accuracy and sensitivity. 20,21,23,28,36,37 We further improved this method by minimizing the number of exponential PCR cycles used for template generation and by including an internal control sample with a relatively high number of quantitatively defined VIS. The internal control allowed us to define the sensitivity, accuracy, and overall quality of each MiSeq run, and provides a standard for measuring clonal abundance in complex samples.…”

“…20,24,28 This is most likely explained by potential biases in exponential PCR amplification of sonicated fragments from different VIS. While our data support the use of shear site counts in place of read counts, shear site counts could potentially underestimate the absolute abundance of a relatively large dominant clone because the number of different shear sites for a given VIS could be limited by the length of sheared DNA fragments.…”

“…1A), allowing the clonal abundance to be estimated by counting the number of unique shear sites associated with each VIS. 20,24,28 Sheared DNA ends were repaired to form blunt-ended DNA and 3¢ dA tails were then added to the blunt ends. Adapter ligation was performed using the commercially available NEBNext Multiplex Oligos for Illumina kit that allows direct compatibility with the Illumina MiSeq system (Fig.…”

HTLV-I infection: A dynamic struggle between viral persistence and host immunity

“…Nevertheless, it is not clear why the rate of spontaneous viral expression τ would increase over time. Indeed, this rate is thought to be characteristic to each individual and is determined by two primary factors that remain relatively constant over time: the HTLV-I genomic sequence, which is genetically stable, and the dominance of infected T-cell clones, each defined by a specific site of integration into the host DNA Gillet et al, 2011;Meekings et al, 2008). Further studies are needed to resolve these issues and test the validity of our hypothesis.…”