Simian T Lymphotropic Virus 1 Infection of Papio anubis: tax Sequence Heterogeneity and T Cell Recognition.
ABSTRACT: Baboons naturally infected with simian T lymphotropic virus (STLV) are a potentially useful model system for the study of vaccination against human T lymphotropic virus (HTLV). Here we expanded the number of available full-length baboon STLV-1 sequences from one to three and related the T cell responses that recognize the immunodominant Tax protein to the tax sequences present in two individual baboons. Continuously growing T cell lines were established from two baboons, animals 12141 and 12752. Next-generation sequencing (NGS) of complete STLV genome sequences from these T cell lines revealed them to be closely related but distinct from each other and from the baboon STLV-1 sequence in the NCBI sequence database. Overlapping peptides corresponding to each unique Tax sequence and to the reference baboon Tax sequence were used to analyze recognition by T cells from each baboon using intracellular cytokine staining (ICS). Individual baboons expressed more gamma interferon and tumor necrosis factor alpha in response to Tax peptides corresponding to their own STLV-1 sequence than in response to Tax peptides corresponding to the reference baboon STLV-1 sequence. Thus, our analyses revealed distinct but closely related STLV-1 genome sequences in two baboons, extremely low heterogeneity of STLV sequences within each baboon, no evidence for superinfection within each baboon, and a ready ability of T cells in each baboon to recognize circulating Tax sequences. While amino acid substitutions that result in escape from CD8+ T cell recognition were not observed, premature stop codons were observed in 7% and 56% of tax sequences from peripheral blood mononuclear cells from animals 12141 and 12752, respectively.IMPORTANCE It has been estimated that approximately 100,000 people suffer serious morbidity and 10,000 people die each year from the consequences associated with human T lymphotropic virus (HTLV) infection. There are no antiviral drugs and no preventive vaccine. A preventive vaccine would significantly impact the global burden associated with HTLV infections. Here we provide fundamental information on the simian T lymphotropic virus (STLV) naturally transmitted in a colony of captive baboons. The limited viral sequence heterogeneity in individual baboons, the identity of the viral gene product that is the major target of cellular immune responses, the persistence of viral amino acid sequences that are the major targets of cellular immune responses, and the emergence in vivo of truncated variants in the major target of cellular immune responses all parallel what are seen with HTLV infection of humans. These results justify the use of STLV-infected baboons as a model system for vaccine development efforts.
Project description:There are currently 5 million to 10 million human T-lymphotropic virus type 1 (HTLV-1)-infected people, and many of them will develop severe complications resulting from this infection. A vaccine is urgently needed in areas where HTLV-1 is endemic. Many vaccines are best tested in nonhuman primate animal models. As a first step in designing an effective HTLV-1 vaccine, we defined the CD8(+) and CD4(+) T cell response against simian T-lymphotropic virus type 1 (STLV-1), a virus closely related to HTLV-1, in olive baboons (Papio anubis). Consistent with persistent antigenic exposure, we observed that STLV-1-specific CD8(+) T cells displayed an effector memory phenotype and usually expressed CD107a, gamma interferon (IFN-?), and tumor necrosis factor alpha (TNF-?). To assess the viral targets of the cellular immune response in STLV-1-infected animals, we used intracellular cytokine staining to detect responses against overlapping peptides covering the entire STLV-1 proteome. Our results show that, similarly to humans, the baboon CD8(+) T cell response narrowly targeted the Tax protein. Our findings suggest that the STLV-1-infected baboon model may recapitulate some of the important aspects of the human response against HTLV-1 and could be an important tool for the development of immune-based therapy and prophylaxis.HTLV-1 infection can lead to many different and often fatal conditions. A vaccine deployed in areas of high prevalence might reduce the incidence of HTLV-1-induced disease. Unfortunately, there are very few animal models of HTLV-1 infection useful for testing vaccine approaches. Here we describe cellular immune responses in baboons against a closely related virus, STLV-1. We show for the first time that the immune response against STLV-1 in naturally infected baboons is largely directed against the Tax protein. Similar findings in humans and the sequence similarity between the human and baboon viruses suggest that the STLV-1-infected baboon model might be useful for developing a vaccine against HTLV-1.
Project description: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.
Project description:Searching for clues to the evolution of the primate T-lymphotropic viruses (PTLVs), which include the human and the simian T-lymphotropic viruses (HTLV and STLV), we have identified another PTLV, which differs sufficiently from the known PTLV-I and PTLV-II types to be designated here PTLV-L. The virus was isolated from a wild-born baboon (Papio hamadryas) from Eritrea. In a cDNA library a 1802-bp-long fragment was identified that extends from the env region, including the complete transmembrane protein gene, to part of the tax/rex gene. Homologies at the nucleotide sequence level of PTLV-L, prototype simian T-lymphotropic virus-PH969, with HTLV-I and -II, respectively, were 62% and 64% overall, 65% and 70% in the env region, and 80% and 80% in the partial tax/rex sequence. In the 5' part of the pX region a significant homology was seen only with HTLV-II (52%). Phylogenetic analysis based on the gene encoding the transmembrane protein indicates that PTLV-L represents a PTLV type with a long independent evolution, longer than any strain within the PTLV-I or PTLV-II groups. The finding of another PTLV type in African baboons is further evidence of the wide variety of PTLV found on this continent. Whether PTLV-L resembles PTLV-I and PTLV-II in the extension of its host range to other primates, including humans, remains to be seen.
Project description:Two young female baboons naturally infected with simian T-lymphotropic virus type 1 (STLV1) were euthanized due to chronic respiratory disease that was unresponsive to treatment. Massive lymphocytic infiltration of the lung interstitium suggested a diagnosis of STLV-associated lymphoma. In each case, the diagnosis was confirmed through inverse PCR (IPCR) that detected monoclonally integrated STLV1 provirus in cellular DNA extracted from lymphoma tissue and peripheral blood cells (PBC). One dominant STLV1-infected T-cell clone and 3 minor clones were detected in PBC from each baboon. Using archived PBC DNA and primers within the proviral genome and chromosomal DNA flanking the STLV1 integration sites in PCR analyses, we determined that the dominant clone in one baboon had first appeared approximately 8 mo after infection and had circulated for 4 y before clinical disease developed. ELISA testing of archived serum revealed that both baboons seroconverted to the p19 and p24 gag proteins and the envelope gp46 protein but not to the viral tax protein. Titers to p24 and gp46 rose significantly after infection and remained relatively constant until death, whereas titers to p19 increased with time. Although spontaneous STLV1-associated lymphomas have been described in baboons, the STLV1-associated lymphomas described here occurred in 2 relatively young baboons, both of whom had become infected with STLV at 3 to 4 y of age and developed lymphoma within 5 y of infection.
Project description:Among 65 samples obtained from a primate rescue center located in Cameroon, two female adult red-capped mangabeys (Cercocebus torquatus) (CTO-602 and CTO-604), of wild-caught origin, had a peculiar human T-cell lymphotropic virus type 2 (HTLV-2)-like Western blot seroreactivity (p24, RGD21, +/-K55). Analyses of the simian T-cell lymphotropic virus type 3 (STLV-3)/CTO-604 complete proviral sequence (8,919 bp) indicated that this novel strain was highly divergent from HTLV-1 (60% nucleotide similarity), HTLV-2 (62%), or STLV-2 (62%) prototypes. It was, however, related to STLV-3/PH-969 (87%), a divergent STLV strain previously isolated from an Eritrean baboon. The STLV-3/CTO-604 sequence possesses the major open reading frames corresponding to the structural, enzymatic, and regulatory proteins. However, its long terminal repeat is shorter, with only two 21-bp repeats. Furthermore, as demonstrated by reverse transcriptase PCR, this new STLV exhibits significant differences from STLV-3/PH-969 at the mRNA splice junction position level. In all phylogenetic analyses, STLV-3/CTO-604 and STLV-3/PH-969 clustered in a highly supported single clade, indicating an evolutionary lineage independent from primate T-lymphotropic virus type 1 (PTLV-1) and PTLV-2. Nevertheless, the nucleotide divergence between STLV-3/PH-969 and STLV-3/CTO-604 is equivalent to or higher than the divergence observed between the different HTLV-1 or HTLV-2 subtypes. Thus, the STLV-3/CTO-604 strain can be considered the prototype of a second subtype in the PTLV-3 type. The presence of two related viruses in evolutionarily distantly related African monkeys species, living in two opposite ecosystems (rain forest versus desert), reinforces the possible African origin of PTLV and opens new avenues regarding the search for a possible human counterpart of these viruses in individuals exhibiting such HTLV-2-like seroreactivities.
Project description:The recent discovery of human T-lymphotropic virus type 3 (HTLV-3) in Cameroon highlights the importance of expanded surveillance to better understand the prevalence and public health impact of this new retrovirus. HTLV diversity was investigated in 408 persons in rural Cameroon who reported simian exposures. Plasma from 29 persons (7.2%) had reactive serology. HTLV tax sequences were detected in 3 persons. Phylogenetic analysis confirmed HTLV-1 infection in two individuals and HTLV-3 infection in a third person (Cam2013AB). The complete proviral genome from Cam2013AB shared 98% identity and clustered tightly in distinct lineage with simian T-lymphotropic virus type 3 (STLV-3) subtype D recently identified in two guenon monkeys near this person's village. These results document a fourth HTLV-3 infection with a new and highly divergent strain we designate HTLV-3 (Cam2013AB) subtype D demonstrating the existence of a broad HTLV-3 diversity likely originating from multiple zoonotic transmissions of divergent STLV-3.
Project description:We and others have recently uncovered the existence of human T-cell lymphotropic virus type 3 (HTLV-3), the third member of the HTLV family. We have now sequenced the full-length HTLV-3Pyl43 provirus. As expected, HTLV-3Pyl43 contains open reading frames corresponding to the gag, pol, env, tax, and rex genes. Interestingly, its long terminal repeat (LTR) includes only two Tax-responsive elements, as is the case for type 3 simian T-cell lymphotropic viruses (STLV-3). Phylogenetic analyses reveal that HTLV-3Pyl43 is closely related to central African STLV-3. Unexpectedly, the proximal pX region of HTLV-3Pyl43 lacks 366 bp compared to its STLV-3 counterpart. Because of this deletion, the previously described RorfII sequence is lacking. At the amino acid level, Tax3Pyl43 displays strong similarities with HTLV-1 Tax, including the sequence of a PDZ class I binding motif. In transient-transfection assays, Tax3Pyl43 activates the transcriptions from HTLV-3, HTLV-1, and HTLV-2 LTRs. Mutational analysis indicates that two functional domains (M22 and M47) important for transactivation through the CREB/ATF or NF-kappaB pathway are similar but not identical in Tax1 and Tax3Pyl43. We also show that Tax3Pyl43 transactivates the human interleukin-8 and Bcl-XL promoters through the induction of the NF-kappaB pathway. On the other hand, Tax3Pyl43 represses the transcriptional activity of the p53 tumor suppressor protein as well as the c-Myb promoter. Altogether, these results demonstrate that although HTLV-3 and HTLV-1 have only 60% identity, Tax3Pyl43 is functionally closely related to the transforming protein Tax1 and suggest that HTLV-3, like HTLV-1, might be pathogenic in vivo.
Project description:Human T-lymphotropic virus type 3 (HTLV-3) is a new virus recently identified in two primate hunters in Central Africa. Limited sequence analysis shows that HTLV-3 is distinct from HTLV-1 and HTLV-2 but is genetically similar to simian T-lymphotropic virus type 3 (STLV-3). We report here the first complete HTLV-3 sequence obtained by PCR-based genome walking using uncultured peripheral blood lymphocytes from an HTLV-3-infected person. The HTLV-3(2026ND) genome is 8,917 bp long and is genetically equidistant from HTLV-1 and HTLV-2, sharing about 62% identity. Phylogenetic analysis of all gene regions confirms this relationship and shows that HTLV-3 falls within the diversity of STLV-3, suggesting a primate origin. However, HTLV-3(2026ND) is unique, sharing only 87% to 92% sequence identity with STLV-3. SimPlot and phylogenetic analysis did not reveal any evidence of genetic recombination with either HTLV-1, HTLV-2, or STLV-3. Molecular dating estimates that the ancestor of HTLV-3 is as old as HTLV-1 and HTLV-2, with an inferred divergence time of 36,087 to 54,067 years ago. HTLV-3 has a prototypic genomic structure, with all enzymatic, regulatory, and structural proteins preserved. Like STLV-3, HTLV-3 is missing a third 21-bp transcription element found in the long terminal repeats of HTLV-1 and HTLV-2 but instead contains a unique activator protein-1 transcription factor upstream of the 21-bp repeat elements. A PDZ motif, like that in HTLV-1, which is important for cellular signal transduction and transformation, is present in the C terminus of the HTLV-3 Tax protein. A basic leucine zipper region located in the antisense strand of HTLV-1, believed to play a role in viral replication and oncogenesis, was also found in the complementary strand of HTLV-3. The ancient origin of HTLV-3, the broad distribution of STLV-3 in Africa, and the propensity of STLVs to cross species into humans all suggest that HTLV-3 may be prevalent and support the need for expanded surveillance for this virus.
Project description:Four types of human T cell lymphotropic viruses (HTLV) have been described (HTLV-1 to HTLV-4) with three of them having closely related simian virus analogues named STLV-1, -2, and -3. To assess the risk of cross-species transmissions of STLVs from nonhuman primates to humans in the Democratic Republic of Congo, a total of 330 samples, derived from primate bushmeat, were collected at remote forest sites where people rely on bushmeat for subsistence. STLV prevalences and genetic diversity were estimated by PCR and sequence analysis of tax-rex and LTR fragments. Overall, 7.9% of nonhuman primate bushmeat is infected with STLVs. We documented new STLV-1 and STLV-3 variants in six out of the seven species tested and showed for the first time STLV infection in C. mona wolfi, C. ascanius whitesidei, L. aterrimus aterrimus, C. angolensis, and P. tholloni. Our results provide increasing evidence that the diversity and geographic distribution of PTLVs are much greater than previously thought.
Project description: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.