Simian T-Cell Lymphotropic Virus Type 1 from Mandrillus sphinx as a Simian Counterpart of Human T-Cell Lymphotropic Virus Type 1 Subtype D

A recent serological and molecular survey of a semifree-ranging colony of mandrills (Mandrillus sphinx) living in Gabon, central Africa, indicated that 6 of 102 animals, all males, were infected with simian T-cell lymphotropic virus type 1 (STLV-1). These animals naturally live in the same forest area as do human inhabitants (mostly Pygmies) who are infected by the recently described human T-cell lymphotropic virus type 1 (HTLV-1) subtype D. We therefore investigated whether these mandrills were infected with an STLV-1 related to HTLV-1 subtype D. Nucleotide and/or amino acid sequence analyses of complete or partial long terminal repeat (LTR), env, and rex regions showed that HTLV-1 subtype D-specific mutations were found in three of four STLV-1-infected mandrills, while the remaining monkey was infected by a different STLV-1 subtype. Phylogenetic studies conducted on the LTR as well as on the env gp21 region showed that these three new STLV-1 strains from mandrills fall in the same monophyletic clade, supported by high bootstrap values, as do the sequences of HTLV-1 subtype D. These data show, for the first time, the presence of the same subtype of primate T-cell lymphotropic virus type 1 in humans and wild-caught monkeys originating from the same geographical area. This strongly supports the hypothesis that mandrills are the natural reservoir of HTLV-1 subtype D, although the possibility that another monkey species living in the same area could be the original reservoir of both human and mandrill viruses cannot be excluded. Due to the quasi-identity of both human and monkey viruses, interspecies transmission episodes leading to such a clade may have occurred recently.     

Human T-Lymphocyte Transformation with Human T-Cell Lymphotropic Virus Type 2

Human T-cell lymphotrophic virus type 2 (HTLV-2), a common infection of intravenous drug users and subpopulations of Native Americans, is uncommon in the general population. In contrast with the closely related HTLV-1, which is associated with both leukemia and neurologic disorders, HTLV-2 lacks a strong etiologic association with disease. HTLV-2 does shares many properties with HTLV-1, including in vitro lymphocyte transformation capability. To better assess the ability of HTLV-2 to transform lymphocytes, a limiting dilution assay was used to generate clonal, transformed lymphocyte lines. As with HTLV-1, the transformation efficiency of HTLV-2 producer cells was proportionately related to the number of lethally irradiated input cells and was comparable to HTLV-1-mediated transformation efficiency. HTLV-2-infected cells were reproducibly isolated and had markedly increased growth potential compared to uninfected cells; HTLV-2 transformants required the continued presence of exogenous interleukin 2 for growth for several months and were maintained for over 2 years in culture. All HTLV-2-transformed populations were CD2 and/or CD3 positive and B1 negative and were either CD4⁺ or CD8⁺ populations or a mixture of CD4⁺ and CD8⁺ lymphocytes. Clonality of the HTLV-2 transformants was confirmed by Southern blot analysis of T-cell receptor β chain rearrangement. Southern blot analysis revealed a range of integrated full-length genomes from one to multiple. In situ hybridization analysis of HTLV-2 integration revealed no obvious chromosomal integration pattern.     

Barefoot Plantar Pressure Indicates Progressive Neurological Damage in Patients with Human T-Cell Lymphotropic Virus Type 1 Infection

Background

The human T-Cell Lymphotropic Virus Type 1 (HTLV-1) is a retrovirus associated with neurological alterations; individuals with HTLV-1 infection may develop HTLV-1 associated myelopathy / tropical spastic paraparesis (HAM/TSP). Frequent neurological complaints include foot numbness and leg weakness. In this study, we compared the distribution of the body weight on different areas of the foot in HTLV-1 patients with HAM/TSP, asymptomatic HTLV-1 patients, and healthy individuals.

Methodology

We studied 36 HTLV-1 infected patients, who were divided in two groups of 18 patients each based on whether or not they had been diagnosed with HAM/TSP, and 17 control subjects. The evaluation included an interview on the patient’s clinical history and examinations of the patient’s reflexes, foot skin tactile sensitivity, and risk of falling. The pressure distribution on different areas of the foot was measured with baropodometry, using a pressure platform, while the patients had their eyes open or closed.

Main Findings

The prevalence of neurological disturbances—altered reflexes and skin tactile sensitivity and increased risk of falling—was higher in HTLV-1 HAM/TSP patients than in HTLV-1 asymptomatic patients. The medium and maximum pressure values were higher in the forefoot than in the midfoot and hindfoot in both HTLV-1 groups. In addition, the pressure on the hindfoot was lower in HAM/TSP patients compared to control subjects.

Conclusions

The neurological disturbances associated with HTLV-1 infection gradually worsened from HTLV-1 asymptomatic patients to HAM/TSP patients. Baropodometry is a valuable tool to establish the extent of neurological damage in patients suffering from HTLV-1 infection.     

Complete Genome Sequence of Central Africa Human T-Cell Lymphotropic Virus Subtype 1b

Human T-lymphotropic virus type 1 (HTLV-1) has a global spread, and it is estimated that around 20 million persons are infected. Seven major genetic subtypes are recognized. However, there are complete genomes only from the HTLV-1a (cosmopolitan) and HTLV-1c (Melanesian) subtypes. Here, the first full-length genome of an HTLV-1b strain, a subtype so far restricted to Central African countries, is revealed. The genome size of HTLV-1b SF26, a strain isolated in Brazil, was determined to be 8,267 bp. The genomic analysis showed that all characteristic regions and genes of a prototypic HTLV-1 virus are conserved. This genome can provide information for further studies on the evolutionary history and pathogenic potential of this human oncovirus.     

Palmitoylation and p8-Mediated Human T-Cell Leukemia Virus Type 1 Transmission

The orf-I gene of human T-cell leukemia type 1 (HTLV-1) encodes p8 and p12 and has a conserved cysteine at position 39. p8 and p12 form disulfide-linked dimers, and only the monomeric forms of p8 and p12 are palmitoylated. Mutation of cysteine 39 to alanine (C39A) abrogated dimerization and palmitoylation of both proteins. However, the ability of p8 to localize to the cell surface and to increase cell adhesion and viral transmission was not affected by the C39A mutation.     

71-Kilodalton Heat Shock Cognate Protein Acts as a Cellular Receptor for Syncytium Formation Induced by Human T-Cell Lymphotropic Virus Type 1

We previously reported that the region corresponding to amino acids 197 to 216 of the gp46 surface glycoprotein (gp46-197) served as a binding domain for the interaction between gp46 and trypsin-sensitive membrane components of the target cell, leading to syncytium formation induced by human T-cell lymphotropic virus type 1 (HTLV-1)-bearing cells. Our new evidence shows that the 71-kDa heat shock cognate protein (HSC70) acts as a cellular receptor for syncytium formation. Using affinity chromatography with the peptide gp46-197, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, we isolated three components (bands A, B, and C) from MOLT-4 cell lysate which exhibited specific interactions with gp46 and inhibitory activities for syncytium formation induced by HTLV-1-bearing cells. Band A and B components were identified as HSC70 and β-actin, respectively, through amino acid sequencing by tandem mass spectrometry and immunostaining with specific monoclonal antibodies. Band C is likely to be a nonprotein component, because full activity for syncytium formation was seen after extensive trypsin digestion. Anti-HSC70 monoclonal antibody clearly blocked syncytium formation in a coculture of HTLV-1-bearing cells and indicator cells, whereas no inhibition was seen with anti-β-actin monoclonal antibody. Furthermore, flow cytometric analysis indicated that anti-HSC70 antibody reacted with MOLT-4 cells. Thus, we propose that HSC70 expressed on the target cell surface acts as a cellular acceptor to gp46 exposed on the HTLV-1-infected cell for syncytium formation, thereby leading to cell-to-cell transmission of HTLV-1.     

Human Immunodeficiency Virus Type 1 Nucleocapsid p1 Confers ESCRT Pathway Dependence

To facilitate the release of infectious progeny virions, human immunodeficiency virus type 1 (HIV-1) exploits the Endosomal Sorting Complex Required for Transport (ESCRT) pathway by engaging Tsg101 and ALIX through late assembly (L) domains in the C-terminal p6 domain of Gag. However, the L domains in p6 are known to be dispensable for efficient particle production by certain HIV-1 Gag constructs that have the nucleocapsid (NC) domain replaced by a foreign dimerization domain to substitute for the assembly function of NC. We now show that one such L domain-independent HIV-1 Gag construct (termed Z_WT) that has NC-p1-p6 replaced by a leucine zipper domain is resistant to dominant-negative inhibitors of the ESCRT pathway that block HIV-1 particle production. However, Z_WT became dependent on the presence of an L domain when NC-p1-p6 was restored to its C terminus. Furthermore, when the NC domain was replaced by a leucine zipper, the p1-p6 region, but not p6 alone, conferred sensitivity to inhibition of the ESCRT pathway. In an authentic HIV-1 Gag context, the effect of an inhibitor of the ESCRT pathway on particle production could be alleviated by deleting a portion of the NC domain together with p1. Together, these results indicate that the ESCRT pathway dependence of HIV-1 budding is determined, at least in part, by the NC-p1 region of Gag.Human immunodeficiency virus type 1 (HIV-1) and other retroviruses hijack the cellular Endosomal Sorting Complex Required for Transport (ESCRT) pathway to promote the detachment of virions from the cell surface and from each other (3, 21, 42, 44, 47). The ESCRT pathway was initially identified based on its requirement for the sorting of ubiquitinated cargo into multivesicular bodies (MVB) (50, 51). During MVB biogenesis, the ESCRT pathway drives the membrane deformation and fission events required for the inward vesiculation of the limiting membrane of this organelle (26, 29, 50, 51). More recently, it emerged that the ESCRT pathway is also essential for the normal abscission of daughter cells during the final stage of cell division (10, 43). Most of the components of the ESCRT pathway are involved in the formation of four heteromeric protein complexes termed ESCRT-0, ESCRT-I, ESCRT-II, and ESCRT-III. Additional components include ALIX, which interacts both with ESCRT-I and ESCRT-III, and the AAA ATPase Vps4, which mediates the disassembly of ESCRT-III (29, 42).The deformation and scission of endocytic membranes is thought to be mediated by ESCRT-III, which, together with Vps4, constitutes the most conserved element of the pathway (23, 26, 42). Indeed, it was recently shown that purified yeast ESCRT-III induces membrane deformation (52), and in another study three subunits of yeast ESCRT-III were sufficient to promote the formation of intralumenal vesicles in an in vitro assay (61). In mammals, ESCRT-III is formed by the charged MVB proteins (CHMPs), which are structurally related and tightly regulated through autoinhibition (2, 33, 46, 53, 62). The removal of an inhibitory C-terminal domain induces polymerization and association with endosomal membranes and converts CHMPs into potent inhibitors of retroviral budding (34, 46, 53, 60, 62). Alternatively, CHMPs can be converted into strong inhibitors of the ESCRT pathway and of HIV-1 budding through the addition of a bulky tag such as green fluorescent protein (GFP) or red fluorescent protein (RFP) (27, 36, 39, 54). Retroviral budding in general is also strongly inhibited by catalytically inactive Vps4 (22, 41, 55), or upon Vsp4B depletion (31), confirming the crucial role of ESCRT-III.Retroviruses engage the ESCRT pathway through the activity of so-called late assembly (L) domains in Gag. In the case of HIV-1, the primary L domain maps to a conserved PTAP motif in the C-terminal p6 domain of Gag (24, 28) and interacts with the ESCRT-I component Tsg101 (15, 22, 40, 58). HIV-1 p6 also harbors an auxiliary L domain of the LYPx_nL type, which interacts with the V domain of ALIX (20, 35, 39, 54, 59, 63). Interestingly, Tsg101 binding site mutants of HIV-1 can be fully rescued through the overexpression of ALIX, and this rescue depends on the ALIX binding site in p6 (20, 56). In contrast, the overexpression of a specific splice variant of the ubiquitin ligase Nedd4-2 has been shown to rescue the release and infectivity of HIV-1 mutants lacking all known L domains in p6 (12, 57). Nedd4 family ubiquitin ligases had previously been implicated in the function of PPxY-type L domains, which also depend on an intact ESCRT pathway for function (4, 32, 38). However, HIV-1 Gag lacks PPxY motifs, and the WW domains of Nedd4-2, which mediate its interaction with PPxY motifs, are dispensable for the rescue of HIV-1 L domain mutants (57).ALIX also interacts with the nucleocapsid (NC) region of HIV-1 Gag (18, 49), which is located upstream of p6 and the p1 spacer peptide. ALIX binds HIV-1 NC via its Bro1 domain, and the capacity to interact with NC and to stimulate the release of a minimal HIV-1 Gag construct is shared among widely divergent Bro1 domain proteins (48). Based on these findings and the observation that certain mutations in NC cause a phenotype that resembles that of L domain mutants, it has been proposed that NC cooperates with p6 to recruit the machinery required for normal HIV-1 budding (18, 49).NC also plays a role in Gag polyprotein multimerization, and this function of NC depends on its RNA-binding activity (5-8). It has been proposed that the role of the NC-nucleic acid interaction during assembly is to promote the formation of Gag dimers (37), and HIV-1 assembly in the absence of NC can indeed be efficiently rescued by leucine zipper dimerization domains (65). Surprisingly, in this setting the L domains in p6 also became dispensable, since particle production remained efficient even when the entire NC-p1-p6 region of HIV-1 Gag was replaced by a leucine zipper (1, 65). These findings raised the possibility that the reliance of wild-type (WT) HIV-1 Gag on a functional ESCRT pathway is, at least in part, specified by NC-p1-p6. However, it also remained possible that the chimeric Gag constructs engaged the ESCRT pathway in an alternative manner.In the present report, we provide evidence supporting the first of those two possibilities. Particle production became independent of ESCRT when the entire NC-p1-p6 region was replaced by a leucine zipper, and reversion to ESCRT dependence was shown to occur as a result of restoration of p1-p6 but not of p6 alone. Furthermore, although the deletion of p1 alone had little effect in an authentic HIV-1 Gag context, the additional removal of a portion of NC improved particle production in the presence of an inhibitor of the ESCRT pathway. Together, these data imply that the NC-p1 region plays an important role in the ESCRT-dependence of HIV-1 particle production.     

A Lysine-to-Arginine Change Found in Natural Alleles of the Human T-Cell Lymphotropic/Leukemia Virus Type 1 p12I Protein Greatly Influences Its Stability

The HTLV-1 singly spliced open reading frame I protein, p12(I), is highly unstable and appears to be necessary for persistent infection in rabbits. Here we demonstrate that p12(I) forms dimers through two putative leucine zipper domains and that its stability is augmented by specific proteasome inhibitors. p12(I) is ubiquitylated, and mutations of its unique carboxy-terminus lysine residue to an arginine greatly enhance its stability. Interestingly, analysis of 53 independent HTLV-1 strains revealed that the natural p12(I) alleles found in ex vivo samples of tropical spastic paraparesis-HTLV-1-associated myelopathy patients contain a Lys at position 88 in some cases, whereas arginine is consistently found at position 88 in HTLV-1 strains from all adult T-cell leukemia-lymphoma (ATLL) cases and healthy carriers studied. This apparent segregation of different alleles in tropical spastic paraparesis-HTLV-associated myelopathy and ATLL or healthy carriers may be relevant in vivo, since p12(I) binds the interleukin-2 receptor beta and gammac chains, raising the possibility that the two natural alleles might affect differently the regulation of these molecules.     

Human T Lymphotropic Virus Type 1 protein Tax reduces histone levels

Background

Human immunodeficiency virus type 1 (HIV-1) infects cells by means of ligand-receptor interactions. This lentivirus uses the CD4 receptor in conjunction with a chemokine coreceptor, either CXCR4 or CCR5, to enter a target cell. HIV-1 is characterized by high sequence variability. Nonetheless, within this extensive variability, certain features must be conserved to define functions and phenotypes. The determination of coreceptor usage of HIV-1, from its protein envelope sequence, falls into a well-studied machine learning problem known as classification. The support vector machine (SVM), with string kernels, has proven to be very efficient for dealing with a wide class of classification problems ranging from text categorization to protein homology detection. In this paper, we investigate how the SVM can predict HIV-1 coreceptor usage when it is equipped with an appropriate string kernel.

Results

Three string kernels were compared. Accuracies of 96.35% (CCR5) 94.80% (CXCR4) and 95.15% (CCR5 and CXCR4) were achieved with the SVM equipped with the distant segments kernel on a test set of 1425 examples with a classifier built on a training set of 1425 examples. Our datasets are built with Los Alamos National Laboratory HIV Databases sequences. A web server is available at http://genome.ulaval.ca/hiv-dskernel.

Conclusion

We examined string kernels that have been used successfully for protein homology detection and propose a new one that we call the distant segments kernel. We also show how to extract the most relevant features for HIV-1 coreceptor usage. The SVM with the distant segments kernel is currently the best method described.     

Syncytium-Inhibiting Monoclonal Antibodies Produced against Human T-Cell Lymphotropic Virus Type 1-Infected Cells Recognize Class II Major Histocompatibility Complex Molecules and Block by Protein Crowding

Four new monoclonal antibodies (MAbs) that inhibit human T-cell lymphotropic virus type 1 (HTLV-1)-induced syncytium formation were produced by immunizing BALB/c mice with HTLV-1-infected MT2 cells. Immunoprecipitation studies and binding assays of transfected mouse cells showed that these MAbs recognize class II major histocompatibility complex (MHC) molecules. Previously produced anti-class II MHC antibodies also blocked HTLV-1-induced cell fusion. Coimmunoprecipitation and competitive MAb binding studies indicated that class II MHC molecules and HTLV-1 envelope glycoproteins are not associated in infected cells. Anti-MHC antibodies had no effect on human immunodeficiency virus type 1 (HIV-1) syncytium formation by cells coinfected with HIV-1 and HTLV-1, ruling out a generalized disruption of cell membrane function by the antibodies. High expression of MHC molecules suggested that steric effects of bound anti-MHC antibodies might explain their inhibition of HTLV-1 fusion. An anti-class I MHC antibody and a polyclonal antibody consisting of several nonblocking MAbs against other molecules bound to MT2 cells at levels similar to those of class II MHC antibodies, and they also blocked HTLV-1 syncytium formation. Dose-response experiments showed that inhibition of HTLV-1 syncytium formation correlated with levels of antibody bound to the surface of infected cells. The results show that HTLV-1 syncytium formation can be blocked by protein crowding or steric effects caused by large numbers of immunoglobulin molecules bound to the surface of infected cells and have implications for the structure of the cellular HTLV-1 receptor(s).Human T-cell lymphotropic virus type 1 (HTLV-1) is a type C retrovirus and the etiologic agent of adult T-cell leukemia (43, 56, 59) and HTLV-1-associated myelopathy or tropical spastic paraparesis (15, 17, 49, 61). Although HTLV-1 shows tropism primarily for T cells, it can infect a variety of cell types including cells from some nonhuman species (6, 9, 27, 46, 48, 60, 62). Infection by free HTLV-1 tends to be highly inefficient, and the virus appears to be transmitted primarily by the cell-to-cell route (37). The HTLV-1 envelope glycoprotein is synthesized as a 61-kDa precursor which is cleaved into surface (gp46) and transmembrane (gp21) proteins (40, 57). gp46 is thought to serve as the virus attachment protein, as does gp120 for human immunodeficiency virus (HIV) (40, 57). Although previous reports have identified host cell molecules which might potentially mediate virus binding (9, 14), the cellular receptor for HTLV-1 has not been definitively identified. A recent study in which affinity chromatography was carried out with a gp46 peptide has provided evidence that the heat shock protein HSC70 binds directly to gp46 and may serve as a virus receptor (47).gp21 contains an N-terminal hydrophobic fusion domain and likely serves as a fusion protein similar to HIV gp41 (12, 61). Like many other retroviruses, HTLV-1 can induce syncytium formation between infected cells and certain uninfected cell types (28, 39). However, there are no data to indicate that virus transmission or virus persistence in vivo depends on syncytium formation. It is thought that cell-cell fusion involves the same receptors and occurs in a manner similar to virus-cell fusion. For this reason, HTLV-1 syncytium assays have been used to screen for cell surface molecules that may serve as virus receptors (13, 14, 25, 29). Monoclonal antibodies (MAbs) against a number of membrane proteins including members of the tetraspanner family (30, 31) have been found to block syncytium formation. My colleagues and I recently reported that expression of the cell adhesion molecule vascular cell adhesion molecule 1 (VCAM-1) on uninfected cells can confer sensitivity to HTLV-1-mediated syncytium formation (25). In this previous study, we were not able to block HTLV-1 cell fusion with MAbs against the major VCAM-1 counterreceptor VLA-4 (25). Others have reported that MAbs to other adhesion molecules including intercellular adhesion molecule 3 (ICAM-3) also block HTLV-1 syncytium formation (29). We have demonstrated that adhesion molecules also facilitate HIV type 1 (HIV-1) infection and syncytium formation (16, 24). Thus, adhesion molecules may be important accessory molecules for retroviruses generally.Earlier studies on accessory molecules involved in HTLV-1 biology have been extended by immunizing mice with HTLV-1-infected cells and screening for MAbs that block VCAM-1-supported HTLV-1 syncytium formation. Four new MAbs that completely block HTLV-1-mediated cell fusion have been generated. The MAbs were all determined to be specific for class II major histocompatibility complex (MHC) molecules. These MAbs had no effect on syncytium formation induced by HIV-1. Studies on the mechanism by which the MAbs mediate this effect have revealed a novel mode of antibody blockade of virus-induced cell fusion: protein crowding at the infected cell surface resulting in steric blockade of critical receptor-ligand interactions.     

Human T-Cell Lymphotropic Virus Type 1 Subtype C Molecular Variants among Indigenous Australians: New Insights into the Molecular Epidemiology of HTLV-1 in Australo-Melanesia

Background

HTLV-1 infection is endemic among people of Melanesian descent in Papua New Guinea, the Solomon Islands and Vanuatu. Molecular studies reveal that these Melanesian strains belong to the highly divergent HTLV-1c subtype. In Australia, HTLV-1 is also endemic among the Indigenous people of central Australia; however, the molecular epidemiology of HTLV-1 infection in this population remains poorly documented.

Findings

Studying a series of 23 HTLV-1 strains from Indigenous residents of central Australia, we analyzed coding (gag, pol, env, tax) and non-coding (LTR) genomic proviral regions. Four complete HTLV-1 proviral sequences were also characterized. Phylogenetic analyses implemented with both Neighbor-Joining and Maximum Likelihood methods revealed that all proviral strains belong to the HTLV-1c subtype with a high genetic diversity, which varied with the geographic origin of the infected individuals. Two distinct Australians clades were found, the first including strains derived from most patients whose origins are in the North, and the second comprising a majority of those from the South of central Australia. Time divergence estimation suggests that the speciation of these two Australian clades probably occurred 9,120 years ago (38,000–4,500).

Conclusions

The HTLV-1c subtype is endemic to central Australia where the Indigenous population is infected with diverse subtype c variants. At least two Australian clades exist, which cluster according to the geographic origin of the human hosts. These molecular variants are probably of very ancient origin. Further studies could provide new insights into the evolution and modes of dissemination of these retrovirus variants and the associated ancient migration events through which early human settlement of Australia and Melanesia was achieved.