Perturbation of Human T-Cell Leukemia Virus Type 1 Particle Morphology by Differential Gag Co-Packaging.

Human T-cell leukemia virus type 1 (HTLV-1) is an important cancer-causing human retrovirus that has infected approximately 15 million individuals worldwide. Many aspects of HTLV-1 replication, including virus particle structure and assembly, are poorly understood. Group-specific antigen (Gag) proteins labeled at the carboxy terminus with a fluorophore protein have been used extensively as a surrogate for fluorescence studies of retroviral assembly. How these tags affect Gag stoichiometry and particle morphology has not been reported in detail. In this study, we used an HTLV-1 Gag expression construct with the yellow fluorescence protein (YFP) fused to the carboxy-terminus as a surrogate for the HTLV-1 Gag-Pol to assess the effects of co-packaging of Gag and a Gag-YFP on virus-like particle (VLP) morphology and analyzed particles by cryogenic transmission electron microscopy (cryo-TEM). Scanning transmission electron microscopy (STEM) and fluorescence fluctuation spectroscopy (FFS) were also used to determine the Gag stoichiometry. We found that ratios of 3:1 (Gag:Gag-YFP) or greater resulted in a particle morphology indistinguishable from that of VLPs produced with the untagged HTLV-1 Gag, i.e., a mean diameter of ~113 nm and a mass of 220 MDa as determined by cryo-TEM and STEM, respectively. Furthermore, FFS analysis indicated that HTLV-1 Gag-YFP was incorporated into VLPs in a predictable manner at the 3:1 Gag:Gag-YFP ratio. Both STEM and FFS analyses found that the Gag copy number in VLPs produced with a 3:1 ratio of Gag:Gag-YFP was is in the range of 1500-2000 molecules per VLP. The observations made in this study indicate that biologically relevant Gag-Gag interactions occur between Gag and Gag-YFP at ratios of 3:1 or higher and create a Gag lattice structure in VLPs that is morphologically indistinguishable from that of VLPs produced with just untagged Gag. This information is useful for the quantitative analysis of Gag-Gag interactions that occur during virus particle assembly and in released immature particles.

Distinct Morphology of Human T-Cell Leukemia Virus Type 1-Like Particles.

The Gag polyprotein is the main retroviral structural protein and is essential for the assembly and release of virus particles. In this study, we have analyzed the morphology and Gag stoichiometry of human T-cell leukemia virus type 1 (HTLV-1)-like particles and authentic, mature HTLV-1 particles by using cryogenic transmission electron microscopy (cryo-TEM) and scanning transmission electron microscopy (STEM). HTLV-1-like particles mimicked the morphology of immature authentic HTLV-1 virions. Importantly, we have observed for the first time that the morphology of these virus-like particles (VLPs) has the unique local feature of a flat Gag lattice that does not follow the curvature of the viral membrane, resulting in an enlarged distance between the Gag lattice and the viral membrane. Other morphological features that have been previously observed with other retroviruses include: (1) a Gag lattice with multiple discontinuities; (2) membrane regions associated with the Gag lattice that exhibited a string of bead-like densities at the inner leaflet; and (3) an arrangement of the Gag lattice resembling a railroad track. Measurement of the average size and mass of VLPs and authentic HTLV-1 particles suggested a consistent range of size and Gag copy numbers in these two groups of particles. The unique local flat Gag lattice morphological feature observed suggests that HTLV-1 Gag could be arranged in a lattice structure that is distinct from that of other retroviruses characterized to date.

Analysis of human T-cell leukemia virus type 1 particles by using cryo-electron tomography.

The particle structure of human T-cell leukemia virus type 1 (HTLV-1) is poorly characterized. Here, we have used cryo-electron tomography to analyze HTLV-1 particle morphology. Particles produced from MT-2 cells were polymorphic, roughly spherical, and varied in size. Capsid cores, when present, were typically poorly defined polyhedral structures with at least one curved region contacting the inner face of the viral membrane. Most of the particles observed lacked a defined capsid core, which likely impacts HTLV-1 particle infectivity.

Human T-cell leukemia virus type 1 p8 protein increases cellular conduits and virus transmission.

The human T-cell leukemia virus type 1 (HTLV-1) is the cause of adult T-cell leukemia/lymphoma as well as tropical spastic paraparesis/HTLV-1-associated myelopathy. HTLV-1 is transmitted to T cells through the virological synapse and by extracellular viral assemblies. Here, we uncovered an additional mechanism of virus transmission that is regulated by the HTLV-1-encoded p8 protein. We found that the p8 protein, known to anergize T cells, is also able to increase T-cell contact through lymphocyte function-associated antigen-1 clustering. In addition, p8 augments the number and length of cellular conduits among T cells and is transferred to neighboring T cells through these conduits. p8, by establishing a T-cell network, enhances the envelope-dependent transmission of HTLV-1. Thus, the ability of p8 to simultaneously anergize and cluster T cells, together with its induction of cellular conduits, secures virus propagation while avoiding the host's immune surveillance. This work identifies p8 as a viral target for the development of therapeutic strategies that may limit the expansion of infected cells in HTLV-1 carriers and decrease HTLV-1-associated morbidity.

Biophysical analysis of HTLV-1 particles reveals novel insights into particle morphology and Gag stochiometry.

BACKGROUND: Human T-lymphotropic virus type 1 (HTLV-1) is an important human retrovirus that is a cause of adult T-cell leukemia/lymphoma. While an important human pathogen, the details regarding virus replication cycle, including the nature of HTLV-1 particles, remain largely unknown due to the difficulties in propagating the virus in tissue culture. In this study, we created a codon-optimized HTLV-1 Gag fused to an EYFP reporter as a model system to quantitatively analyze HTLV-1 particles released from producer cells. RESULTS: The codon-optimized Gag led to a dramatic and highly robust level of Gag expression as well as virus-like particle (VLP) production. The robust level of particle production overcomes previous technical difficulties with authentic particles and allowed for detailed analysis of particle architecture using two novel methodologies. We quantitatively measured the diameter and morphology of HTLV-1 VLPs in their native, hydrated state using cryo-transmission electron microscopy (cryo-TEM). Furthermore, we were able to determine HTLV-1 Gag stoichiometry as well as particle size with the novel biophysical technique of fluorescence fluctuation spectroscopy (FFS). The average HTLV-1 particle diameter determined by cryo-TEM and FFS was 71 ± 20 nm and 75 ± 4 nm, respectively. These values are significantly smaller than previous estimates made of HTLV-1 particles by negative staining TEM. Furthermore, cryo-TEM reveals that the majority of HTLV-1 VLPs lacks an ordered structure of the Gag lattice, suggesting that the HTLV-1 Gag shell is very likely to be organized differently compared to that observed with HIV-1 Gag in immature particles. This conclusion is supported by our observation that the average copy number of HTLV-1 Gag per particle is estimated to be 510 based on FFS, which is significantly lower than that found for HIV-1 immature virions. CONCLUSIONS: In summary, our studies represent the first quantitative biophysical analysis of HTLV-1-like particles and reveal novel insights into particle morphology and Gag stochiometry.

Human T-lymphotropic virus-1 visualized at the virological synapse by electron tomography.

Human T-lymphotropic virus 1 (HTLV-1) is transmitted directly between cells via an organized cell-cell contact called a virological synapse (VS). The VS has been studied by light microscopy, but the ultrastructure of the VS and the nature of the transmitted viral particle have remained unknown. Cell-free enveloped virions of HTLV-1 are undetectable in the serum of individuals infected with the human T-lymphotropic virus 1 (HTLV-1) and during in vitro culture of naturally infected lymphocytes. However, the viral envelope protein is required for infectivity of HTLV-1, suggesting that complete, enveloped HTLV-1 virions are transferred across the synapse. Here, we use electron tomography combined with immunostaining of viral protein to demonstrate the presence of enveloped HTLV-1 particles within the VS formed between naturally infected lymphocytes. We show in 3D that HTLV-1 particles can be detected in multiple synaptic clefts at different locations simultaneously within the same VS. The synaptic clefts are surrounded by the tightly apposed plasma membranes of the two cells. HTLV-1 virions can contact the recipient cell membrane before detaching from the infected cell. The results show that the HTLV-1 virological synapse that forms spontaneously between lymphocytes of HTLV-1 infected individuals allows direct cell-cell transmission of the virus by triggered, directional release of enveloped HTLV-1 particles into confined intercellular spaces.

DC-SIGN facilitates fusion of dendritic cells with human T-cell leukemia virus type 1-infected cells.

Interactions between the oncogenic retrovirus human T-cell leukemia virus type 1 (HTLV-1) and dendritic cells (DCs) are poorly characterized. We show here that monocyte-derived DCs form syncytia and are infected upon coculture with HTLV-1-infected lymphocytes. We examined the role of DC-specific ICAM-3-grabbing nonintegrin (DC-SIGN), a C-type lectin expressed in DCs, in HTLV-1-induced syncytium formation. DC-SIGN is known to bind with high affinity to various viral envelope glycoproteins, including human immunodeficiency virus (HIV) and hepatitis C virus, as well as to the cellular receptors ICAM-2 and ICAM-3. After cocultivating DCs and HTLV-1-infected cells, we found that anti-DC-SIGN monoclonal antibodies (MAbs) were able to decrease the number and size of HTLV-1-induced syncytia. Moreover, expression of the lectin in epithelial-cell lines dramatically enhanced the ability to fuse with HTLV-1-positive cells. Interestingly, in contrast to the envelope (Env) glycoproteins of HIV and other viruses, that of HTLV-1 does not bind directly to DC-SIGN. The facilitating role of the lectin in HTLV-1 syncytium formation is mediated by its interaction with ICAM-2 and ICAM-3, as demonstrated by use of MAbs directed against these adhesion molecules. Altogether, our results indicate that DC-SIGN facilitates HTLV-1 infection and fusion of DCs through an ICAM-dependent mechanism.

Role of the human T-cell leukemia virus type 1 PTAP motif in Gag targeting and particle release.

Human T-cell leukemia virus type 1 (HTLV-1) Gag is targeted to the plasma membrane for particle assembly and release. How HTLV-1 Gag targeting occurs is not well understood. The PPPY and PTAP motifs were previously shown to be involved in HTLV-1 particle release with PTAP playing a more subtle role in virus budding. These L domains function through the interaction with host cellular proteins normally involved in multivesicular body (MVB) morphogenesis. The plasma membrane pathway rather than the MVB pathway was found to be the primary pathway for HTLV-1 particle release in HeLa cells. Intriguingly, disruption of the PTAP motif led to a defect in the targeting of Gag from the plasma membrane to CD63-positive MVBs. Particles or particle buds were observed to be associated with MVBs by electron microscopy, implying that Gag targeting to the MVB resulted in particle budding. Blocking clathrin-dependent endocytosis was found not to influence localization of the HTLV-1 Gag PTAP mutant, indicating that Gag did not reach the MVBs through clathrin-dependent endocytosis. Our observations imply that the interaction between Gag and TSG101 is not required for Gag targeting to the MVB. Overexpression of dynamitin p50 increased particle release, suggesting that there was an increase in the intracellular transport of MVBs to the cell periphery by the utilization of the dynein-dynactin motor complex. Intriguingly, virus particle release with this mutant was reduced by 20-fold compared to that of wild type in HeLa cells, which is in marked contrast to the less-than-twofold defect observed for particle production of the HTLV-1 Gag PTAP mutant from 293T cells. These results indicate that the role of the PTAP motif in L domain function is cell type dependent.

Thymus-derived leukemia-lymphoma in mice transgenic for the Tax gene of human T-lymphotropic virus type I.

Adult T-cell leukemia-lymphoma (ATLL) is a group of T-cell malignancies caused by infection with human T-lymphotropic virus type I (HTLV-I). Although the pathogenesis of ATLL remains incompletely understood, the viral regulatory protein Tax is centrally involved in cellular transformation. Here we describe the generation of HTLV-I Tax transgenic mice using the Lck proximal promoter to restrict transgene expression to developing thymocytes. After prolonged latency periods, transgenic mice developed diffuse large-cell lymphomas and leukemia with clinical, pathological and immunological features characteristic of acute ATLL. Transgenic mice were functionally immunocompromised and they developed opportunistic infections. Fulminant disease also developed rapidly in SCID mice after engraftment of lymphomatous cells from transgenic mice. Flow cytometry showed that the cells were CD4(-) and CD8(-), but CD44(+), CD25(+) and cytoplasmic CD3(+). This phenotype is indicative of a thymus-derived pre-T-cell phenotype, and disease development was associated with the constitutive activation of NF-kappaB. Our model accurately reproduces human disease and will provide a tool for analysis of the molecular events in transformation and for the development of new therapeutics.

HTLV: virus linfotrópico de células T humanas. Historia de un virus milenario y la importancia en el descubrimiento de las migraciones humanas / HTLV: lymphotropic virus of human t-cells. The history of an ancient virus and its significance of human migrations

El virus HTLV I/II es un retrovirus, muy antiguo, cuyo origen posiblemente sea su homólogo en los simios, quienes lo transmiten al humano y luego se difunde entre nuestra especie. Gracias a los adelantos científicos, se han podido establecer las tres probables entradas o rutas migratorias de nuestros antepasados entre los distintos continentes. Ellos lamentablemente desconocían que estaban infectados. En nuestros tiempos, este insignificante y tan pequeño virus presenta áreas endémicas en todo el mundo (inclusive Argentina) y es el responsable de dos enfermedades, la Paraparesia Espástica Tropical y la Leucemia T del adulto, ambas incurables. Se ha podido prevenir la diseminación del mismo por el desarrollo de pruebas de tamizaje o selección (enzimoinmunoensayo) y suplementarias (Western Blot), como también por el asesoramiento médico de los individuos infectados.

Both the PPPY and PTAP motifs are involved in human T-cell leukemia virus type 1 particle release.

In retroviruses, the late (L) domain has been defined as a conserved motif in the Gag polyprotein precursor that, when mutated, leads to the emergence of virus particles that fail to pinch off from the plasma membrane. These domains have been observed to contain the PPXY, PTAP, or YXXL motifs. The deltaretroviruses, which include bovine leukemia virus (BLV) and human T-cell leukemia virus type 1 (HTLV-1) and HTLV-2, have a conserved PPPY motif in the C-terminal region of the matrix (MA) domain of Gag, while HTLV-1 also encodes a PTAP motif in MA. In this study, we analyzed the roles of the PPPY and PTAP motifs in the C terminus of MA in HTLV-1 particle release. Mutation of either motif (i.e., PPPY changed to APPY or PTAP changed to PTRP) reduced budding efficiencies. Particle buds and electron-dense regions of plasma membrane were observed by electron microscopy. When the locations of PPPY and PTAP were switched, particle release was eliminated. Intriguingly, the replacement of the PTAP motif with either the PPPY or YPDL motifs did not influence the release of virus particles, but the replacement of the PPPY motif with either PTAP or YPDL eliminated particle production. This indicates that the role that PPPY plays in HTLV-1 budding cannot be replaced with either PTAP or YPDL. A similar observation was made with the BLV PPPY motif. Finally, HTLV-1 particle release was found to be sensitive to proteasome inhibitors, implicating a role for ubiquitin in HTLV-1 budding. In summary, our observations indicate that (i) the PPPY motif plays a crucial role in virus budding and (ii) the PTAP motif plays a more subtle role in HTLV-1 particle release. Each of these motifs may play an important role in virus release from specific cell types and therefore be important in efficient virus spread and transmission.

Differential budding efficiencies of human T-cell leukemia virus type I (HTLV-I) Gag and Gag-Pro polyproteins from insect and mammalian cells.

In this study, we examined the ability of human T-cell leukemia virus type I (HTLV-I) Gag and Gag-Pro to assemble immature virus-like particles (VLPs) and bud from insect and mammalian cells. Transmission electron microscopy of insect cells infected with a recombinant baculovirus carrying the entire gag gene revealed that Pr53(Gag) is targeted to the plasma membrane, where it extensively accumulates and forms electron-dense evaginations. However, no particles could be detected either inside the cells or in the culture supernatants. With the Gag-Pro-expressing construct, we observed HTLV-I-specific cytoplasmic proteolysis of the Gag precursor, but again no particle released in the culture supernatants. Transmission electron microscopic analysis of insect cells expressing Gag-Pro polyprotein revealed large vacuoles in the cytoplasm and no budding particles at the plasma membrane. In contrast, human immunodeficiency virus type 1 Gag polyprotein expressed in insect cells is able to release VLPs. These data showed that unlike other retroviruses, Pr53(Gag) is unable to be released as immature VLPs from insect cells. To determine whether the block in particle budding and release is due to an intrinsic property of Pr53(Gag) or the absence of essential cellular factors in insect cells, we expressed Gag and Gag-Pro polyproteins in human 293 cells. The results indicate that Pr53(Gag) and p24 capsid are released within particles into the culture supernatants of human 293 cells. We found that the myristylation of the N-terminal glycine residue is essential for Gag release. Altogether, these results strongly suggest that the proper assembly of HTLV-I particles is dependent on mammalian host cell factors.

Visualisation of phenotypically mixed HIV-1 and HTLV-I virus particles by electron microscopy.

Virions produced after HIV-1 infection of HTLV-I transformed cells have an expanded tropism that has been attributed to the presence of HTLV-I glycoproteins in the envelope. This report now directly identifies these phenotypically mixed virions by immunogold labelling electron microscopy. Furthermore we estimate there are 2% of these in cell-free supernatant, which represents up to 1 x 10(7) particles/ml from an in vitro infection. HTLV-1 envelope labelling was localised to a single region, suggesting a defined event in packaging of foreign envelope proteins into HIV-1 virus particles.

[A virus called HTLV-1. Virological and molecular aspects]. / Un virus appelé HTLV-1. Aspects virologiques et moléculaires.

A VIRAL PATHOGEN: HTLV-1, the first oncogenic retrovirus discovered in man carries, in addition to the genes coding for its structural protein, genes for capside and matrix (gag), reverse transcriptase and integrase (pol), protease and envelope (env), a unique region (termed pX) of about 2 kb situated at the position 3' of the env gene that encodes for Tax and Rex regulator proteins. TAX AND REX: These two proteins play a role in the viral cycle. Rex acts at the post-transcriptional level; Its capacity to modulate Tax expression could allow the virus to establish a chronic infection with little expression. Tax stimulates viral transcription and also plays a fundamental role in leukecernogenesis by modifying the expression of several genes crucial for cell survival and proliferation. GENETIC STABILITY: The HTLV-1 genome sequence appears to be very stable. Specifically leukernogeneic or neurotrophic strains are unlikely to exist. However, despite this genetic stability, there are molecular variants related to the geographical origin of the virus.

Isolation of a retrovirus from multiple sclerosis patients in self-generated Iodixanol gradients.

The use of Iodixanol, a relatively new iodinated gradient medium, is described for isolation of a retrovirus, which was harvested from the supernatant of lymphoid cell lines originating from patients with multiple sclerosis (MS). The virus is produced in low amounts and has been shown to be fragile, as manifested in a loss of surface glycoproteins when purified in other gradient media. The gradient fractions were analysed after centrifugation in Iodixanol by incorporation of 3H-UTP, reverse transcriptase (RT) assays and electron microscopy (EM) and it was found that Iodixanol does not cause the degree of damage to the particles observed previously. These more favourable conditions are probably due to low viscosity and almost iso-osmotic conditions even in high concentrations. Furthermore, these advantages go together with higher reproducibility in self-forming gradients, easier handling and shorter centrifugation time. Iodixanol can also be used for preparation of HTLV-1.

Electron microscopic observation of a Brazilian HTLV-I isolated.

Several studies carried out in Brazil have shown a high incidence of HTLV-I infection among the general population and in different groups, such as blood donors, hemophiliacs, hematological and neurological patients. Cases of adult T-cell leukemia/lymphoma as well as tropical spastic paraparesis/HTLV-I associated myelopathy have already been described. Therefore, it is important to characterize Brazilian HTLV strains using different technical approaches. The purpose of this paper is to characterize and recognize HTLV particles employing routine and immunoelectron microscopy in lymphocyte cocultures. Ultrastructural analysis showed typical large and small virus particles in close relation with the lymphocyte membrane. Immunoelectron microscopy, carried out with HTLV positive sera, allowed the identification of the virus as a type C oncovirus, group HTLV-BLV. The first interaction events between virus and lymphocyte membrane have also been analysed and structures related to the endocytic route for HTLV entrance were pointed out.

Infection of choroid plexus cells by human T cell leukaemia virus type I.

Very little is known about the factors that determine the outcome of infection by human T cell leukaemia virus type I (HTLV-I) and the neurotropism of this virus is still a controversial point. In transgenic mice, the HTLV-I LTR is active mainly in the central nervous system (CNS), in parenchyma as well as in ependymal and choroid plexus cells. The latter are of particular interest and could represent the way of entry of the virus into the CNS. In this study we show that primary cultures of sheep choroid plexus can be infected with HTLV-I, leading to characteristic multinucleated syncytial cells containing virus RNA and proteins. HTLV-I p24 Gag protein was detected in the culture medium and the presence of virus particles was observed by electron microscopy 40 days after infection. At this time post-infection HTLV-I could be transmitted to human cord blood cells.

Establishment of novel lymphoid cell lines dually infected with human T cell lymphotropic viruses types I and II.

With the goal of establishing an in vitro system of dual infection with human T cell lymphotropic viruses (HTLV) types I and II, rabbit lymphocytes were cocultured with a mixture of lethally irradiated HTLV-I-producing Ra-1 and HTLV-II-producing RII cell lines. This gave rise to a lymphoid cell line, RW-1, that was dually infected with HTLV-I and -II as detected by immunofluorescence staining, electron microscopy, and polymerase chain reaction using primers specific for the pol and env regions of each virus and by Southern blot hybridization. Two clonal cell lines derived from RW-1 were also coinfected with the viruses, indicating that dual infection had occurred at the single cell level. The coinfection could be readily propagated to fresh lymphocytes by coculture with RW-1. In contrast, attempts to superinfect HTLV-I-infected lymphoid cell lines with HTLV-II and vice versa were consistently unsuccessful, suggesting receptor interference between HTLV-I and -II.

Ultrastructural aspects of the human T-lymphotropic virus type I (HTLV-I) in Brazil

Nowadays, Brazil may be considered an endemic area for HTLV-I infection. Several studies showed a high incidence of HTLV-I infection among the general population and different groups such as blood donors, hemophiliacs, hematological and neurological patients. Cases of adult T-cell leukemia/lymphoma as well as tropical spastic paraparesis/HTLV-I associated myelopathy have been already described. Therefore the use of different technical approaches, to characterize Brazilian HTLV strains has become important. HTLV particles were characterized and recognized by the use of transmission electron microscopy in lymphocyte cocultures. The ultra-structural analysis revealed typical virus particles close to the lymphocyte membrane. The immunoelectron microscopy allowed the identification of the virus as HTLV, a type C oncovirus, group HTLV-BLV. The ethanol phosphotungstic acid technique showed structures similar to the virus budding process and the routine preparations have contributed to the analysis of the first virus-cell interaction events. Structures related to the endocytic route HTLV entrance are also pointed out.

The cutaneous T cell lymphoma, mycosis fungoides, is a human T cell lymphotropic virus-associated disease. A study of 50 patients.

For nearly two decades it has been suspected that the cutaneous T cell lymphoma, mycosis fungoides (MF), and its leukemic variant, the Sézary syndrome, are caused by the human T lymphotropic virus (HTLV-I/II). Arguments against this concept included the finding that only a small number of MF patients have antibodies to HTLV-I/II and that attempts to detect proviral sequences by mere Southern hybridization of extracted DNA usually met with failure. However, we have reported repeatedly that HTLV-like particles emerge in blood mononuclear cell (PBMC) cultures of practically all patients with this disease. In several instances, the particles were identified as HTLV by immunoelectron microscopy as well as biomolecular analysis. With the assumptions that the virus in MF patients may have become detection by Southern hybridization alone, the extracts of freshly isolated PBMC of 50 consecutive patients were subjected to combined PCR/Southern analysis. Here we report the presence of HTLV pol and/or tax proviral sequences in 46 out of 50 (92%) of the patients tested. In addition, five of the patients, who lacked antibodies to HTLV-I/II structural proteins, were found to be seropositive for tax. It thus seems reasonable to conclude that MF/Sézary syndrome is an HTLV-associated disease and that lack of an immune response does not preclude infection with this type of virus.