Precise excision of HTLV-1 provirus with a designer-recombinase.

The human T cell leukemia virus type 1 (HTLV-1) is a pathogenic retrovirus that persists as a provirus in the genome of infected cells and can lead to adult T cell leukemia (ATL). Worldwide, more than 10 million people are infected and approximately 5% of these individuals will develop ATL, a highly aggressive cancer that is currently incurable. In the last years, genome editing tools have emerged as promising antiviral agents. In this proof-of-concept study, we use substrate-linked directed evolution (SLiDE) to engineer Cre-derived site-specific recombinases to excise the HTLV-1 proviral genome from infected cells. We identified a conserved loxP-like sequence (loxHTLV) present in the long terminal repeats of the majority of virus isolates. After 181 cycles of SLiDE, we isolated a designer-recombinase (designated RecHTLV), which efficiently recombines the loxHTLV sequence in bacteria and human cells with high specificity. Expression of RecHTLV in human Jurkat T cells resulted in antiviral activity when challenged with an HTLV-1 infection. Moreover, expression of RecHTLV in chronically infected SP cells led to the excision of HTLV-1 proviral DNA. Our data suggest that recombinase-mediated excision of the HTLV-1 provirus represents a promising approach to reduce proviral load in HTLV-1-infected individuals, potentially preventing the development of HTLV-1-associated diseases.

Transcriptional Targeting of Dendritic Cells Using an Optimized Human Fascin1 Gene Promoter.

Deeper knowledge about the role of the tumor microenvironment (TME) in cancer development and progression has resulted in new strategies such as gene-based cancer immunotherapy. Whereas some approaches focus on the expression of tumoricidal genes within the TME, DNA-based vaccines are intended to be expressed in antigen-presenting cells (e.g., dendritic cells, DCs) in secondary lymphoid organs, which in turn induce anti-tumor T cell responses. Besides effective delivery systems and the requirement of appropriate adjuvants, DNA vaccines themselves need to be optimized regarding efficacy and selectivity. In this work, the concept of DC-focused transcriptional targeting was tested by applying a plasmid encoding for the luciferase reporter gene under the control of a derivative of the human fascin1 gene promoter (pFscnLuc), comprising the proximal core promoter fused to the normally more distantly located DC enhancer region. DC-focused activity of this reporter construct was confirmed in cell culture in comparison to a standard reporter vector encoding for luciferase under the control of the strong ubiquitously active cytomegalovirus promoter and enhancer (pCMVLuc). Both plasmids were also compared upon intravenous administration in mice. The organ- and cell type-specific expression profile of pFscnLuc versus pCMVLuc demonstrated favorable activity especially in the spleen as a central immune organ and within the spleen in DCs.

Transfer of HTLV-1 p8 and Gag to target T-cells depends on VASP, a novel interaction partner of p8.

The Human T-cell leukemia virus type 1 (HTLV-1) orf I-encoded accessory protein p8 is cleaved from its precursor p12, and both proteins contribute to viral persistence. p8 induces cellular protrusions, which are thought to facilitate transfer of p8 to target cells and virus transmission. Host factors interacting with p8 and mediating p8 transfer are unknown. Here, we report that vasodilator-stimulated phosphoprotein (VASP), which promotes actin filament elongation, is a novel interaction partner of p8 and important for p8 and HTLV-1 Gag cell-to-cell transfer. VASP contains an Ena/VASP homology 1 (EVH1) domain that targets the protein to focal adhesions. Bioinformatics identified a short stretch in p8 (amino acids (aa) 24-45) which may mediate interactions with the EVH1 domain of VASP. Co-immunoprecipitations confirmed interactions of VASP:p8 in 293T, Jurkat and HTLV-1-infected MT-2 cells. Co-precipitation of VASP:p8 could be significantly blocked by peptides mimicking aa 26-37 of p8. Mutational studies revealed that the EVH1-domain of VASP is necessary, but not sufficient for the interaction with p8. Further, deletion of the VASP G- and F-actin binding domains significantly diminished co-precipitation of p8. Imaging identified areas of partial co-localization of VASP with p8 at the plasma membrane and in protrusive structures, which was confirmed by proximity ligation assays. Co-culture experiments revealed that p8 is transferred between Jurkat T-cells via VASP-containing conduits. Imaging and flow cytometry revealed that repression of both endogenous and overexpressed VASP by RNA interference or by CRISPR/Cas9 reduced p8 transfer to the cell surface and to target Jurkat T-cells. Stable repression of VASP by RNA interference in chronically infected MT-2 cells impaired both p8 and HTLV-1 Gag transfer to target Jurkat T-cells, while virus release was unaffected. Thus, we identified VASP as a novel interaction partner of p8, which is important for transfer of HTLV-1 p8 and Gag to target T-cells.

Latency Reversing Agents: Kick and Kill of HTLV-1?

Human T-cell leukemia virus type 1 (HTLV-1), the cause of adult T-cell leukemia/lymphoma (ATLL), is a retrovirus, which integrates into the host genome and persistently infects CD4+ T-cells. Virus propagation is stimulated by (1) clonal expansion of infected cells and (2) de novo infection. Viral gene expression is induced by the transactivator protein Tax, which recruits host factors like positive transcription elongation factor b (P-TEFb) to the viral promoter. Since HTLV-1 gene expression is repressed in vivo by viral, cellular, and epigenetic mechanisms in late phases of infection, HTLV-1 avoids an efficient CD8+ cytotoxic T-cell (CTL) response directed against the immunodominant viral Tax antigen. Hence, therapeutic strategies using latency reversing agents (LRAs) sought to transiently activate viral gene expression and antigen presentation of Tax to enhance CTL responses towards HTLV-1, and thus, to expose the latent HTLV-1 reservoir to immune destruction. Here, we review strategies that aimed at enhancing Tax expression and Tax-specific CTL responses to interfere with HTLV-1 latency. Further, we provide an overview of LRAs including (1) histone deacetylase inhibitors (HDACi) and (2) activators of P-TEFb, that have mainly been studied in context of human immunodeficiency virus (HIV), but which may also be powerful in the context of HTLV-1.

Characterizing the Interaction between the HTLV-1 Transactivator Tax-1 with Transcription Elongation Factor ELL2 and Its Impact on Viral Transactivation.

The human T-cell leukemia virus type 1 (HTLV-1)-encoded transactivator and oncoprotein Tax-1 is essential for HTLV-1 replication. We recently found that Tax-1 interacts with transcription elongation factor for RNA polymerase II 2, ELL2, which enhances Tax-1-mediated transactivation of the HTLV-1 promotor. Here, we characterize the Tax-1:ELL2 interaction and its impact on viral transactivation by confocal imaging, co-immunoprecipitation, and luciferase assays. We found that Tax-1 and ELL2 not only co-precipitate, but also co-localize in dot-like structures in the nucleus. Tax-1:ELL2 complex formation occurred independently of Tax-1 point mutations, which are crucial for post translational modifications (PTMs) of Tax-1, suggesting that these PTMs are irrelevant for Tax-1:ELL2 interaction. In contrast, Tax-1 deletion mutants lacking either N-terminal (aa 1-37) or C-terminal regions (aa 150-353) of Tax-1 were impaired in interacting with ELL2. Contrary to Tax-1, the related, non-oncogenic Tax-2B from HTLV-2B did not interact with ELL2. Finally, we found that ELL2-R1 (aa 1-353), which carries an RNA polymerase II binding domain, and ELL2-R3 (aa 515-640) are sufficient to interact with Tax-1; however, only ELL2-truncations expressing R1 could enhance Tax-1-mediated transactivation of the HTLV-1 promoter. Together, this study identifies domains in Tax-1 and ELL2 being required for Tax-1:ELL2 complex formation and for viral transactivation.

A novel positive feedback-loop between the HTLV-1 oncoprotein Tax and NF-κB activity in T-cells.

BACKGROUND: Human T-cell leukemia virus type 1 (HTLV-1) infects primarily CD4+ T-lymphocytes and evoques severe diseases, predominantly Adult T-Cell Leukemia/ Lymphoma (ATL/L) and HTLV-1-associated Myelopathy/ Tropical Spastic Paraparesis (HAM/TSP). The viral transactivator of the pX region (Tax) is important for initiating malignant transformation, and deregulation of the major signaling pathway nuclear factor of kappa B (NF-κB) by Tax represents a hallmark of HTLV-1 driven cancer. RESULTS: Here we found that Tax mutants which are defective in NF-κB signaling showed diminished protein expression levels compared to Tax wildtype in T-cells, whereas Tax transcript levels were comparable. Strikingly, constant activation of NF-κB signaling by the constitutive active mutant of inhibitor of kappa B kinase (IKK2, IKK-ß), IKK2-EE, rescued protein expression of the NF-κB defective Tax mutants M22 and K1-10R and even increased protein levels of Tax wildtype in various T-cell lines while Tax transcript levels were only slightly affected. Using several Tax expression constructs, an increase of Tax protein occurred independent of Tax transcripts and independent of the promoter used. Further, Tax and M22 protein expression were strongly enhanced by 12-O-Tetradecanoylphorbol-13-Acetate [TPA; Phorbol 12-myristate 13-acetate (PMA)]/ ionomycin, inducers of NF-κB and cytokine signaling, but not by tumor necrosis factor alpha (TNF-α). On the other hand, co-expression of Tax with a dominant negative inhibitor of κB, IκBα-DN, or specific inhibition of IKK2 by the compound ACHP, led to a vast decrease in Tax protein levels to some extent independent of Tax transcripts in transiently transfected and Tax-transformed T-cells. Cycloheximide chase experiments revealed that co-expression of IKK2-EE prolongs the half-life of M22, and constant repression of NF-κB signaling by IκBα-DN strongly reduces protein stability of Tax wildtype suggesting that NF-κB activity is required for Tax protein stability. Finally, protein expression of Tax and M22 could be recovered by NH4Cl and PYR-41, inhibitors of the lysosome and the ubiquitin-activating enzyme E1, respectively. CONCLUSIONS: Together, these findings suggest that Tax's capability to induce NF-κB is critical for protein expression and stabilization of Tax itself. Overall, identification of this novel positive feedback loop between Tax and NF-κB in T-cells improves our understanding of Tax-driven transformation.

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.

The tumor marker Fascin is induced by the Epstein-Barr virus-encoded oncoprotein LMP1 via NF-κB in lymphocytes and contributes to their invasive migration.

BACKGROUND: The actin-bundling protein Fascin (FSCN1) is a tumor marker that is highly expressed in numerous types of cancer including lymphomas and is important for migration and metastasis of tumor cells. Fascin has also been detected in B lymphocytes that are freshly-infected with Epstein-Barr virus (EBV), however, both the inducers and the mechanisms of Fascin upregulation are still unclear. RESULTS: Here we show that the EBV-encoded oncoprotein latent membrane protein 1 (LMP1), a potent regulator of cellular signaling and transformation, is sufficient to induce both Fascin mRNA and protein in lymphocytes. Fascin expression is mainly regulated by LMP1 via the C-terminal activation region 2 (CTAR2). Block of canonical NF-κB signaling using a chemical inhibitor of IκB kinase ß (IKKß) or cotransfection of a dominant-negative inhibitor of IκBα (NFKBIA) reduced not only expression of p100, a classical target of the canonical NF-κB-pathway, but also LMP1-induced Fascin expression. Furthermore, chemical inhibition of IKKß reduced both Fascin mRNA and protein levels in EBV-transformed lymphoblastoid cell lines, indicating that canonical NF-κB signaling is required for LMP1-mediated regulation of Fascin both in transfected and transformed lymphocytes. Beyond that, chemical inhibition of IKKß significantly reduced invasive migration of EBV-transformed lymphoblastoid cells through extracellular matrix. Transient transfection experiments revealed that Fascin contributed to LMP1-mediated enhancement of invasive migration through extracellular matrix. While LMP1 enhanced the number of invaded cells, functional knockdown of Fascin by two different small hairpin RNAs resulted in significant reduction of invaded, non-attached cells. CONCLUSIONS: Thus, our data show that LMP1-mediated upregulation of Fascin depends on NF-κB and both NF-κB and Fascin contribute to invasive migration of LMP1-expressing lymphocytes.

Intercellular Transport of Viral Proteins.

Viruses are vehicles to exchange genetic information and proteins between cells and organisms by infecting their target cells either cell-free, or depending on cell-cell contacts. Several viruses like certain retroviruses or herpesviruses transmit by both mechanisms. However, viruses have also evolved the properties to exchange proteins between cells independent of viral particle formation. This exchange of viral proteins can be directed to target cells prior to infection to interfere with restriction factors and intrinsic immunity, thus, making the target cell prone to infection. However, also bystander cells, e.g. immune cell populations, can be targeted by viral proteins to dampen antiviral responses. Mechanistically, viruses exploit several routes of cell-cell communication to exchange viral proteins like the formation of extracellular vesicles or the formation of long-distance connections like tunneling nanotubes. Although it is known that viral nucleic acids can be transferred between cells as well, this chapter concentrates on viral proteins of human pathogenic viruses covering all Baltimore classes and summarizes our current knowledge on intercellular transport of viral proteins between cells.

APOBEC3A, APOBEC3B, and APOBEC3H haplotype 2 restrict human T-lymphotropic virus type 1.

The human APOBEC3 family consists of seven cytidine deaminases (A3A to A3H), some of which display potent antiretroviral activity against HIV-1 and other retroviruses. Studies that analyzed the effect of A3G on human T-lymphotropic virus type 1 (HTLV-1) infectivity resulted in conflicting findings, and our knowledge of HTLV-1 restriction by other A3 proteins remains limited. Since HTLV-1, much like HIV, targets CD4(+) T cells, we hypothesized that A3 proteins other than A3G restrict HTLV-1. All seven human A3 proteins were tested in HTLV-1 reporter and HIV-1 infectivity assays. We show that A3A, A3B, and A3H haplotype 2 (A3H hapII) acted as potent inhibitors of HTLV-1. Wild-type HIV-1, in contrast, was restricted by A3B and A3H hapII, but not by A3A. Catalytic site mutants of A3A, A3B, and A3H hapII showed that A3A and A3B restriction of HTLV-1 required deaminase activity. However, A3H hapII acted in a deaminase-independent manner when restricting HTLV-1, while requiring deaminase activity for HIV-1 restriction. We also analyzed A3 editing of HTLV-1 in five T-cell lines obtained from HTLV-1-infected patients. These cell lines contained extensively edited HTLV-1 sequences with G-to-A mutations in dinucleotide contexts suggestive of APOBEC3 mutagenesis. Comparison of the A3-induced mutations from reporter cells and the patient-derived cell lines indicate that A3G but also other A3 members, possibly A3A and A3B, affect HTLV-1 in vivo. Taken together, our data indicate that HTLV-1 is a likely target for multiple A3 proteins.

The tumor marker Fascin is strongly induced by the Tax oncoprotein of HTLV-1 through NF-kappaB signals.

Oncogenic transformation of CD4(+) T cells by human T-cell lymphotropic virus type 1 (HTLV-1) is understood as the initial step to adult T-cell leukemia/lymphoma, a process that is mainly initiated by perturbation of cellular signaling by the viral Tax oncoprotein, a potent transcriptional regulator. In search of novel biomarkers with relevance to oncogenesis, we identified the tumor marker and actin-bundling protein Fascin (FSCN1) to be specifically and strongly up-regulated in both HTLV-1-transformed and adult T-cell leukemia/lymphoma patient-derived CD4(+) T cells. Fascin is important for migration and metastasis in various types of cancer. Here we report that a direct link can exist between a single viral oncoprotein and Fascin expression, as the viral oncoprotein Tax was sufficient to induce high levels of Fascin. Nuclear factor-κB signals were important for Tax-mediated transcriptional regulation of Fascin in T cells. This suggests that Fascin up-regulation by Tax contributes to the development of HTLV-1-associated pathogenesis.

Milk Transmission of Mammalian Retroviruses.

The transmission of viruses from one host to another typically occurs through horizontal or vertical pathways. The horizontal pathways include transmission amongst individuals, usually through bodily fluids or excretions, while vertical transmission transpires from mother to their offspring, either during pregnancy, childbirth, or breastfeeding. While there are more than 200 human pathogenic viruses to date, only a small number of them are known to be transmitted via breast milk, including cytomegalovirus (CMV), human immunodeficiency virus type 1 (HIV-1), and human T cell lymphotropic virus type 1 (HTLV-1), the latter two belonging to the family Retroviridae. Breast milk transmission is a common characteristic among mammalian retroviruses, but there is a lack of reports summarizing our knowledge regarding this route of transmission of mammalian retroviruses. Here, we provide an overview of the transmission of mammalian exogenous retroviruses with a focus on Orthoretrovirinae, and we highlight whether they have been described or suspected to be transmitted through breast milk, covering various species. We also elaborate on the production and composition of breast milk and discuss potential entry sites of exogenous mammalian retroviruses during oral 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.

Alternative NF-κB Signaling Discriminates Induction of the Tumor Marker Fascin by the Viral Oncoproteins Tax-1 and Tax-2 of Human T-Cell Leukemia Viruses.

Transcriptional regulation of the actin-bundling protein and tumor marker Fascin is highly diverse depending on cell and tumor type. Previously, we discovered that the viral oncoprotein Tax-1 of human T-cell leukemia virus type 1 (HTLV-1) considerably enhances Fascin expression in T-cells, depending on classical NF-κB signaling. In this study, we asked if the non-oncogenic Tax-2 of the related HTLV-2 is still able to induce Fascin by using luciferase assays, immunoblot, and qPCR. We found that Tax-2 only slightly induces Fascin expression compared to Tax-1; however, both Tax-1 and Tax-2 comparably activated a 1.6 kb fragment in the human Fascin promoter including Tax-responsive elements. Furthermore, we identified a link between Tax-induced activity of the alternative NF-κB pathway and Fascin induction. While treatment with the second mitochondria-derived activator of caspases (SMAC)-mimetic AZD5582, a compound known to robustly activate alternative NF-κB signaling, did not induce Fascin, combination of AZD5582 with activation of classical NF-κB signaling by Tax-2 significantly induced Fascin expression. In conclusion, our data demonstrate that both classical and alternative NF-κB activity are necessary for strong Fascin induction by the viral Tax oncoproteins, thus, shedding new light on the regulation of Fascin in T-cells and during viral transformation.

HDAC inhibitors Panobinostat and Romidepsin enhance tax transcription in HTLV-1-infected cell lines and freshly isolated patients' T-cells.

The viral transactivator Tax plays a key role in HTLV-1 reactivation and de novo infection. Previous approaches focused on the histone deacetylase inhibitor (HDACi) Valproate as a latency-reversing agent to boost Tax expression and expose infected cells to the host's immune response. However, following treatment with Valproate proviral load decreases in patients with HAM/TSP were only transient. Here, we hypothesize that other compounds, including more potent and selective HDACi, might prove superior to Valproate in manipulating Tax expression. Thus, a panel of HDACi (Vorinostat/SAHA/Zolinza, Panobinostat/LBH589/Farydak, Belinostat/PXD101/Beleodaq, Valproate, Entinostat/MS-275, Romidepsin/FK228/Istodax, and MC1568) was selected and tested for toxicity and potency in enhancing Tax expression. The impact of the compounds was evaluated in different model systems, including transiently transfected T-cells, chronically HTLV-1-infected T-cell lines, and freshly isolated PBMCs from HTLV-1 carriers ex vivo. We identified the pan-HDACi Panobinostat and class I HDACi Romidepsin as particularly potent agents at raising Tax expression. qRT-PCR analysis revealed that these inhibitors considerably boost tax and Tax-target gene transcription. However, despite this significant increase in tax transcription and histone acetylation, protein levels of Tax were only moderately enhanced. In conclusion, these data demonstrate the ability of Panobinostat and Romidepsin to manipulate Tax expression and provide a foundation for further research into eliminating latently infected cells. These findings also contribute to a better understanding of conditions limiting transcription and translation of viral gene products.

Elevated cyclic AMP levels in T lymphocytes transformed by human T-cell lymphotropic virus type 1.

Human T-cell lymphotropic virus type 1 (HTLV-1), the cause of adult T-cell leukemia/lymphoma (ATLL), transforms CD4(+) T cells to permanent growth through its transactivator Tax. HTLV-1-transformed cells share phenotypic properties with memory and regulatory T cells (T-reg). Murine T-reg-mediated suppression employs elevated cyclic AMP (cAMP) levels as a key regulator. This led us to determine cAMP levels in HTLV-1-transformed cells. We found elevated cAMP concentrations as a consistent feature of all HTLV-1-transformed cell lines, including in vitro-HTLV-1-transformed, Tax-transformed, and patient-derived cells. In transformed cells with conditional Tax expression, high cAMP levels coincided with the presence of Tax but were lost without it. However, transient ectopic expression of Tax alone was not sufficient to induce cAMP. We found specific downregulation of the cAMP-degrading phosphodiesterase 3B (PDE3B) in HTLV-1-transformed cells, which was independent of Tax in transient expression experiments. This is in line with the notion that PDE3B transcripts and cAMP levels are inversely correlated. Overexpression of PDE3B led to a decrease of cAMP in HTLV-1-transformed cells. Decreased expression of PDE3B was associated with inhibitory histone modifications at the PDE3B promoter and the PDE3B locus. In summary, Tax transformation and its continuous expression contribute to elevated cAMP levels, which may be regulated through PDE3B suppression. This shows that HTLV-1-transformed cells assume biological features of long-lived T-cell populations that potentially contribute to viral persistence.

Strong induction of 4-1BB, a growth and survival promoting costimulatory receptor, in HTLV-1-infected cultured and patients' T cells by the viral Tax oncoprotein.

Human T-cell leukemia virus type 1 (HTLV-1), the cause of adult T-cell leukemia, stimulates the growth of infected T cells in cultures and in nonleukemic patients. In the latter, HTLV-1 is found in long-term persisting T-cell clones. The persistence of normal T cells is controlled by the growth-stimulating and antiapoptotic functions of costimulatory receptors, while the growth-stimulating HTLV-1 functions are mediated by the viral oncoprotein Tax. Here we analyzed the impact of Tax on costimulatory receptors in T cells with repressible Tax and found that among these receptors 4-1BB (TNFRSF9/CD137/ILA) was induced most strongly. Up-regulated 4-1BB expression was a consistent feature of all HTLV-1-infected cell lines, whether patient-derived or in vitro transformed. Tax was sufficient to induce the expression of the endogenous 4-1BB gene in uninfected T cells, and it strongly activated (45-fold) the 4-1BB promoter via a single NF-kappaB site. The ligand of 4-1BB was also found on transformed T-cell lines, opening up the possibility of autostimulation. Moreover, 4-1BB expression in patients' lymphocytes ex vivo correlated with Tax expression, strongly suggesting Tax-mediated 4-1BB activation in vivo. Thus, 4-1BB up-regulation by Tax could contribute to growth, survival, and clonal expansion of the infected cells during persistence and disease.

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.

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.