Transmission Of Human T-cell Leukemia Virus Type 1 Research Articles

Establishment of a novel human T-cell leukemia virus type 1 infection model using cell-free virus.

The primary mode of infection by human T-cell leukemia virus type 1 (HTLV-1) is cell-to-cell transmission during contact between infected cells and target cells. Cell-free HTLV-1 infections are known to be less efficient than infections with other retroviruses, and transmission of free HTLV-1 is considered not to occur in vivo. However, it has been demonstrated that cell-free HTLV-1 virions can infect primary lymphocytes and dendritic cells in vitro, and that virions embedded in biofilms on cell membranes can contribute to transmission. The establishment of an efficient cell-free HTLV-1 infection model would be a useful tool for analyzing the replication process of HTLV-1 and the clonal expansion of infected cells. We first succeeded in obtaining supernatants with high-titer cell-free HTLV-1 using a highly efficient virus-producing cell line. The HTLV-1 virions retained the structural characteristics of retroviruses. Using this cell-free infection model, we confirmed that a variety of cell lines and primary cultured cells can be infected with HTLV-1 and demonstrated that the provirus was randomly integrated into all chromosomes in the target cells. The provirus-integrated cell lines were HTLV-1-productive. Furthermore, we demonstrated for the first time that cell-free HTLV-1 is infectious in vivo using a humanized mouse model. These results indicate that this cell-free infection model recapitulates the HTLV-1 life cycle, including entry, reverse transcription, integration into the host genome, viral replication, and secondary infection. The new cell-free HTLV-1 infection model is promising as a practical resource for studying HTLV-1 infection.IMPORTANCECo-culture of infected and target cells is frequently used for studying HTLV-1 infection. Although this method efficiently infects HTLV-1, the cell mixture is complex, and it is extremely difficult to distinguish donor infected cells from target cells. In contrast, cell-free HTLV-1 infection models allow for more strict experimental conditions. In this study, we established a novel and efficient cell-free HTLV-1 infection model. Using this model, we successfully evaluated the infectivity titers of cell-free HTLV-1 as proviral loads (copies per 100 cells) in various cell lines, primary cultured cells, and a humanized mouse model. Interestingly, the HTLV-1-associated viral biofilms played an important role in enhancing the infectivity of the cell-free infection model. This cell-free HTLV-1 infection model reproduces the replication cycle of HTLV-1 and provides a simple, powerful, and alternative tool for researching HTLV-1 infection.

Cost-effectiveness of human T-cell leukemia virus type 1 (HTLV-1) antenatal screening for prevention of mother-to-child transmission.

Human T-cell leukemia virus type 1 (HTLV-1) causes adult T-cell leukemia-lymphoma (ATL) and HTLV-1-associated myelopathy-tropical spastic paraparesis (HAM/TSP) with a poor prognosis. This study aimed to evaluate the cost-effectiveness and health impact of HTLV-1 antenatal screening. A state-transition model was developed for HTLV-1 antenatal screening and no screening over a lifetime horizon from a healthcare payer perspective. A hypothetical cohort of 30-year-old individuals was targeted. The main outcomes were costs, quality-adjusted life-years (QALYs), life expectancy life-years (LYs), incremental cost-effectiveness ratios (ICERs), HTLV-1 carriers, ATL cases, HAM/TSP cases, ATL-associated deaths, and HAM/TSP-associated deaths. The willingness-to-pay (WTP) threshold was set at US$50,000 per QALY gained. In the base-case analysis, HTLV-1 antenatal screening (US$76.85, 24.94766 QALYs, 24.94813 LYs, ICER; US$40,100 per QALY gained) was cost-effective compared with no screening (US$2.18, 24.94580 QALYs, 24.94807 LYs). Cost-effectiveness was sensitive to the maternal HTLV-1 seropositivity rate, HTLV-1 transmission rate with long-term breastfeeding from HTLV-1 seropositive mothers to children, and the cost of the HTLV-1 antibody test. HTLV-1 antenatal screening was cost-effective when the maternal HTLV-1 seropositivity rate was greater than 0.0022 and the cost of the HTLV-1 antibody test was lower than US$94.8. Probabilistic sensitivity analysis using a second-order Monte-Carlo simulation showed that HTLV-1 antenatal screening was 81.1% cost-effective at a WTP threshold of US$50,000 per QALY gained. For 10,517,942 individuals born between 2011 and 2021, HTLV-1 antenatal screening costs US$785 million, increases19,586 QALYs and 631 LYs, and prevents 125,421 HTLV-1 carriers, 4,405 ATL cases, 3,035 ATL-associated deaths, 67 HAM/TSP cases, and 60 HAM/TSP-associated deaths, compared with no screening over a lifetime. HTLV-1 antenatal screening is cost-effective and has the potential to reduce ATL and HAM/TSP morbidity and mortality in Japan. The findings strongly support the recommendation for HTLV-1 antenatal screening as a national infection control policy in HTLV-1 high-prevalence countries.

Increasing horizontal transmission of human T-cell leukemia virus type 1 in adolescents and young adults in Japan.

Human T-cell leukemia virus type 1 (HTLV-1) is a causative agent of the life-threatening diseases, adult T-cell leukemia/lymphoma and HTLV-1-associated myelopathy. Following implementation of antenatal screening in Japan, novel transmission of HTLV-1 in adolescent and adult generations is expected to replace vertical transmission as the main route for transmission. To obtain the current status of HTLV-1 horizontal infection and to assess the fluctuation of transmission occurring among adolescents and adults in Japan. We followed-up 5,017,916 eligible repeat blood donors for 8 years from 2013 to 2021. We evaluated HTLV-1 transmission rate by age group (16-69 years-old), and calculated the total number of novel transmissions in Japan using demographic statistics published by the government of Japan. We identified 457 seroconverters (men, 203; women, 254) in a total of 19,244,604 person-years during the study period. The number of seroconversions per 100,000 person-years was 1.54 for men and 4.21 for women. An increase in the number of novel infections was observed in both sexes in adolescent and young adult generations despite the health bias of blood donors. We estimate that more than 2,800 new HTLV-1 infections occur annually in Japan. It is a serious concern that without immediate measures against new HTLV-1 infections, such as guideline formulation, an inclusion of HTLV as routine screening in sexual health services, an information campaign, and surveillance of the general population, novel HTLV-1 infection could continue to increase in Japan and be a source of global transmission.

Milk Transmission of HTLV-1 and the Need for Innovative Prevention Strategies.

Breastfeeding is recommended by the World Health Organization for at least 6 months up to 2 years of age, and breast milk protects against several diseases and infections. Intriguingly, few viruses are transmitted via breastfeeding including Human T-cell leukemia virus Type 1 (HTLV-1). HTLV-1 is a highly oncogenic yet neglected retrovirus, which primarily infects CD4+ T-cells in vivo and causes incurable diseases like HTLV-1-associated inflammatory conditions or Adult T-cell leukemia/lymphoma (ATLL) after lifelong viral persistence. Worldwide, at least 5–10 million people are HTLV-1-infected and most of them are unaware of their infection posing the risk of silent transmissions. HTLV-1 is transmitted via cell-containing body fluids such as blood products, semen, and breast milk, which constitutes the major route of mother-to-child transmission (MTCT). Risk of transmission increases with the duration of breastfeeding, however, abstinence from breastfeeding as it is recommended in some endemic countries is not an option in resource-limited settings or underrepresented areas and populations. Despite significant progress in understanding details of HTLV-1 cell-to-cell transmission, it is still not fully understood, which cells in which organs get infected via the oral route, how these cells get infected, how breast milk affects this route of infection and how to inhibit oral transmission despite breastfeeding, which is an urgent need especially in underrepresented areas of the world. Here, we review these questions and provide an outlook how future research could help to uncover prevention strategies that might ultimately allow infants to benefit from breastfeeding while reducing the risk of HTLV-1 transmission.

Human T-cell leukemia virus type 1 infection among Japanese immigrants and their descendants living in Southeast Brazil: A call for preventive and control responses.

Human T-cell leukemia virus type 1 (HTLV-1) has worldwide distribution and is considered endemic in southwestern Japan. HTLV-1 infection has been associated with adult T-cell leukemia/lymphoma (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) besides other diseases. This cross-sectional study aimed to investigate the prevalence, risk factors and molecular characterization of HTLV-1, among the world’s largest population of Japanese immigrants and their descendants outside of Japan, in São Paulo, Southeast Brazil, as well as to analyze the phylogenetic relationship among isolates of HTLV-1. From July to December 2017, 2,139 individuals from five Japanese associations were interviewed and submitted to blood collection. All serum samples were first tested for the presence of anti-HTLV-1/2 antibodies by ELISA and then peripheral blood from individuals with positive serological results were analyzed for the presence of HTLV-1 5’LTR proviral DNA. Partial sequencing of the 5’LTR region of HTLV-1 proviral DNA was performed by Sanger. The prevalence of HTLV-1 infection was 5.1% (CI 95%: 4.2–6.0). In the multiple logistic regression model, HTLV-1 infection was associated with age ≥ 45 years, female sex, being first and second-generation Japanese immigrants, and having sexual partners with history of blood transfusion. The phylogenetic analysis revealed that all HTLV-1 were classified as Cosmopolitan (1a) subtype. Of them, 47.8% were classified as Transcontinental (A) subgroup and 52.2% as belonging to the Japanese (B) subgroup. Although most HTLV-1-infected patients were asymptomatic (97.3%), blurred vision was associated with HTLV-1 infection. The high prevalence of HTLV-1 infection found in this studied population and especially the intra- and interfamily HTLV-1 transmission presents an urgent call for preventive and control responses of this infection in Brazil.

A Nationwide Antenatal Human T-Cell Leukemia Virus Type-1 Antibody Screening in Japan.

Japan has been running a nationwide antenatal human T-cell leukemia virus type-1 (HTLV-1) antibody screening program since 2010 for the prevention of HTLV-1 mother-to-child transmission. As part of the program, pregnant women are invited to take an HTLV-1 antibody screening test, usually within the first 30 weeks of gestation, during regular pregnancy checkups. Pregnant women tested positive on the antibody screening test undergo a confirmatory test, either western blotting or line immunoassay. In indeterminate case, polymerase chain reaction (PCR) is used as a final test to diagnose infection. Pregnant women tested positive on a confirmatory or PCR test are identified as HTLV-1 carriers. As breastfeeding is a predominant route of postnatal HTLV-1 mother-to-child transmission, exclusive formula feeding is widely used as a postnatal preventive measure. Although there is insufficient evidence that short-term breastfeeding during ≤3 months does not increase the risk of mother-to-child transmission compared to exclusive formula feeding, this feeding method is considered if the mother is eager to breastfeed her child. However, it is important that mothers and family members fully understand that there is an increase in the risk of mother-to-child transmission when breastfeeding would be prolonged. As there are only a few clinical studies on the protective effect of frozen-thawed breastmilk feeding on mother-to-child transmission of HTLV-1, there is little evidence to recommend this feeding method. Further study on the protective effects of these feeding methods are needed. It is assumed that the risk of anxiety or depression may increase in the mothers who selected exclusive formula feeding or short-term breastfeeding. Thus, an adequate support and counseling for these mothers should be provided. In addition to raising public awareness of HTLV-1 infection, epidemiological data from the nationwide program needs to be collected and analyzed. In most cases, infected children are asymptomatic, and it is necessary to clarify how these children should be followed medically.

Collagen IV (COL4A1, COL4A2), a Component of the Viral Biofilm, Is Induced by the HTLV-1 Oncoprotein Tax and Impacts Virus Transmission.

Human T-cell leukemia virus type 1 (HTLV-1) is the etiologic agent for Adult T-Cell Leukemia/Lymphoma (ATLL) and HTLV-1-Associated Myelopathy/Tropical Spastic Paraparesis (HAM/TSP). HTLV-1 infects CD4+ T-cells via cell-to-cell transmission requiring reorganization of the cytoskeleton and expression of the viral transactivator and oncoprotein Tax. Viruses spread at the virological synapse (VS), a virus-induced specialized cell-cell contact, by polarized budding into synaptic clefts, and by cell surface transfer of viral biofilms (VBs). Since little is known about Tax’s role in formation of the VB, we asked which component of the VB is regulated by Tax and important for HTLV-1 transmission. Collagens are not only structural proteins of the extracellular matrix and basal membrane but also represent an important component of the VB. Here, we report that among the collagens known to be present in VBs, COL4 is specifically upregulated in the presence of HTLV-1 infection. Further, we found that transient expression of Tax is sufficient to induce COL4A1 and COL4A2 transcripts in Jurkat and CCRF-CEM T-cells, while robust induction of COL4 protein requires continuous Tax expression as shown in Tax-transformed T-cell lines. Repression of Tax led to a significant reduction of COL4A1/A2 transcripts and COL4 protein. Mechanistically, luciferase-based promoter studies indicate that Tax activates the COL4A2 and, to a less extent, the COL4A1 promoter. Imaging showing partial co-localization of COL4 with the viral Gag protein in VBs at the VS and transfer of COL4 and Gag to target cells suggests a role of COL4 in VB formation. Strikingly, in chronically infected C91-PL cells, knockout of COL4A2 impaired Gag transfer between infected T-cells and acceptor T-cells, while release of virus-like particles was unaffected. Taken together, we identified COL4 (COL4A1, COL4A2) as a component of the VB and a novel cellular target of Tax with COL4A2 appearing to impact virus transmission. Thus, this study is the first to provide a link between Tax’s activity and VB formation by hijacking COL4 protein functions.

Pentosan Polysulfate Demonstrates Anti-human T-Cell Leukemia Virus Type 1 Activities In Vitro and In Vivo.

Human T-cell leukemia virus type 1 (HTLV-1) infection causes T-cell leukemia and inflammatory diseases, most notably including HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). The underlying mechanism for the pathogenesis of HAM/TSP remains unclear. According to a recent clinical trial, a humanized antibody that targets CCR4+ cells ameliorates inflammation by reducing the number of infected cells in the central nervous system; this result suggests that the transmigration of HTLV-1-infected cells plays a crucial role in HAM/TSP. Partly due to the blood-brain barrier, current treatments for HAM/TSP are mostly palliative. Pentosan polysulfate (PPS), a semisynthetic glycosaminoglycan, has recently been used to treat HAM/TSP and was found to alleviate the symptoms. In this study, we investigated the effect of PPS on HTLV-1-infected cells and provide evidence for its efficacy in HAM/TSP. PPS was cytotoxic to certain HTLV-1-infected cells and significantly suppressed HTLV-1 virion production. PPS also efficiently inhibited HTLV-1 cell-cell transmission in T cells. In addition, PPS blocked HTLV-1 infection of primary endothelial cells (human umbilical vascular endothelial cells) and suppressed the subsequent induction of proinflammatory cytokine expression. Furthermore, PPS was found to inhibit the adhesion and transmigration of HTLV-1-infected cells. We also confirmed the anti-HTLV-1 effect of PPS in vivo using two mouse models. PPS blocked HTLV-1 infection in a mouse model with peripheral blood mononuclear cell (PBMC)-humanized NOD-scid IL2Rgammanull (huPBMC NSG) mice. PPS was also found to suppress the development of dermatitis and lung damage in HTLV-1 bZIP factor (HBZ)-transgenic (HBZ-Tg) mice, an HTLV-1 transgenic mouse model in which the mice develop systemic inflammation.IMPORTANCE HTLV-1 is the first human retrovirus to have been identified and is endemic in certain areas worldwide. HTLV-1 infection leads to the development of an inflammatory disease called HAM/TSP, a myelopathy characterized by slowly progressive spastic paraparesis. There have been no effective therapeutics available for HAM/TSP, but recently, a semisynthetic glycosaminoglycan, named pentosan polysulfate (PPS), has been found to alleviate the symptoms of HAM/TSP. Here we conducted a comprehensive study on the effect of PPS both in vitro and in vivo PPS demonstrated anti-HTLV-1 potential in infected cell lines, as shown by its suppressive effects on HTLV-1 replication and transmission and on the transmigration of infected T cells. Moreover, results obtained from two HTLV-1 mouse models demonstrate that PPS inhibits HTLV-1 infection and inflammation development in vivo Our work offers insights into the treatment of HAM/TSP by PPS and also suggests its possible use for treating other HTLV-1-induced inflammatory diseases.

Conflicting effects of poly(ADP-ribose) polymerase inhibitor on cell-mediated and virion-mediated HTLV-1 infection

Adult T-cell leukemia and human T-cell leukemia virus type 1 (HTLV-1) – associated myelopathy/tropical spastic paraparesis, which develop after HTLV-1 infection, are difficult to cure. In particular, the mode of HTLV-1 propagation is not well understood. Poly (ADP-ribose) polymerase-1 is reported to be a co-activator of HTLV-1 Tax protein; however, the effects of polyADP-ribosylation on infectivity of HTLV-1 have not been fully clarified. We studied the effects of a PARP inhibitor on two modes of HTLV-1 transmission: through cell adhesion between MT-2 cells (an HTLV-1-infected cell line) and uninfected cells and through virus particles produced by HTLV-1-producing c77 cells. Although the PARP inhibitor decreased HTLV-1 infection through cell adhesion, it increased HTLV-1 infection through virion production and caused apoptosis of HTLV-1-infected cells. Thus, careful consideration is required for clinical application of PARP inhibitors in HTLV-1 patients.

The Botanical Glycoside Oleandrin Inhibits Human T-cell Leukemia Virus Type-1 Infectivity and Env-Dependent Virological Synapse Formation.

At present, there are no antiretroviral drugs that inhibit incorporation of the envelope glycoprotein into newly-synthesized virus particles. The botanical glycoside, oleandrin, derived from extracts of Nerium oleander, has previously been shown to reduce the levels of the gp120 envelope glycoprotein on human immunodeficiency virus type-1 (HIV-1) particles and inhibit HIV-1 infectivity in vitro. We therefore tested whether oleandrin or an extract from N. oleander could also inhibit the infectivity of the human T-cell leukemia virus type-1 (HTLV-1): A related enveloped retrovirus and emerging tropical infectious agent. The treatment of HTLV-1+ lymphoma T-cells with either oleandrin or a N. oleander extract did not significantly inhibit viral replication or the release of p19Gag-containing particles into the culture supernatants. However, the collected virus particles from treated cells exhibited reduced infectivity on primary human peripheral blood mononuclear cells (huPBMCs). Unlike HIV-1, extracellular HTLV-1 particles are poorly infectious and viral transmission typically occurs via direct intercellular interactions across a virological synapse. We therefore investigated whether oleandrin or a N. oleander extract could inhibit virus transmission from a GFP-expressing HTLV-1+ lymphoma T-cell-line to huPBMCs in co-culture assays. These results demonstrated that both oleandrin and the crude phytoextract inhibited the formation of virological synapses and the transmission of HTLV-1 in vitro. Importantly, these findings suggest oleandrin may have broad antiviral activity against enveloped viruses by reducing the incorporation of the envelope glycoprotein into mature particles, a stage of the infection cycle not targeted by modern HAART.

Inhibition of Tunneling Nanotube (TNT) Formation and Human T-cell Leukemia Virus Type 1 (HTLV-1) Transmission by Cytarabine

The human T-cell leukemia virus type 1 (HTLV-1) is highly dependent on cell-to-cell interaction for transmission and productive infection. Cell-to-cell interactions through the virological synapse, biofilm-like structures and cellular conduits have been reported, but the relative contribution of each mechanism on HTLV-1 transmission still remains vastly unknown. The HTLV-1 protein p8 has been found to increase viral transmission and cellular conduits. Here we show that HTLV-1 expressing cells are interconnected by tunneling nanotubes (TNTs) defined as thin structures containing F-actin and lack of tubulin connecting two cells. TNTs connected HTLV-1 expressing cells and uninfected T-cells and monocytes and the viral proteins Tax and Gag localized to these TNTs. The HTLV-1 expressing protein p8 was found to induce TNT formation. Treatment of MT-2 cells with the nucleoside analog cytarabine (cytosine arabinoside, AraC) reduced number of TNTs and furthermore reduced TNT formation induced by the p8 protein. Intercellular transmission of HTLV-1 through TNTs provides a means of escape from recognition by the immune system. Cytarabine could represent a novel anti-HTLV-1 drug interfering with viral transmission.

Quantitating the Transfer of the HTLV-1 p8 Protein Between T-Cells by Flow Cytometry.

The Human T-cell leukemia virus type 1 (HTLV-1)-encoded accessory protein p8 is cleaved from the precursor protein p12 encoded by the HTLV-1 open reading frame I. Both p12 and p8 are thought to contribute to efficient viral persistence. Mechanistically, p8 induces T-cell conjugates and cellular conduits. The latter are considered to facilitate transfer of p8 to target cells and virus transmission. Transfer of p8 between p8-expressing T-cells and recipient cells has been analyzed by immunofluorescence and live imaging. However, automatic quantitation of p8-transfer between cells has not been studied yet. Here we developed a novel method allowing time saving quantitation of p8 transfer between cells by flow cytometry. After establishing a protocol for the detection of intracellular p8 by flow cytometry and validation of p8 protein expression by western blot and immunofluorescence, we set up a co-culture assay between p8-expressing donor Jurkat T-cells and recipient Jurkat T-cells that had been prestained with a well-retained live cell dye. Upon quantitating the amount of p8 positive recipient cells with regard to the percentage of p8 expressing donor cells, time course experiments confirmed that p8 is rapidly transferred between Jurkat T-cells. We found that p8 enters approximately 5% of recipient T-cells immediately upon co-culture for 5 min. Prolonged co-culture for up to 24 h revealed an increase of relative p8 transfer to approximately 23% of the recipient cells. Immunofluorescence analysis of co-culture experiments and manual quantitation of p8 expression in fluorescence images confirmed the validity of the flow cytometry based assay. Application of the new assay revealed that manipulation of actin polymerization significantly decreased p8 transfer between Jurkat T-cells suggesting an important role of actin dynamics contributing to p8 transfer. Further, transfer of p8 to co-cultured T-cells varies between different donor cell types since p8 transfer could hardly been detected in co-cultures of 293T donor cells with Jurkat acceptor cells. In summary, our novel assay allows automatic and rapid quantitation of p8 transfer to target cells and might thus contribute to a better understanding of cellular processes and dynamics regulating p8 transfer and HTLV-1 transmission.

Natural Course of Human T-Cell Leukemia Virus Type 1 Proviral DNA Levels in Carriers During Pregnancy.

The measurement of human T-cell leukemia virus type 1 (HTLV-1) proviral DNA levels by using polymerase chain reaction has been beneficial for confirming HTLV-1 infection during pregnancy. However, the influence of pregnancy on HTLV-1 infection and proviral DNA levels among pregnant women with HTLV-1 has not been clarified. We prospectively gathered blood samples from 36 pregnant women in whom HTLV-1 carriage was previously diagnosed and sequentially measured their proviral DNA levels. The HTLV-1 proviral DNA levels remained at a plateau during pregnancy but were elevated after delivery. Moreover, flow cytometry and serological analyses revealed that the regulatory T-cell population and soluble interleukin 2 receptor levels were similarly elevated after birth in comparison with those in control pregnant women. This study is the first to provide data on sequential changes in HTLV-1 proviral DNA levels during and after pregnancy. These findings will guide the establishment of a better program to prevent mother-to-child transmission of HTLV-1.

HTLV-1 bZIP Factor Enhances T-Cell Proliferation by Impeding the Suppressive Signaling of Co-inhibitory Receptors.

Human T-cell leukemia virus type 1 (HTLV-1) causes adult T-cell leukemia-lymphoma (ATL) and inflammatory diseases. To enhance cell-to-cell transmission of HTLV-1, the virus increases the number of infected cells in vivo. HTLV-1 bZIP factor (HBZ) is constitutively expressed in HTLV-1 infected cells and ATL cells and promotes T-cell proliferation. However, the detailed mechanism by which it does so remains unknown. Here, we show that HBZ enhances the proliferation of expressing T cells after stimulation via the T-cell receptor. HBZ promotes this proliferation by influencing the expression and function of multiple co-inhibitory receptors. HBZ suppresses the expression of BTLA and LAIR-1 in HBZ expressing T cells and ATL cells. Expression of T cell immunoglobulin and ITIM domain (TIGIT) and Programmed cell death 1 (PD-1) was enhanced, but their suppressive effect on T-cell proliferation was functionally impaired. HBZ inhibits the co-localization of SHP-2 and PD-1 in T cells, thereby leading to impaired inhibition of T-cell proliferation and suppressed dephosphorylation of ZAP-70 and CD3ζ. HBZ does this by interacting with THEMIS, which associates with Grb2 and SHP-2. Thus, HBZ interacts with the SHP containing complex, impedes the suppressive signal from PD-1 and TIGIT, and enhances the proliferation of T cells. Although HBZ was present in both the nucleus and the cytoplasm of T cells, HBZ was localized largely in the nucleus by suppressed expression of THEMIS by shRNA. This indicates that THEMIS is responsible for cytoplasmic localization of HBZ in T cells. Since THEMIS is expressed only in T-lineage cells, HBZ mediated inhibition of the suppressive effects of co-inhibitory receptors accounts for how HTLV-1 induces proliferation only of T cells in vivo. This study reveals that HBZ targets co-inhibitory receptors to cause the proliferation of infected cells.

The Tax-Inducible Actin-Bundling Protein Fascin Is Crucial for Release and Cell-to-Cell Transmission of Human T-Cell Leukemia Virus Type 1 (HTLV-1).

The delta-retrovirus Human T-cell leukemia virus type 1 (HTLV-1) preferentially infects CD4+ T-cells via cell-to-cell transmission. Viruses are transmitted by polarized budding and by transfer of viral biofilms at the virological synapse (VS). Formation of the VS requires the viral Tax protein and polarization of the host cytoskeleton, however, molecular mechanisms of HTLV-1 cell-to-cell transmission remain incompletely understood. Recently, we could show Tax-dependent upregulation of the actin-bundling protein Fascin (FSCN-1) in HTLV-1-infected T-cells. Here, we report that Fascin contributes to HTLV-1 transmission. Using single-cycle replication-dependent HTLV-1 reporter vectors, we found that repression of endogenous Fascin by short hairpin RNAs and by Fascin-specific nanobodies impaired gag p19 release and cell-to-cell transmission in 293T cells. In Jurkat T-cells, Tax-induced Fascin expression enhanced virus release and Fascin-dependently augmented cell-to-cell transmission to Raji/CD4+ B-cells. Repression of Fascin in HTLV-1-infected T-cells diminished virus release and gag p19 transfer to co-cultured T-cells. Spotting the mechanism, flow cytometry and automatic image analysis showed that Tax-induced T-cell conjugate formation occurred Fascin-independently. However, adhesion of HTLV-1-infected MT-2 cells in co-culture with Jurkat T-cells was reduced upon knockdown of Fascin, suggesting that Fascin contributes to dissemination of infected T-cells. Imaging of chronically infected MS-9 T-cells in co-culture with Jurkat T-cells revealed that Fascin’s localization at tight cell-cell contacts is accompanied by gag polarization suggesting that Fascin directly affects the distribution of gag to budding sites, and therefore, indirectly viral transmission. In detail, we found gag clusters that are interspersed with Fascin clusters, suggesting that Fascin makes room for gag in viral biofilms. Moreover, we observed short, Fascin-containing membrane extensions surrounding gag clusters and clutching uninfected T-cells. Finally, we detected Fascin and gag in long-distance cellular protrusions. Taken together, we show for the first time that HTLV-1 usurps the host cell factor Fascin to foster virus release and cell-to-cell transmission.

Modes of Human T Cell Leukemia Virus Type 1 Transmission, Replication and Persistence.

Human T-cell leukemia virus type 1 (HTLV-1) is a retrovirus that causes cancer (Adult T cell Leukemia, ATL) and a spectrum of inflammatory diseases (mainly HTLV-associated myelopathy—tropical spastic paraparesis, HAM/TSP). Since virions are particularly unstable, HTLV-1 transmission primarily occurs by transfer of a cell carrying an integrated provirus. After transcription, the viral genomic RNA undergoes reverse transcription and integration into the chromosomal DNA of a cell from the newly infected host. The virus then replicates by either one of two modes: (i) an infectious cycle by virus budding and infection of new targets and (ii) mitotic division of cells harboring an integrated provirus. HTLV-1 replication initiates a series of mechanisms in the host including antiviral immunity and checkpoint control of cell proliferation. HTLV-1 has elaborated strategies to counteract these defense mechanisms allowing continuous persistence in humans.

The neutralizing function of the anti-HTLV-1 antibody is essential in preventing in vivo transmission of HTLV-1 to human T cells in NOD-SCID/γcnull (NOG) mice.

BackgroundHuman T-cell leukemia virus type 1 (HTLV-1) causes both neoplastic and inflammatory diseases, including adult T-cell leukemia and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Because these life-threatening and disabling diseases are not yet curable, it is important to prevent new HTLV-1 infections.FindingsIn this study, we have established a simple humanized mouse model of HTLV-1 infection for evaluating prophylactic and therapeutic interventions. In this model, HTLV-1-negative normal human peripheral blood mononuclear cells (PBMCs) are transplanted directly into the spleens of severely immunodeficient NOD-SCID/γcnull (NOG) mice, together with mitomycin-treated HTLV-1-producing T cells. Using this model, we tested the efficacy of monoclonal antibodies (mAbs) specific to HTLV-1 as well as human IgG isolated from HAM/TSP patients (HAM-IgG) in preventing HTLV-1-infection. One hour before and 24 h after transplantation of the human cells, each antibody sample was inoculated intraperitoneally. On day 14, human PBMCs isolated from the mouse spleens were tested for HTLV-1 infection. Whereas fresh CD4-positive and CD8-positive T cells isolated from untreated mice or mice treated with isotype control mAb, HTLV-1 non-neutralizing mAbs to envelope gp46, gag p19, and normal human IgG were all infected with HTLV-1; the mice treated with either HTLV-1 neutralizing anti-gp46 mAb or HAM-IgG did not become infected.ConclusionsOur data indicate that the neutralizing function of the antibody, but not the antigen specificity, is essential for preventing the in vivo transmission of HTLV-1. The present animal model will also be useful for the in vivo evaluation of the efficacy of candidate molecules to be used as prophylactic and therapeutic intervention against HTLV-1 infection.Electronic supplementary materialThe online version of this article (doi:10.1186/s12977-014-0074-z) contains supplementary material, which is available to authorized users.

The neutralizing function of the anti-HTLV-1 antibody is essential in preventing in vivo transmission of HTLV-1 to human T cells in NOD-SCID/¿cnull (NOG) mice

Human T-cell leukemia virus type 1 (HTLV-1) causes both neoplastic and inflammatory diseases, including adult T-cell leukemia and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Because these life-threatening and disabling diseases are not yet curable, it is important to prevent new HTLV-1 infections. In this study, we have established a simple humanized mouse model of HTLV-1 infection for evaluating prophylactic and therapeutic interventions. In this model, HTLV-1-negative normal human peripheral blood mononuclear cells (PBMCs) are transplanted directly into the spleens of severely immunodeficient NOD-SCID/γcnull (NOG) mice, together with mitomycin-treated HTLV-1-producing T cells. Using this model, we tested the efficacy of monoclonal antibodies (mAbs) specific to HTLV-1 as well as human IgG isolated from HAM/TSP patients (HAM-IgG) in preventing HTLV-1-infection. One hour before and 24 h after transplantation of the human cells, each antibody sample was inoculated intraperitoneally. On day 14, human PBMCs isolated from the mouse spleens were tested for HTLV-1 infection. Whereas fresh CD4-positive and CD8-positive T cells isolated from untreated mice or mice treated with isotype control mAb, HTLV-1 non-neutralizing mAbs to envelope gp46, gag p19, and normal human IgG were all infected with HTLV-1; the mice treated with either HTLV-1 neutralizing anti-gp46 mAb or HAM-IgG did not become infected. Our data indicate that the neutralizing function of the antibody, but not the antigen specificity, is essential for preventing the in vivo transmission of HTLV-1. The present animal model will also be useful for the in vivo evaluation of the efficacy of candidate molecules to be used as prophylactic and therapeutic intervention against HTLV-1 infection.

Animal Models Utilized in HTLV-1 Research.

Since the isolation and discovery of human T-cell leukemia virus type 1 (HTLV-1) over 30 years ago, researchers have utilized animal models to study HTLV-1 transmission, viral persistence, virus-elicited immune responses, and HTLV-1-associated disease development (ATL, HAM/TSP). Non-human primates, rabbits, rats, and mice have all been used to help understand HTLV-1 biology and disease progression. Non-human primates offer a model system that is phylogenetically similar to humans for examining viral persistence. Viral transmission, persistence, and immune responses have been widely studied using New Zealand White rabbits. The advent of molecular clones of HTLV-1 has offered the opportunity to assess the importance of various viral genes in rabbits, non-human primates, and mice. Additionally, over-expression of viral genes using transgenic mice has helped uncover the importance of Tax and Hbz in the induction of lymphoma and other lymphocyte-mediated diseases. HTLV-1 inoculation of certain strains of rats results in histopathological features and clinical symptoms similar to that of humans with HAM/TSP. Transplantation of certain types of ATL cell lines in immunocompromised mice results in lymphoma. Recently, “humanized” mice have been used to model ATL development for the first time. Not all HTLV-1 animal models develop disease and those that do vary in consistency depending on the type of monkey, strain of rat, or even type of ATL cell line used. However, the progress made using animal models cannot be understated as it has led to insights into the mechanisms regulating viral replication, viral persistence, disease development, and, most importantly, model systems to test disease treatments.

Forefront studies on HTLV-1 oncogenesis

Almost 40 years ago, Takatsuki et al. recognized the existence of a peculiar T cell leukemia in Kyoto, Japan that they named Adult T Leukemia (ATL). They reported a series of 13 patients in 1976 (Uchiyama et al., 1977). In 1980, the group of Gallo reported the discovery of a human oncogenic retrovirus that they named Human T cell Leukemia Virus type 1 (HTLV-1) in cells obtained from two US patients classified as mycosis fungoides and Sezary syndrome (Poiesz et al., 1980), but who were, in retrospect, probably suffering from ATL [for an historical perspective see (Takatsuki, 2005)]. Shortly after, the groups of Hinuma (Miyoshi et al., 1981) and of Yoshida (Yoshida et al., 1982) uncovered the presence of HTLV-1 in cells obtained from ATL patients. In 1985–1986, two groups independently reported that a neurological disease named HTLV-1 Associated Myelopathy/Tropical Spastic Paraparesis (HAM/TSP) was also caused by HTLV-1 (Gessain et al., 1985; Osame et al., 1986). Since then, other inflammatory diseases (uveitis, infective dermatitis) have also been linked to this viral infection. Other members of the HTLV family (i.e., HTLV-2, HTLV-3, and HTLV-4 have also now been reported, none of them being clearly associated so far with an oncogenic process or a neurodegenerative disease (Kalyanaraman et al., 1982; Calattini et al., 2005; Wolfe et al., 2005). Almost 10 years ago, ours colleagues Kuan Teh Jeang and Mitsuaki Yoshida organized a special issue on HTLV infection in Oncogene. In setting up this issue, we cannot forget the memory our friend Teh. We called upon the expertise of different research groups from Europe, Japan, and USA. However, we regret that the format of this issue prevented us from soliciting many other colleagues. The following reviews will deal with many fascinating aspects of viral cycle, but summarizes also new approaches that should allow a better integrated research. A first group of articles provides information about HTLV-1 epidemiology and associated-pathogenesis. The article from Gessain and Cassar provides an updated view on HTLV-1 distribution, based on data obtained from 1.5 billion individuals originating from endemic areas (Gessain and Cassar, 2012). Iwanaga et al. focused their review on ATL epidemiology and show its peculiar characteristic [age at onset, risk factor, proviral load, etc. (Iwanaga et al., 2012)]. Yamano and Sato provide an interesting perspective on HAM/TSP physiopathology, and remind us that optimal therapeutic treatments are still lacking for those patients (Yamano and Sato, 2012). The review by Kamoi ad Mochizuki summarizes our current knowledge on HTLV-1 uveitis, which is the most common cause of uveitis in endemic areas (Kamoi and Mochizuki, 2012). Going deeper in the pathological mechanisms linked to HTLV-1 infection, Yamagishi and Watanabe summarize recent data showing that ATL cells express abnormally low levels of a cellular oncosuppressor miRNA and display some epigenetic changes on the promoter of genes critical for cell cycle (Yamagishi and Watanabe, 2012). A second group of articles summarizes the interaction between the virus and the host's cells. Before causing diseases, HTLV-1 has to enter the cell. However, the mechanisms of HTLV-1 transmission and cell entry have remained elusive for a long period of time. Pique and Jones have summarized recent insights about those mechanisms both at the cell level but also between individuals (Pique and Jones, 2012). HTLV-1 associated diseases are linked to the fact that HTLV-1 evades both adaptative and innate immune responses. Kannagi et al. provide us with an exciting review, which explains us how the virus evades the interferon response, but also that dysfunction of the CTL response might be a risk factor for disease development in infected carriers (Kannagi et al., 2012). A third group of articles reports data on individual viral proteins that play important roles in the viral cycle and/or in pathogenesis. Nakano and Watanabe remind us the important role played by Rex, which uses cellular pathways to export unspliced or singly spliced viral mRNAs in the cell cytoplasm, therefore allowing expressing of structural proteins (Nakano and Watanabe, 2012). Currer et al., Zhao and Matsuoka focused their attention on Tax and HBZ, two viral proteins that play important roles in the control of viral transcription and oncogenesis (Currer et al., 2012; Zhao and Matsuoka, 2012). Finally, Bai and Nicot provide an overview on 4 auxiliary viral proteins (p12, p8, p30, and p13), which are required for establishing a persistent infection in vivo (Bai and Nicot, 2012). Finally, Duc Dodon and colleagues remind us that studying HTLV-1 pathogenesis requires animal models (Dodon et al., 2012). Rabbits, rats, transgenic mice, and monkeys have been used in the past. However, recent approaches using humanized mice might represent an interesting alternative for studying HTLV-1 associated diseases.