Simian T-cell leukemia virus (STLV) infection in wild primate populations in Cameroon: evidence for dual STLV type 1 and type 3 infection in agile mangabeys (Cercocebus agilis)

Three types of human T-cell leukemia virus (HTLV)-simian T-cell leukemia virus (STLV) (collectively called primate T-cell leukemia viruses [PTLVs]) have been characterized, with evidence for zoonotic origin from primates for HTLV type 1 (HTLV-1) and HTLV-2 in Africa. To assess human exposure to STLVs in western Central Africa, we screened for STLV infection in primates hunted in the rain forests of Cameroon. Blood was obtained from 524 animals representing 18 different species. All the animals were wild caught between 1999 and 2002; 328 animals were sampled as bush meat and 196 were pets. Overall, 59 (11.2%) of the primates had antibodies cross-reacting with HTLV-1 and/or HTLV-2 antigens; HTLV-1 infection was confirmed in 37 animals, HTLV-2 infection was confirmed in 9, dual HTLV-1 and HTLV-2 infection was confirmed in 10, and results for 3 animals were indeterminate. Prevalences of infection were significantly lower in pets than in bush meat, 1.5 versus 17.0%, respectively. Discriminatory PCRs identified STLV-1, STLV-3, and STLV-1 and STLV-3 in HTLV-1-, HTLV-2-, and HTLV-1- and HTLV-2-cross-reactive samples, respectively. We identified for the first time STLV-1 sequences in mustached monkeys (Cercopithecus cephus), talapoins (Miopithecus ogouensis), and gorillas (Gorilla gorilla) and confirmed STLV-1 infection in mandrills, African green monkeys, agile mangabeys, and crested mona and greater spot-nosed monkeys. STLV-1 long terminal repeat (LTR) and env sequences revealed that the strains belonged to different PTLV-1 subtypes. A high prevalence of PTLV infection was observed among agile mangabeys (Cercocebus agilis); 89% of bush meat was infected with STLV. Cocirculation of STLV-1 and STLV-3 and STLV-1-STLV-3 coinfections were identified among the agile mangabeys. Phylogenetic analyses of partial LTR sequences indicated that the agile mangabey STLV-3 strains were more related to the STLV-3 CTO604 strain isolated from a red-capped mangabey (Cercocebus torquatus) from Cameroon than to the STLV-3 PH969 strain from an Eritrean baboon or the PPA-F3 strain from a baboon in Senegal. Our study documents for the first time that (i) a substantial proportion of wild-living monkeys in Cameroon is STLV infected, (ii) STLV-1 and STLV-3 cocirculate in the same primate species, (iii) coinfection with STLV-1 and STLV-3 occurs in agile mangabeys, and (iv) humans are exposed to different STLV-1 and STLV-3 subtypes through handling primates as bush meat.     

Divergent simian T-cell lymphotropic virus type 3 (STLV-3) in wild-caught Papio hamadryas papio from Senegal: widespread distribution of STLV-3 in Africa

Among eight samples obtained from a French primatology research center, six adult guinea baboons (Papio hamadryas papio), caught in the wild in Senegal, had a peculiar human T-cell leukemia virus type 2 (HTLV-2)-like Western blot seroreactivity (p24(+), GD21(+), K55(+/-)). Partial sequence analyses of the tax genes (433 bp) indicated that these baboons were infected by a novel divergent simian T-cell lymphotropic virus (STLV). Analyses of the complete proviral sequence (8,892 bp) for one of these strains (STLV-3/PPA-F3) indicate that this STLV was highly divergent from the HTLV-1 (61.6% of nucleotide similarity), HTLV-2 (61.2%), or STLV-2 (60.6%) prototype. It was, however, much more closely related to the few other known STLV-3 strains, exhibiting 87 and 89% of nucleotide similarity with STLV-3/PHA-PH969 (formerly PTLV-L/PH969) and STLV-3/CTO-604, respectively. The STLV-3/PPA-F3 sequence possesses the major HTLV or STLV open reading frames corresponding to the structural, enzymatic, and regulatory proteins. However, its long terminal repeat comprises only two 21-bp repeats. In all phylogenetic analyses, STLV-3/PPA-F3 clustered together in a highly supported single clade with the other known strains of STLV-3, indicating an independent evolution from primate T-cell lymphotropic virus type 1 (PTLV-1) and PTLV-2. The finding of a new strain of STLV-3 in a West African monkey (Guinea baboon) greatly enlarges the geographical distribution and the host range of species infected by this PTLV type in the African continent. The recent discovery of several different STLV-3 strains in many different African monkey species, often in contact with humans, strongly suggests potential interspecies transmission events, as it was described for STLV-1, between nonhuman primates but also to humans.     

Subsets of T cells in healthy rhesus macaques (Macaca mulatta) infected with simian T-lymphotropic virus type 1

Simian T-lymphotropic virus type 1 (STLV-1) is a C-type retrovirus of nonhuman primates that is genetically and antigenically related to human T-lymphotropic virus type 1 (HTLV-1). Infection with STLV-1 has been reported in many species of Old World monkeys and apes, including rhesus macaques (Macaca mulatta). Similar to HTLV infection in humans, STLV infection has been associated with T-cell lymphoproliferative disease or lymphoma in a small proportion of infected animals, predominantly African species. There are conflicting reports of T-cell subset alterations in healthy HTLV-1 carriers. To the authors' knowledge, analysis of T-cell subsets in healthy STLV-1 carrier rhesus macaques has not been reported. Subsets of T cells in peripheral blood from healthy, STLV-1-seropositive rhesus macaques (n = 17) and seronegative controls matched for age and sex (n = 17) were determined by use of fluorescence-activated cell sorter analysis. Parameters measured included CD3, CD4, CD8, CD25, CD28, CD38, and HLA-DR cell sets. Significant differences in T-cell subsets or hematologic parameters were not observed between healthy STLV-seropositive and seronegative groups.     

Complete nucleotide sequence of the new simian T-lymphotropic virus, STLV-PH969 from a Hamadryas baboon, and unusual features of its long terminal repeat.

A third type of primate T-lymphotropic virus, PTLV-L, with STLV-PH969 as a prototype, has recently been isolated from an African baboon (Papio hamadryas). Classification of this virus has been based on partial sequence analysis of cDNA from a virus-producing cell line, PH969. We obtained the complete nucleotide sequence of this virus with a proviral genome of 8,916 bp. All major genes, homologous in all human T-cell lymphotropic virus (HTLV)-related viruses, and their corresponding mRNAs, including appropriate splicing, were identified. One additional nonhomologous open reading frame in the proximal pX region is accessible for translation through alternative splicing. Sequence comparison shows that STLV-PH969 is equidistantly related to HTLV type 1 (HTLV-1) and HTLV-2. In all coding regions, the similarity tends to be the lowest between STLV-PH969 and HTLV-1. However, in the long terminal repeat (LTR) region, the lowest similarity was found between STLV-PH969 and HTLV-2. The U3-R and R-U5 boundaries of the STLV-PH969 LTR were experimentally determined at nucleotides 268 and 524, respectively. This 695-bp LTR is 60 and 73 bp shorter than the LTRs of HTLV-1 and HTLV-2, respectively, but its general organization is similar to the one found in the HTLV-bovine leukemia virus genus. In the long region between the polyadenylation signal and the poly(A) site, sequence similarity with the HTLV-1 Rex-responsive element (RexRE) core and secondary structure prediction suggest the presence of a RexRE. The presence of three 21-bp repeats is conserved within the U3 region of HTLV-1, HTLV-2, and BLV. Only two direct repeats with similarity to these Tax-responsive elements were found in the STLV-PH969 LTR, which might suggest differences in the Tax-mediated transactivation of this virus. We conclude that STLV-PH969 has all the genes and genomic regions to suggest a replication cycle comparable to that of HTLV-1 and HTLV-2.     

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.     

The genome organization of STLV-3 is similar to that of the AIDS virus except for a truncated transmembrane protein

Nucleotide sequence analysis of the 3' portion of the genome of simian T-lymphotropic virus type 3 from African green monkeys (STLV-3agm) reveals that it has the same general genome structure as the human immunodeficiency virus (HIV-1), the etiologic agent of AIDS. Short segments of strong amino acid homology and similar predicted protein structure characterize the tat and art/trs open reading frames (orf) of both viruses. Strong conservation of 3' orf and of another, cs-orf, for which no protein product has been identified suggests that they both encode proteins important to the life cycle of these viruses. The extracellular glycoproteins of STLV-3 and HIV-1 share a similar backbone structure and 50%-55% amino acid homology in constant domains of the HIV-1 protein. The most evident departure in structural organization is truncation of the transmembrane glycoproteins in two STLV-3agm clones and a biologically active, noncytopathic clone of HTLV-4.     

HIV-1 Tat interaction with Dicer: requirement for RNA

Four primate (PTLV), human (HTLV) and simian (STLV) T-cell leukemia virus types, have been characterized thus far, with evidence of a simian zoonotic origin for HTLV-1, HTLV-2 and HTLV-3 in Africa. The PTLV envelope glycoprotein surface component (SUgp46) comprises a receptor-binding domain (RBD) that alternates hypervariable and highly conserved sequences. To further delineate highly conserved motifs in PTLV RBDs, we investigated the intrahost variability of HTLV-1 and STLV-1 by generating and sequencing libraries of DNA fragments amplified within the RBD of the SUgp46 env gene. Using new and highly cross-reactive env primer pairs, we observed the presence of Env quasispecies in HTLV-1 infected individuals and STLV-1 naturally infected macaques, irrespective of the clinical status. These intrahost variants helped us to define highly conserved residues and motifs in the RBD. The new highly sensitive env PCR described here appears suitable for the screening of all known variants of the different PTLV types and should, therefore, be useful for the analysis of seroindeterminate samples.     

The simian T-lymphotropic/leukemia virus from Pan paniscus belongs to the type 2 family and infects Asian macaques.

The proviral DNA of the simian T-leukemia/lymphotropic virus (STLV) isolate, originally obtained from a captive colony of pygmy chimpanzees (Pan paniscus) (STLV(pan-p)), was cloned from the DNA of the chronically infected human T-cell line L93-79B. The entire proviral DNA sequence was obtained and compared with sequences of the known genotypes of STLV and human T-leukemia/lymphotropic virus types 1 and 2 (HTLV-1 and -2). Phylogenetic analysis indicates that STLV-2(pan-p) is an early divergence within the type 2 lineage and should be referred to as STLV-2(pan-p). Since STLV-2(pan-p) has been found in African nonhuman primates, we investigated its infectiousness and pathogenicity in Asian monkeys. Pigtailed macaques were inoculated with human cells harboring STLV(pan-p), and infection was assessed by virus isolation, PCR analysis of peripheral blood mononuclear cells, and seroconversion against viral antigens in HTLV-1/HTLV-2 and Western blot assay. Pigtailed macaques became persistently infected by STLV-2(pan-p), and the virus could be transferred by blood transfusion from an infected pigtailed macaque to a rhesus macaque. In addition, like HTLV-1 and HTLV-2, STLV-2(pan-p) was infectious in rabbits. In summary, STLV-2(pan-p) is a novel retrovirus distantly related to HTLV-2 and displays a host range similar to that demonstrated for other HTLV and STLV strains.     

Molecular epidemiology of 58 new African human T-cell leukemia virus type 1 (HTLV-1) strains: identification of a new and distinct HTLV-1 molecular subtype in Central Africa and in Pygmies.

To gain new insights on the origin, evolution, and modes of dissemination of human T-cell leukemia virus type I (HTLV-1), we performed a molecular analysis of 58 new African HTLV-1 strains (18 from West Africa, 36 from Central Africa, and 4 from South Africa) originating from 13 countries. Of particular interest were eight strains from Pygmies of remote areas of Cameroon and the Central African Republic (CAR), considered to be the oldest inhabitants of these regions. Eight long-term activated T-cell lines producing HTLV-1 gag and env antigens were established from peripheral blood mononuclear cell cultures of HTLV-1 seropositive individuals, including three from Pygmies. A fragment of the env gene encompassing most of the gp21 transmembrane region was sequenced for the 58 new strains, while the complete long terminal repeat (LTR) region was sequenced for 9 strains, including 4 from Pygmies. Comparative sequence analyses and phylogenetic studies performed on both the env and LTR regions by the neighbor-joining and DNA parsimony methods demonstrated that all 22 strains from West and South Africa belong to the widespread cosmopolitan subtype (also called HTLV-1 subtype A). Within or alongside the previously described Zairian cluster (HTLV-1 subtype B), we discovered a number of new HTLV-1 variants forming different subgroups corresponding mainly to the geographical origins of the infected persons, Cameroon, Gabon, and Zaire. Six of the eight Pygmy strains clustered together within this Central African subtype, suggesting a common origin. Furthermore, three new strains (two originating from Pygmies from Cameroon and the CAR, respectively, and one from a Gabonese individual) were particularly divergent and formed a distinct new phylogenetic cluster, characterized by specific mutations and occupying in most analyses a unique phylogenetic position between the large Central African genotype (HTLV-1 subtype B) and the Melanesian subtype (HTLV-1 subtype C). We have tentatively named this new HTLV-1 genotype HTLV-1 subtype D. While the HTLV-1 subtype D strains were not closely related to any known African strain of simian T-cell leukemia virus type 1 (STLV-1), other Pygmy strains and some of the new Cameroonian and Gabonese HTLV-1 strains were very similar (>98% nucleotide identity) to chimpanzee STLV-1 strains, reinforcing the hypothesis of interspecies transmission between humans and monkeys in Central Africa.     

Isolation and characterization of a new simian T-cell leukemia virus type 1 from naturally infected celebes macaques (Macaca tonkeana): complete nucleotide sequence and phylogenetic relationship with the Australo-Melanesian human T-cell leukemia virus type 1.

A study of simian T-cell leukemia virus type 1 (STLV-1) infection in a captive colony of 23 Macaca tonkeana macaques indicated that 17 animals had high human T-cell leukemia virus type 1 (HTLV-1) antibody titers. Genealogical analysis suggested mainly a mother-to-offspring transmission of this STLV-1. Three long-term T-cell lines, established from peripheral blood mononuclear cell cultures from three STLV-1-seropositive monkeys, produced HTLV-1 Gag and Env antigens and retroviral particles. The first complete nucleotide sequence of an STLV-1 (9,025 bp), obtained for one of these isolates, indicated an overall genetic organization similar to that of HTLV-1 but with a nucleotide variability for the structural genes ranging from 7.8 to 13.1% compared with the HTLV-1 ATK and STLV-1 PTM3 Asian prototypes. The Tax and Rex regulatory proteins were well conserved, while the pX region, known to encode new proteins in HTLV-1 (open reading frames I and II), was more divergent than that in the ATK strain. Furthermore, a fragment of 522 bp of the gp21 env gene from uncultured peripheral blood mononuclear cell DNAs from five of the STLV-1-infected monkeys was sequenced. Phylogenetic trees constructed with the long terminal repeat and env (gp46 and gp21) regions demonstrated that this new STLV-1 occupies a unique position within the Asian STLV-1 and HTLV-1 isolates, being, by most analyses, related more to the Australo-Melanesian HTLV-1 topotype than to any other Asian STLV-1. These data raise new hypotheses on the possible interspecies viral transmission between monkeys carrying STLV-1 and early Australoid settlers, ancestors of the present day Australo-Melanesian inhabitants, during their migrations from the Southeast Asian land mass to the greater Australian continent.     

Human and Simian T-Cell Leukemia Viruses Type 2 (HTLV-2 and STLV-2pan-p) Transform T Cells Independently of Jak/STAT Activation

Human T-lymphotropic virus type 1 (HTLV-1) and HTLV-2 differ in pathogenicity in vivo. HTLV-1 causes leukemia and neurologic and inflammatory diseases, whereas HTLV-2 is less clearly associated with human disease. Both retroviruses transform human T cells in vitro, and transformation by HTLV-1 was found to be associated with the constitutive activation of the Jak/STAT pathway. To assess whether HTLV-2 transformation may also result in constitutive activation of the Jak/STAT pathway, six interleukin-2-independent, HTLV-2-transformed T-cell lines were analyzed for the presence of activated Jak and STAT proteins by electrophoretic mobility shift assay. In addition, the phosphorylation status of Jak and STAT proteins was assessed directly by immunoprecipitation and immunoblotting with an antiphosphotyrosine antibody. Jak/STAT proteins were not found to be constitutively activated in any of the T-cell lines infected by the type 2 human and nonhuman primate viruses, suggesting that HTLV-2 and the cognate virus simian T-lymphotropic virus type 2 from Pan paniscus transform T cells in vitro by mechanisms at least partially different from those used by HTLV-1.     

猴T淋巴细胞趋向性病毒1型双抗原夹心ELISA检测试剂盒的研制

目的研制灵敏度和特异性高的检测实验猴血清中T淋巴细胞趋向性病毒-1型（STLV-1型）／E体的双抗原夹心ELISA（dsELISA）检测试剂盒。方法采用经原核表达系统表达并纯化的人T淋巴细胞白血病病毒-1型（HTLV-1型）的Env蛋白作为包被用抗原,建立了检测STLV-1的dsELISA诊断方法。通过优化反应条件和筛选试剂,确定了dsELISA诊断试剂盒的相关条件,并经敏感性、特异性和重复性试验考查该试剂盒质量。结果试剂盒特异性好,批内重复试验变异系数（CV）〈7％,批间重复试验CV〈10％。对200份猴血清进行随机检测,与国际公认的诊断试剂盒（美国BioReliance公司）的符合率为97％。结论本试剂盒可初步应用于临床上实验猴STLV-1型抗体的检测。     

Characterization and sequencing of prototypic human T-lymphotropic virus type 1 (HTLV-1) from an HTLV-1/2 seroindeterminate patient

Serological screening for human T-lymphotropic virus type 1 (HTLV-1) parallels the standard screening process for human immunodeficiency virus (HIV), in which samples found positive by enzyme-linked immunosorbent assay (ELISA) are confirmed with a modified Western blot procedure. There are a significant number of cases in which HTLV-1/2 ELISA-positive specimens demonstrate an incomplete banding pattern on this Western blot. Individuals providing these atypical antibody responses are categorized as seroindeterminate for HTLV-1/2. Although HTLV-1 genomic sequences are readily detectable in the peripheral blood lymphocytes (PBL) of seropositive individuals, previous studies have repeatedly demonstrated that PBL from the vast majority of HTLV-1/2 seroindeterminate individuals are PCR negative for HTLV-1. As a result, identification of the agent responsible for this indeterminate reactivity has been of interest. We have generated an HTLV-1-positive B-cell line (SI-1 B) from one of these seroindeterminate individuals. Previous screening for HTLV-1 in PBL from this patient had been routinely negative by primary PCR; however, HTLV-1 tax had been periodically detected by nested PCR. DNA sequence data generated with genomic DNA from the SI-1 B cell line and HTLV-1-specific primers demonstrated the presence of a full-length viral genome with >97% homology to the Cosmopolitan form of HTLV-1. A 12-bp deletion was identified in the 3'-gag/5'-prot region, which would predict translation of altered or nonfunctional proteins from these genes. We propose that this HTLV-1/2-seroindeterminate patient is infected with a prototypic form of HTLV-1 at an extremely low viral load and that this finding may explain HTLV-1/2 seroindeterminate reactivity in at least a subset of these individuals.     

Molecular characterization and phylogenetic analyses of a new, highly divergent simian T-cell lymphotropic virus type 1 (STLV-1marc1) in Macaca arctoides.

A serological survey of a captive colony of Asian monkeys indicated that six Macaca arctoides had antibodies to human T-cell leukemia/lymphotropic viruses (HTLV). Over a 4-year interval, sera from these animals continued to exhibit a peculiar Western blot (WB) pattern resembling an HTLV-2 pattern (p24gag reactivity of equal or greater intensity than that of p19gag and a strong reactivity to recombinant gp21) but also exhibiting, in five of six cases, a reactivity against MTA-1, an HTLV-1 gp46 peptide. PCR experiments on DNA extracted from peripheral blood mononuclear cells using HTLV-1- or HTLV-2-specific long terminal repeat, gag, pol, env, and tax primers yielded negative results. However, highly conserved primers successfully amplified three different gene segments of env, tax, and env-tax. The results of comparative sequence analysis demonstrated that STLV-1marc1 was not closely related to any known STLV-1 strain, was the most divergent strain of the HTLV-1-STLV-1 group, and lacked the ATG initiation codons corresponding to the p12 and p13 proteins of HTLV-1. Phylogenetic analyses incorporating representative strains of all known HTLV-STLV clades consistently depicted STLV-1marc1 within the HTLV-1-STLV-1 type 1 lineage, but it probably diverged early, since its position is clearly different from all known viral strains of this group and it had a bootstrap resampling value of 100%. Genetic distance estimates between STLV-1marc1 and all other type 1 viruses were of the same order of magnitude as those between STLV-2PanP and all other type 2 viruses. In light of the recent demonstration of interspecies transmission of some STLV-1 strains, our results suggest the existence in Asia of HTLV-1 strains related to this new divergent STLV-1marc1 strain, which may be derived from a common ancestor early in the evolution of the type 1 viruses and could be therefore considered a prototype of a new HTLV-STLV clade.     

Interspecies transmission of macaque simian T-cell leukemia/lymphoma virus type 1 in baboons resulted in an outbreak of malignant lymphoma.

An outbreak of malignant lymphoma has been observed in one of the baboon (Papio hamadryas) stocks of Sukhumi Primate Center. More than 300 cases in this "high-lymphoma stock" have been registered since 1967. Human T-cell lymphotropic virus type 1 (HTLV-1)-related virus was implicated as the etiologic agent of Sukhumi baboon lymphoma. The origin of this virus remained unclear. Two possibilities were originally considered: the origin could be baboon simian T-cell leukemia/lymphoma virus type 1 (STLV-1) or HTLV-1 (before the outbreak started, some Sukhumi baboons were inoculated with human leukemic material). The third possibility entered recently: interspecies transmission of rhesus macaque STLV-1 to baboons. It was prompted by the finding of very close similarity between STLV-1 991-1cc (the strain isolated from a non-Sukhumi baboon inoculated with material from a Sukhumi lymphomatous baboon) and rhesus STLV-1. To test this hypothesis, we investigated 37 Sukhumi STLV-1 isolates from baboons of high-lymphoma stock by PCR discriminating rhesus type and baboon type STLV-1 isolates. All of them were proved to be rhesus type STLV-1. In contrast, all six STLV-1 isolates from baboons belonging to other stocks or populations were of baboon type. The PCR results were fully confirmed by DNA sequence data. The partial env gene gene sequences of all four STLV-1 isolates from Sukhumi lymphomatous baboons were 97 to 100% similar to the sequence of known rhesus STLV-1 and only 85% homologous with the sequence of conventional baboon STLV-1. Thus, interspecies transmission of STLV-1 from rhesus macaques (or closely related species) to baboons occurred at Sukhumi Primate Center. Most probably this event initiated the outbreak of lymphoma in Sukhumi baboons.     

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.     

Functional identification of nucleotides conferring substrate specificity to retroviral integrase reactions.

The long terminal repeats (LTRs) that flank the retroviral DNA genome play a distinct role in the integration process by acting as specific substrates for the integrase (IN). The role of LTR sequences in providing substrate recognition and specificity to integration reactions was investigated for INs from human immunodeficiency virus type 1 (HIV-1), Moloney murine leukemia virus (M-MuLV), human T-cell leukemia virus type 1 (HTLV-1), and human T-cell leukemia virus type 2 (HTLV-2). Overall, these INs required specific LTR sequences for optimal catalysis of 3'-processing reactions, as opposed to strand transfer and disintegration reactions. It is of particular note that in strand transfer reactions the sites of integration were similar among the four INs. In the 3'-processing reaction, sequence specificity for each IN was traced to the three nucleotides proximal to the conserved CA. Reactions catalyzed by M-MuLV IN were additionally influenced by upstream regions. The nucleotide requirements for optimal catalysis differed for each IN. HIV-1 IN showed a broad range of substrate specificities, while HTLV-1 IN and HTLV-2 IN had more defined sequence requirements. M-MuLV IN exhibited greater activity with the heterologous LTR substrates than with its own wild-type substrate. This finding was further substantiated by the high levels of activity catalyzed by the IN on modified M-MuLV LTRs. This work suggests that unlike the other INs examined, M-MuLV IN has evolved with an IN-LTR interaction that is suboptimal.