HTLV-1 biofilm polarization maintained by tetraspanin CD82 is required for efficient viral transmission.

The human T-lymphotropic virus type 1 (HTLV-1) is an oncogenic retrovirus whose transmission relies primarily on cell-to-cell contacts as cell-free viruses are poorly infectious. Among the intercellular transmission routes described, HTLV-1 biofilms are adhesive structures polarized at the cell surface that confine virions in a protective environment, which is believed to promote their simultaneous delivery during infection. Here, we show that several tetraspanins are enriched in HTLV-1 biofilms and incorporated into the viral envelope. However, we report that only the tetraspanin CD82 interacts with HTLV-1 Gag proteins which initiates their polarization into viral biofilms. Also, we demonstrate that CD82 maintains HTLV-1 biofilm polarization and favors viral transmission, as its silencing induces a complete reorganization of viral clusters at the cell surface and reduces the ability of infected T-cells to transmit the virus. Our results highlight the crucial role of CD82 and its glycosylation state in the architectural organization of HTLV-1 biofilms and their subsequent transfer through intercellular contacts.IMPORTANCEIn the early stages of infection, human T-lymphotropic virus type 1 (HTLV-1) dissemination within its host is believed to rely mostly on cell-to-cell contacts. Past studies unveiled a novel mechanism of HTLV-1 intercellular transmission based on the remodeling of the host-cell extracellular matrix and the generation of cell-surface viral assemblies whose structure, composition, and function resemble bacterial biofilms. These polarized aggregates of infectious virions, identified as viral biofilms, allow the bulk delivery of viruses to target cells and may help to protect virions from immune attacks. However, viral biofilms' molecular and functional description is still in its infancy, although it is crucial to fully decipher retrovirus pathogenesis. Here, we explore the function of cellular tetraspanins (CD9, CD81, CD82) that we detect inside HTLV-1 particles within biofilms. Our results demonstrate specific roles for CD82 in the cell-surface distribution and intercellular transmission of HTLV-1 biofilms, which we document as two essential parameters for efficient viral transmission. At last, our findings indicate that N-glycosylation of cell-surface molecules, including CD82, is required for the polarization of HTLV-1 biofilms and for the efficient transmission of HTLV-1 between T-lymphocytes.

How to Control HTLV-1-Associated Diseases: Preventing de Novo Cellular Infection Using Antiviral Therapy.

Five to ten million individuals are infected by Human T-cell Leukemia Virus type 1 (HTLV-1). HTLV-1 is transmitted through prolonged breast-feeding, by sexual contacts and by transmission of infected T lymphocytes through blood transfusion. One to ten percent of infected carriers will develop a severe HTLV-1-associated disease: Adult-T-cell leukemia/lymphoma (ATLL), or a neurological disorder named Tropical Spastic Paraparesis/HTLV-1 Associated Myelopathy (TSP/HAM). In vivo, HTLV-1 is mostly detected in CD4+ T-cells, and to a lesser extent in CD8+ T cells and dendritic cells. There is a strong correlation between HTLV-1 proviral load (PVL) and clinical status of infected individuals. Thus, reducing PVL could be part of a strategy to prevent or treat HTLV-1-associated diseases among carriers. Treatment of ATLL patients using conventional chemotherapy has very limited benefit. Some chronic and acute ATLL patients are, however, efficiently treated with a combination of interferon α and zidovudine (IFN-α/AZT), to which arsenic trioxide is added in some cases. On the other hand, no efficient treatment for TSP/HAM patients has been described yet. It is therefore crucial to develop therapies that could either prevent the occurrence of HTLV-1-associated diseases or at least block the evolution of the disease in the early stages. In vivo, reverse transcriptase (RT) activity is low in infected cells, which is correlated with a clonal mode of viral replication. This renders infected cells resistant to nucleoside RT inhibitors such as AZT. However, histone deacetylase inhibitors (HDACi) associated to AZT efficiently induces viral expression and prevent de novo cellular infection. In asymptomatic STLV-1 infected non-human primates, HDACi/AZT combination allows a strong decrease in the PVL. Unfortunately, rebound in the PVL occurs when the treatment is stopped, highlighting the need for better antiviral compounds. Here, we review previously used strategies targeting HTLV-1 replication. We also tested a series of HIV-1 RT inhibitors in an in vitro anti-HTLV-1 screen, and report that bis-POM-PMEA (adefovir dipivoxil) and bis-POC-PMPA (tenofovir disoproxil) are much more efficient compared to AZT to decrease HTLV-1 cell-to-cell transmission in vitro. Our results suggest that revisiting already established antiviral drugs is an interesting approach to discover new anti-HTLV-1 drugs.

Regulated vesicle fusion generates signaling nanoterritories that control T cell activation at the immunological synapse.

How the vesicular traffic of signaling molecules contributes to T cell receptor (TCR) signal transduction at the immunological synapse remains poorly understood. In this study, we show that the protein tyrosine kinase Lck, the TCRζ subunit, and the adapter LAT traffic through distinct exocytic compartments, which are released at the immunological synapse in a differentially regulated manner. Lck vesicular release depends on MAL protein. Synaptic Lck, in turn, conditions the calcium- and synaptotagmin-7-dependent fusion of LAT and TCRζ containing vesicles. Fusion of vesicles containing TCRζ and LAT at the synaptic membrane determines not only the nanoscale organization of phosphorylated TCRζ, ZAP70, LAT, and SLP76 clusters but also the presence of phosphorylated LAT and SLP76 in interacting signaling nanoterritories. This mechanism is required for priming IL-2 and IFN-γ production and may contribute to fine-tuning T cell activation breadth in response to different stimulatory conditions.

Can viruses form biofilms?

The recent finding that the human T-cell leukemia virus type 1 (HTLV-1) encases itself in a carbohydrate-rich adhesive extracellular 'cocoon', which enables its efficient and protected transfer between cells, unveiled a new infectious entity and a novel mechanism of viral transmission. These HTLV-1 structures are observed at the surface of T cells from HTLV-1-infected patients and are reminiscent of bacterial biofilms. The virus controls the synthesis of the matrix, which surrounds the virions and attaches them to the T cell surface. We propose that, similar to bacterial biofilms, viral biofilms could represent 'viral communities' with enhanced infectious capacity and improved spread compared with 'free' viral particles, and might constitute a key reservoir for chronic infections.

Mycolactone suppresses T cell responsiveness by altering both early signaling and posttranslational events.

Mycolactone is a diffusible lipid toxin produced by Mycobacterium ulcerans, the causative agent of a necrotizing skin disease referred to as Buruli ulcer. Intriguingly, patients with progressive lesions display a systemic suppression of Th1 responses that resolves on surgical excision of infected tissues. In this study, we examined the effects of mycolactone on the functional biology of T cells and identified two mechanisms by which mycolactone suppresses cell responsiveness to antigenic stimulation. At noncytotoxic concentrations, mycolactone blocked the activation-induced production of cytokines by a posttranscriptional, mammalian target of rapamycin, and cellular stress-independent mechanism. In addition, mycolactone triggered the lipid-raft association and activation of the Src-family kinase, Lck. Mycolactone-mediated hyperactivation of Lck resulted in the depletion of intracellular calcium stores and downregulation of the TCR, leading to impaired T cell responsiveness to stimulation. These biochemical alterations were not observed when T cells were exposed to other bacterial lipids, or to structurally related immunosuppressors. Mycolactone thus constitutes a novel type of T cell immunosuppressive agent, the potent activity of which may explain the defective cellular responses in Buruli ulcer patients.

Vesicle traffic to the immunological synapse: a multifunctional process targeted by lymphotropic viruses.

The site of contact between T lymphocytes and antigen-presenting cells becomes, upon antigen recognition, an organized junction named the immunological synapse. Various T cell organelles polarize, together with microtubules, toward the antigen-presenting cell. Among them, intracellular vesicular compartments, such as the Golgi apparatus, the recycling endosomal compartment, or cytotoxic granules help to build the immunological synapse and ensure effector functions, such as polarized secretion of cytokines by helper T cells, or exocytosis of lytic granules by cytotoxic T cells. Lymphotropic retroviruses, such as the human immunodeficiency virus type 1, the human T cell leukemia virus type 1, or the Herpesvirus saimiri, can subvert some of the vesicle traffic mechanisms impeding the generation and function of the immunological synapses. This review focuses on the polarization of vesicle traffic, its regulation, and its role in maintaining the structure and function of the immunological synapse. We discuss how some lymphotropic viruses target the vesicle traffic in T lymphocytes, inhibiting the formation of immunological synapses and modulating the response of infected T cells.

Biofilm-like extracellular viral assemblies mediate HTLV-1 cell-to-cell transmission at virological synapses.

Human T cell leukemia virus type 1 (HTLV-1) is a lymphotropic retrovirus whose cell-to-cell transmission requires cell contacts. HTLV-1-infected T lymphocytes form 'virological synapses', but the mechanism of HTLV-1 transmission remains poorly understood. We show here that HTLV-1-infected T lymphocytes transiently store viral particles as carbohydrate-rich extracellular assemblies that are held together and attached to the cell surface by virally-induced extracellular matrix components, including collagen and agrin, and cellular linker proteins, such as tetherin and galectin-3. Extracellular viral assemblies rapidly adhere to other cells upon cell contact, allowing virus spread and infection of target cells. Their removal strongly reduces the ability of HTLV-1-producing cells to infect target cells. Our findings unveil a novel virus transmission mechanism based on the generation of extracellular viral particle assemblies whose structure, composition and function resemble those of bacterial biofilms. HTLV-1 biofilm-like structures represent a major route for virus transmission from cell to cell.

Recombinant infectious hematopoietic necrosis viruses induce protection for rainbow trout Oncorhynchus mykiss.

Infectious hematopoietic necrosis virus (IHNV) and viral hemorrhagic septicaemia virus (VHSV) are rhabdoviruses that infect salmonids, producing serious economic losses. Two recombinant IHN viruses were generated by reverse genetics. For one (rIHNV GFP) the IHNV NV gene was replaced with the green fluorescent protein (GFP) gene. In the other (rIHNV-Gvhsv GFP) the G gene was also exchanged for that of VHSV. No mortalities, external signs or histological lesions were observed in experimental infections conducted with the recombinant viruses. Neither the rIHNV GFP nor rIHNV-Gvhsv GFP was detected by RT-PCR in any of the examined tissues from experimentally infected fish. In order to assess their potential as vaccines against the wild type viruses, rainbow trout were vaccinated with the recombinant viruses by intraperitoneal injection and challenged 30 d later with virulent IHNV or VHSV. The GFP viruses provided protection against both wild type viruses. None of the recombinant viruses induced antibody production, and the expression of interferon (IFNalpha4) and interferon induced genes such as Mx protein and ISG-15 was not different to that of controls. The rIHNV-Gvhsv GFP did not inhibit cellular apoptosis as it was observed in an IHNV inoculated fish cell line. These studies suggest that the recombinant rIHNV-Gvhsv GFP is a promising candidate as a live recombinant vaccine and also provides a good model to further study viral pathogenicity and the molecular basis of protection against these viral infections.

ZAP-70 kinase regulates HIV cell-to-cell spread and virological synapse formation.

HIV efficiently spreads in lymphocytes, likely through virological synapses (VSs). These cell-cell junctions share some characteristics with immunological synapses, but cellular proteins required for their constitution remain poorly characterized. We have examined here the role of ZAP-70, a key kinase regulating T-cell activation and immunological synapse formation, in HIV replication. In lymphocytes deficient for ZAP-70, or expressing a kinase-dead mutant of the protein, HIV replication was strikingly delayed. We have characterized further this replication defect. ZAP-70 was dispensable for the early steps of viral cycle, from entry to expression of viral proteins. However, in the absence of ZAP-70, intracellular Gag localization was impaired. ZAP-70 was required in infected donor cells for efficient cell-to-cell HIV transmission to recipients and for formation of VSs. These results bring novel insights into the links that exist between T-cell activation and HIV spread, and suggest that HIV usurps components of the immunological synapse machinery to ensure its own spread through cell-to-cell contacts.

Human immunodeficiency virus type-1 infection impairs the formation of the immunological synapse.

HIV-1-infected lymphocytes improperly respond to T cell antigen receptor (TCR) stimulation. To document this phenomenon, we studied the capacity of HIV-1-infected lymphocytes to form immunological synapses. We show here that HIV-1-infected T cells poorly conjugated with antigen-presenting cells, and when they formed conjugates, the synapses were abnormal. TCR and Lck accumulated in the recycling endosomal compartment, and their clustering at the synapse was severely reduced. These phenomena were, to a large extent, caused by Nef, a viral protein affecting intracellular trafficking and signaling pathways. Concomitantly, in HIV-infected cells, tyrosine phosphorylation at the synapse and the patterns of tyrosine phosphorylated proteins were disturbed in a Nef-dependent manner. These findings underscore the importance of Lck and TCR endosomal trafficking in synapse formation and early T cell signaling. Alteration of endocytic and signaling networks at the immunological synapse likely impacts the function and fate of HIV-1-infected cells.

Histological, serological and virulence studies on rainbow trout experimentally infected with recombinant infectious hematopoietic necrosis viruses.

Several recombinant infectious hematopoietic necrosis viruses (IHNV) were produced by reverse genetics and their pathogenicity in trout was evaluated and compared to that of the wild type (wt) viruses IHNV and viral haemorrhagic septicemia virus (VHSV). Recombinant IHNVs used in this study were: rIHNV, identical to the wtIHNV; rIHNV-Gvhsv, a recombinant virus expressing the VHSV G gene instead of the IHNV G gene; rIHNV-Gmut, which possesses 2 targeted mutations in the glycoprotein; and rIHNVmut-Gmut, which is similar to the rIHNV-Gmut, but exhibits additional mutations along the genome. Results obtained in experimental infections showed that the rIHNV and rIHNV-Gmut were the most virulent recombinant viruses. Severity of the lesions induced by the different recombinant viruses was in agreement with mortality data. The kidney and the liver were the organs most affected by the most pathogenic viruses, and the lesions observed resembled those produced by wtIHNV. The introduction of mutations did not alter the tissue tropism of the virus. The recombinant viruses were able to replicate in fish, as shown by immunoperoxidase assay and RT-PCR. Antibodies against IHNV were detected in the fish inoculated with IHNV, rIHNV, rIHNV-Gmut and rIHNVmut-Gmut, and antibodies against VHSV were also found in fish infected with rIHNV-Gvhsv. Finally, antibody production was highest in fish infected with the rIHNVmut-Gmut even though this virus was the least virulent.

The glycoprotein of a fish rhabdovirus profiles the virus-specific T-cell repertoire in rainbow trout.

T-cell responses to viruses are still poorly investigated in lower vertebrates. In rainbow trout, a specific clonal expansion of T cells in response to infection with viral haemorrhagic septicaemia virus (VHSV) was recently identified. Expanded T-cell clones expressed a unique 8 aa Vbeta4-Jbeta1 junction (SSGDSYSE) in different individuals, reminiscent of a typical public response. To get further insight into the nature of this response the modifications of the T-cell repertoire following immunization with plasmid expressing the VHSV external glycoprotein (G), which is the only protein involved in protective immunity, was analysed. After G-based DNA immunization, CDR3-length spectratypes were skewed for several Vbeta-Jbeta combinations, including Vbeta4-Jbeta1. In Vbeta4-Jbeta1, biases consisted of 6 and 8 aa junctions that were detected from day 52, and were still present 3 months after DNA immunization. Sequence analysis of the Vbeta4-Jbeta1 junctions showed that the 8 aa junction (SSGDSYSE) was clearly expanded, indicating that viral G protein was probably the target of the anti-VHSV public response. Additional 6 and 8 aa Vbeta4-Jbeta1 junctions were also expanded in G-DNA-vaccinated fish, showing that significant clonotypic diversity was selected in response to the plasmid-delivered G protein. This higher clonotypic diversity may be related to the demonstrated higher efficiency of G-based DNA vaccines over whole virus immunization. The use of infectious hematopietic necrosis virus (IHNV) recombinant viruses, expressing the VHSV G protein, further substantiated the VHSV G-protein specificity of the 8 aa Vbeta4-Jbeta1 response and designated the 6 aa Vbeta4-Jbeta1 response as potentially directed to a T-cell epitope common to VHSV and IHNV.

Essential role of the NV protein of Novirhabdovirus for pathogenicity in rainbow trout.

Novirhabdovirus, infectious hematopoietic necrosis virus (IHNV), and viral hemorrhagic septicemia virus (VHSV) are fish rhabdoviruses that, in comparison to the other rhabdoviruses, contain an additional gene coding for a small nonvirion (NV) protein of unassigned function. A recombinant IHNV with the NV gene deleted but expressing the green fluorescent protein (rIHNV-Delta NV) has previously been shown to be efficiently recovered by reverse genetics (S. Biacchesi et al., J. Virol. 74:11247-11253, 2000). However, preliminary experiments suggested that the growth in cell culture of rIHNV-Delta NV was affected by the NV deletion. In the present study, we show that the growth in cell culture of rIHNV-Delta NV is indeed severely impaired but that a normal growth of rIHNV-Delta NV can be restored when NV is provided in trans by using fish cell clones constitutively expressing the NV protein. These results indicate that NV is a protein that has a crucial biological role for optimal replication of IHNV in cell culture. Although IHNV and VHSV NV proteins do not share any significant identity, we show here that both NV proteins play a similar role since a recombinant IHNV virus, rIHNV-NV(VHSV), in which the IHNV NV open reading frame has been replaced by that of VHSV, was shown to replicate as well as the wild-type (wt) IHNV into fish cells. Finally, data provided by experimental fish infections with the various recombinant viruses strongly suggest an essential role of the NV protein for the pathogenicity of IHNV. Furthermore, we show that juvenile trout immunized with NV-knockout IHNV were protected against challenge with wt IHNV. That opens a new perspective for the development of IHNV attenuated live vaccines.

High level of Bcl-2 counteracts apoptosis mediated by a live rabies virus vaccine strain and induces long-term infection.

We report here that rabies virus strains, currently used to immunize wildlife against rabies, induce not only caspase-dependent apoptosis in the human lymphoblastoid Jurkat T cell line (Jurkat-vect), but also a caspase-independent pathway involving the apoptosis-inducing factor (AIF). In contrast, a strain of neurotropic RV that does not induce apoptosis did not activate caspases or induce AIF translocation. Bcl-2 overproduction in Jurkat T cells (Jurkat-Bcl-2) abolished both pathways. ERA infection and production were similar in Jurkat-vect and Jurkat-Bcl-2 cells, indicating Bcl-2 has no direct antiviral effects. Bcl-2 production is naturally upregulated by day 3 in ERA-infected Jurkat-vect cultures. The increase in Bcl-2 levels seems to be controlled by the virus infection itself and results in the establishment of long-term, persistently infected cultures that continue to produce virus. Thus, in infections with live RV vaccine strains, infected cells may be productive reservoirs of virus in the long term. This may account for the high efficacy of live rabies vaccines.

Apoptosis inversely correlates with rabies virus neurotropism.

We report that non-neurotropic rabies virus (RV) strains, currently used to immunize wildlife against rabies, induces not only a caspase-dependent apoptosis in the human lymphoblastoid Jurkat T cell line (Jurkat-vect), but also a caspase-independent pathway. Cell redistribution of the apoptosis-inducing factor (AIF) was observed in Jurkat-vect infected with RV vaccine strain. Bcl-2 overproduction in Jurkat T cells (Jurkat-Bcl-2) abolished both caspase activation and AIF distribution. In contrast, strain of neurotropic RV did not induce apoptosis. The inverse correlation of the induction of apoptosis and the capacity of a virus strain to invade the brain suggests that blockage of apoptosis could be a strategy selected by neurotropic virus to favor its progression through the nervous system.