Cytolytic Recombinant Vesicular Stomatitis Viruses Expressing STLV-1 Receptor Specifically Eliminate STLV-1 Env-Expressing Cells in an HTLV-1 Surrogate Model In Vitro.

Human T-cell leukemia virus type 1 (HTLV-1) causes serious and intractable diseases in some carriers after infection. The elimination of infected cells is considered important to prevent this onset, but there are currently no means by which to accomplish this. We previously developed "virotherapy", a therapeutic method that targets and kills HTLV-1-infected cells using a cytolytic recombinant vesicular stomatitis virus (rVSV). Infection with rVSV expressing an HTLV-1 primary receptor elicits therapeutic effects on HTLV-1-infected envelope protein (Env)-expressing cells in vitro and in vivo. Simian T-cell leukemia virus type 1 (STLV-1) is closely related genetically to HTLV-1, and STLV-1-infected Japanese macaques (JMs) are considered a useful HTLV-1 surrogate, non-human primate model in vivo. Here, we performed an in vitro drug evaluation of rVSVs against STLV-1 as a preclinical study. We generated novel rVSVs encoding the STLV-1 primary receptor, simian glucose transporter 1 (JM GLUT1), with or without an AcGFP reporter gene. Our data demonstrate that these rVSVs specifically and efficiently infected/eliminated the STLV-1 Env-expressing cells in vitro. These results indicate that rVSVs carrying the STLV-1 receptor could be an excellent candidate for unique anti-STLV-1 virotherapy; therefore, such antivirals can now be applied to STLV-1-infected JMs to determine their therapeutic usefulness in vivo.

Phylogeny of human T-lymphotropic virus-1 subtypes in Guinea-Bissau.

Background: Human T-cell leukaemia/lymphoma virus type 1 (HTLV-1) was the first human retrovirus discovered and there is an estimate of 15-20 million infected worldwide. Endemic areas are Japan, West Africa, Central Africa, South America, the Caribbean, Middle East, Australia and the Pacific Islands. In Guinea-Bissau, adult HTLV-1 prevalence is 2-3%, and higher among HIV-infected patients. Materials and methods: Blood samples were collected in a recent HIV/HTLV survey in Bissau, the capital of Guinea-Bissau. Initially, participants were tested for HTLV serologically. The p24 and LTR regions of the proviral genome were then attempted sequenced. Sequences were analysed phylogenetically and compared with reference sequences for HTLV-1. Results: A total of 3% (78/2583) participants were positive on chemiluminesent assay, six additional samples came from another study. Of the 84 seropositive participants we successfully performed sequencing on samples, from 66 participants, 17 were positive for LTR only, one for p24 only and 48 for both. Sequences were in subgroup D of HTLV-1a cosmopolitan, while HTLV-1g was present in one participant. Conclusion: HTLV-1a subgroup D and, to a lesser extent HTLV-1g, is present in Guinea-Bissau and sequences are very similar, especially within households. Presence of HTLV-1g indicates monkey-to-man zoonotic events and at least two circulating HTLV strains in Guinea-Bissau. New sequences accession numbers: MG387979-MG388043 for LTR and MG388044-MG388092 for p24.

IL28B gene polymorphisms and Th1/Th2 cytokine levels might be associated with HTLV-associated arthropathy.

The present study is the first investigation of the association between single nucleotide polymorphisms (SNPs - rs8099917, rs12979860 and rs8103142) of the IL28B gene and the development of human T-lymphotropic virus (HTLV)-associated arthropathy (HAA). Individuals with HAA exhibited low interleukin (IL) 6 (p<0.05) and high IL-10 (p<0.05) levels compared with asymptomatic patients. TNF-α/CD4(+) T cell count, TNF-α/CD8(+) T cell count and IFN-γ/proviral load positively correlated in asymptomatic patients. The allelic and genotypic frequencies did not differ between patients with HAA and asymptomatic patients. Seven haplotypes were detected in the investigated population, with haplotype CCT (p<0.05) being the most frequent among the HTLV-infected individuals, while haplotype TTG (p<0.05) was detected in the group with HAA only. Compared with asymptomatic patients, individuals with HAA and genotype TT (rs8099917) exhibited larger numbers of CD8(+) T cells (p<0.05) and higher proviral load levels (p<0.05). Those patients with HAA and genotypes CC (rs12979860) and TT (rs8103142) exhibited high TNF-ß (p<0.05) and IFN-γ (p<0.05) levels. Those patients with HAA and genotype CT/TT (rs12979860) exhibited high IL-10 levels (p<0.05). These results suggest that haplotypes CCT and TTG might be associated with susceptibility to HTLV infection and progression to HAA, respectively. Genotype TT (rs8099917) might be a risk factor for elevation of the proviral load and CD8(+) T cell count. In addition, genotypes CC (rs12979860) and TT (rs8103142) seem to be associated with increased TNF-ß and IFN-γ levels.

Human T-cell lymphotropic virus type 3 (HTLV-3)- and HTLV-4-derived antisense transcripts encode proteins with similar Tax-inhibiting functions but distinct subcellular localization.

The human T-cell lymphotropic virus (HTLV) retrovirus family is composed of the well-known HTLV type 1 (HTLV-1) and HTLV-2 and the most recently discovered HTLV-3 and HTLV-4. Like other retroviruses, HTLV-1 and HTLV-2 gene expression has been thought to be orchestrated through a single transcript. However, recent reports have demonstrated the unique potential of both HTLV-1 and HTLV-2 to produce an antisense transcript. Furthermore, these unexpected and newly identified transcripts lead to the synthesis of viral proteins termed HBZ (HTLV-1 basic leucine zipper) and APH-2 (antisense protein of HTLV-2), respectively. As potential open reading frames are present on the antisense strand of HTLV-3 and HTLV-4, we tested whether in vitro antisense transcription occurred in these viruses and whether these transcripts had a coding potential. Using HTLV-3 and HTLV-4 proviral DNA constructs, antisense transcripts were detected by reverse transcriptase PCR. These transcripts are spliced and polyadenylated and initiate at multiple sites from the 3' long terminal repeat (LTR). The resulting proteins, termed APH-3 and APH-4, are devoid of a typical basic leucine zipper domain but contain basic amino acid-rich regions. Confocal microscopy and Western blotting experiments demonstrated a nucleus-restricted pattern for APH-4, while APH-3 was localized both in the cytoplasm and in the nucleus. Both proteins showed partial colocalization with nucleoli and HBZ-associated structures. Finally, both proteins inhibited Tax1- and Tax3-mediated HTLV-1 and HTLV-3 LTR activation. These results further demonstrate that retroviral antisense transcription is not exclusive to HTLV-1 and HTLV-2 and that APH-3 and APH-4 could impact HTLV-3 and HTLV-4 replication.

HLA-G 14-bp insertion/deletion polymorphism is a risk factor for HTLV-1 infection.

About 95% of HTLV-1 infected patients remain asymptomatic throughout life, and the risk factors associated with the development of related diseases, such as HAM/TSP and ATL, are not fully understood. The human leukocyte antigen-G molecule (HLA-G), a nonclassical HLA class I molecule encoded by MHC, is expressed in several pathological conditions, including viral infection, and is related to immunosuppressive effects that allow the virus-infected cells to escape the antiviral defense of the host. The 14-bp insertion/deletion polymorphism of exon 8 HLA-G gene influences the stability of the transcripts and could be related to HTLV-1-infected cell protection and to the increase of proviral load. The present study analyzed by conventional PCR the 14-bp insertion/deletion polymorphism of exon 8 HLA-G gene in 150 unrelated healthy subjects, 82 HTLV-1 infected patients with symptoms (33 ATL and 49 HAM), and 56 asymptomatic HTLV-1 infected patients (HAC). In addition, the proviral load was determined by quantitative real-time PCR in all infected groups and correlated with 14-bp insertion/deletion genotypes. The heterozygote genotype frequencies were significantly higher in HAM, in the symptomatic group, and in infected patients compared to control (p < 0.05). The proviral load was higher in the symptomatic group than the HAC group (p < 0.0005). The comparison of proviral load and genotypes showed that -14-bp/-14-bp genotype had a higher proviral load than +14-bp/-14-bp and +14-bp/+14-bp genotypes. Although HLA-G 14-bp polymorphism does not appear to be associated with HTLV-1 related disease development, it could be a genetic risk factor for susceptibility to infection.

Aberrant p53 protein expression and function in a panel of hematopoietic cell lines with different p53 mutations.

The p53 gene is one of the most important genes involved in carcinogenesis and its role in part has been clarified by research using cell lines. To know the comprehensive characteristics of 22 hematopoietic cell lines (T, 13 and non-T, nine lines), the relationship between p53 mutational status, its altered functioning, and its mRNA and protein levels were examined. p53 mutations were less frequent in T-cell lines (38% vs. 78%) with mainly single nucleotide substitutions generating missense codons. Of 22 different p53 mutations, 12 (54.5%) resulted in mutated proteins, with the mutations clustering mainly in the sequence-specific DNA-binding site region located from amino acid residues 102 to 292. p53 mRNA and protein assays determined that wild-type cell lines expressed constant levels of both mRNA and protein, but mutated cell lines demonstrated two expression patterns: protein over-expression with reduced mRNA levels, because of missense mutations; and protein under-expression with little mRNA expression, because of other mutations. The resistance to Nutlin (MDM2 inhibitor)-induced apoptosis was associated with p53 mutations independently of MDM2 expression levels. This clarification of the unique associations in cell lines useful for bio-medical studies will contribute to a better understanding of p53-associated carcinogenesis.

HTLV-I Infection: virus structure, immune response to the virus and genetic association studies in HTLV-I-infected individuals.

Although the structure of human T lymphoptropic virus type I (HTLV-I) has been known well, the function of some proteins encoded by HTLV-I PX region is not fully understood. Furthermore, the responses of the immune system to HTLV-I remain still unknown. Most of HTLV-I-infected individuals show a strong and persistently activated cytotoxic T-cell (CTL) response to the virus. The frequency of HTLV-I specific CTL is higher in patients with HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP) compared with HTLV-I carriers. However, the efficacy of the immune response determines the outcome of HTLV-I-associated diseases. Among the risk factors which contribute to the observed differences between HAM/TSP patients and HTLV-I carriers, the interaction between different genes and/or environmental factors seem to be important. These factors may also involve in outcome of HTLV-I infection in infected-individuals.

Emergence of unique primate T-lymphotropic viruses among central African bushmeat hunters.

The human T-lymphotropic viruses (HTLVs) types 1 and 2 originated independently and are related to distinct lineages of simian T-lymphotropic viruses (STLV-1 and STLV-2, respectively). These facts, along with the finding that HTLV-1 diversity appears to have resulted from multiple cross-species transmissions of STLV-1, suggest that contact between humans and infected nonhuman primates (NHPs) may result in HTLV emergence. We investigated the diversity of HTLV among central Africans reporting contact with NHP blood and body fluids through hunting, butchering, and keeping primate pets. We show that this population is infected with a wide variety of HTLVs, including two previously unknown retroviruses: HTLV-4 is a member of a phylogenetic lineage that is distinct from all known HTLVs and STLVs; HTLV-3 falls within the phylogenetic diversity of STLV-3, a group not previously seen in humans. We also document human infection with multiple STLV-1-like viruses. These results demonstrate greater HTLV diversity than previously recognized and suggest that NHP exposure contributes to HTLV emergence. Our discovery of unique and divergent HTLVs has implications for HTLV diagnosis, blood screening, and potential disease development in infected persons. The findings also indicate that cross-species transmission is not the rate-limiting step in pandemic retrovirus emergence and suggest that it may be possible to predict and prevent disease emergence by surveillance of populations exposed to animal reservoirs and interventions to decrease risk factors, such as primate hunting.

High simian T-cell leukemia virus type 1 proviral loads combined with genetic stability as a result of cell-associated provirus replication in naturally infected, asymptomatic monkeys.

Simian T-cell leukemia virus type 1 (STLV-1) is a primate T cell leukemia virus of the group of oncogenic delta retroviruses. Sharing a high level of genetic homology with human T cell leukemia virus type 1 (HTLV-1), it is etiologically linked to the development of simian T cell malignancies that closely resemble HTLV-1 associated leukemias and lymphomas and might thus constitute an interesting model of study. The precise nature of STLV-1 replication in vivo remains unknown. The STLV-1 circulating proviral load of 14 naturally infected Celebes macaques (Macaca tonkeana) was measured by real-time quantitative PCR. The mean proportion of infected peripheral mononuclear cells was 7.9%, ranging from <0.4% to 38.9%. Values and distributions were closely reminiscent of those observed in symptomatic and asymptomatic HTLV-1 infected humans. Sequencing more than 32 kb of LTRs deriving from 2 animals with high proviral load showed an extremely low STLV-1 genetic variability (0.113%). This paradoxical combination of elevated proviral load and remarkable genetic stability was finally explained by the demonstration of a cell-associated dissemination of the virus in vivo. Inverse PCR (IPCR) amplification of STLV-1 integration sites evidenced clones of infected cells in all infected animals. The pattern of STLV-1 replication in these asymptomatic monkeys was indistinguishable from that of HTLV-1 in asymptomatic carriers or in patients with inflammatory diseases. We conclude that, as HTLV-1, STLV-1 mainly replicates by the clonal expansion of infected cells; accordingly, STLV-1 natural monkey infection constitutes an appropriate and promising model for the study of HTLV-1 associated leukemogenesis in vivo.

The influence of ovine MHC class II DRB1 alleles on immune response in bovine leukemia virus infection.

We have reported previously that the alleles of the ovine leukocyte antigen (OLA)-DRB1 gene that encode the Arg-Lys (RK) motif and the Ser-Arg (SR) motif at positions beta70/71 of the OLA-DRbeta1 domain are associated with resistance and susceptibility, respectively, to development of bovine leukemia virus (BLV)-induced ovine lymphoma. Here, to investigate the different immune response in sheep that carried alleles associated with resistance and susceptible for 30 weeks after infection with BLV, we selected sheep that had the RK/RK or SR/SR genotype among the 52 sheep analyzed by polymerase chain reaction-restriction fragment length polymorphism and DNA sequencing of PCR product for the OLA-DRB1 exon 2 and infected them with BLV. Although the number of BLV-infected cells and virus titer had been maintaining low levels throughout the experimental period, the sheep with the RK/RK genotype could induce expansion of CD5- B-cells and rapid production of neutralizing antibody in the early phase of infection. The level of incorporation of [3H]thymidine by peripheral blood mononuclear cells from the sheep with RK/RK genotype gave a strong response to BLV virion antigen and synthetic antigenic peptides that corresponded to T-helper epitope of the BLV envelope glycoprotein gp51. In contrast, the sheep with SR/SR genotype showed a strong response to BLV virion antigen and synthetic antigenic peptides that corresponded to T-cytotoxic and B-cell epitopes. In such cases, the animals with the RK/RK strongly expressed IFN-gamma, the animals with SR/SR genotype strongly expressed IL-2. To determine the proliferating cells, we tried a blocking assay with monoclonal antibodies such as anti-CD4, -CD8 and -DR molecule. We found that these proliferating cells were MHC-restricted CD4+ T-cells.

Lack of sequence variation in sporadic bovine leucosis in regions of tumour suppressor genes p53 and p16.

Regions of the promoter and exons 5-8 of the tumour suppressor gene p53 were analysed in 25 cases of sporadic bovine leucosis. The study included 17 cases of juvenile leucosis, five cases of adult leucosis and three cases of skin leucosis. Exon 2 of tumour suppressor gene p16 was also investigated in the same samples. No sequence variations were present in the analysed areas of the genes. In p53, this fact represents a clear difference in comparison with enzootic bovine leucosis. In p16, no comparative data are available.

Application of an in situ PCR hybridization method to detection of human T-lymphotropic virus type I-infected cells in the lung.

We applied an in situ polymerase chain reaction (PCR) hybridization method in order to detect human T-lymphotropic virus type I-infected cells in routinely-processed paraffin sections of the lung from 13 autopsied patients with adult T-cell leukemia (ATL). Previously reported protocol resulted in somewhat non-specific staining in our sections. Therefore, we used a hot start PCR method using specialized commercially-available polymerase in order to increase the specificity. Of 6 patients with ATL cell invasion into the lungs, 4 exhibited strong positive staining of almost all invading ATL cells. In contrast, 7 patients without ATL cell invasion into the lungs did not demonstrate any significant reactivity. Since the method described here is a relatively simple hot start method and does not yield false-positives, it may allow us to determine whether human T-lymphotropic virus type I (HTLV-I) associated disorders are related to lymphocytes integrating the HTLV-I genome.

Use of a generic polymerase chain reaction assay detecting human T-lymphotropic virus (HTLV) types I, II and divergent simian strains in the evaluation of individuals with indeterminate HTLV serology.

In countries with a low prevalence of human T-lymphotropic virus (HTLV) infection, indeterminate HTLV serologies are a major problem in blood bank screening because of the uncertainties about infection in these cases. The recent discovery of two new types of simian T-lymphotropic viruses (STLV), which give an HTLV-indeterminate serology, raises the question whether indeterminate serologies in humans may be linked to new types of HTLV. Starting from a Tax sequence alignment of all available primate T-cell lymphotropic virus strains (PTLV), including the two new types STLV-PH969 and STLV-PP1664, we developed generic and type-specific nested polymerase chain reactions (PCRs). The generic PCR proved to be highly sensitive and cross-reactive for all four types of PTLV, while the discriminatory PCRs had a high sensitivity and a specificity of 100%. There was no cross-reactivity with human immunodeficiency virus (HIV), ensuring correct interpretation of results from coinfected patients. Among the 77 serologically indeterminate samples tested, 6 were found to be HTLV-1 PCR positive and 1 was HTLV-II PCR positive. Sequencing of one of the HTLV-I PCR positives excluded PCR contamination, and revealed a divergent type of HTLV-I. The majority of the seroindeterminate samples (91%) were however HTLV-PCR negative, and no new types of HTLV were found. This new assay can identify otherwise undetected HTLV-I or HTLV-II infections and is a useful tool of screening for new types of HTLV among seroindeterminate samples.

HTLV-associated diseases: human retroviral infection and cutaneous T-cell lymphomas.

An array of neurologic, oncologic, and autoimmune disorders are associated with infection with the human pathogenic retroviruses human T-cell leukemia virus types I and II (HTLV-I, II), as well as the human immunodeficiency viruses (HIV). The cutaneous T-cell lymphomas, mycosis fungoides (MF) and its hematogenous variant Sezary Syndrome (SS), share similar clinical and pathological features to HTLV-I-associated adult T-cell leukemia (ATL) and speculation of a retroviral link to MF and SS, especially in areas non-endemic for ATL, has lead to an intensified search for HTLV- and HIV-like agents in these diseases. To further explore a potential role for human retroviruses in MF and SS, skin biopsy-derived or peripheral blood mononuclear cell-derived DNA from 17 patients (MF, n = 7; erythrodermic MF (EMF), n = 5; SS, n = 5) from the North Eastern United States were screened using gene amplification by PCR and a liquid hybridization detection assay. Previously published primers and probes for HTLV-I (LTR, gag, pol, env, and pX), and our own primers and probes for HTLV-I (gag, pol, and env), HTLV-II (pol and env) and HIV-I (gag and pol) were employed. Serum antibodies to HTLV-I were negative in all but one EMF patient. The single HTLV-I seropositive patient carrying a diagnosis of EMF generated positive amplified signals for all of the eight HTLV-I regions tested. Ultimately, this individual evolved to exhibit clinical manifestations indistinguishable from ATL. The other 16 patients were negative for all 12 HTLV and HIV retroviral regions. Our findings suggest that none of the known prototypic human retroviruses are associated with seronegative MF and SS. The uniformly positive results for HTLV-I in the seropositive patient suggests that this patient initially presented with a smoldering form of ATL and illustrates the difficulty that sometimes may be encountered in the differential diagnosis of MF, SS, and ATL based solely on clinical and histopathological criteria.

Questions on the evolution of primate T-lymphotropic viruses raised by molecular and epidemiological studies of divergent strains.

In human and simian T-lymphotropic viruses (HTLV and STLV), collectively referred to as primate T-lymphotropic viruses (PTLV), four distinct clades can be distinguished: PTLV-I, PTLV-II, and the newly discovered divergent STLVs isolated from hamadryas baboons and from bonobos (pygmy chimpanzees). The hamadryas STLV is clearly distinct from types I and II, in terms both of sequence divergence and of genomic structure, and would qualify as a separate type, provisionally called PTLV-L. The bonobo STLV is closer to, although clearly distinct from, PTLV-II, at present known only in humans. While PTLV-II, PTLV-L, and the bonobo STLV appear presently to be species specific, PTLV-I has spread during its evolution through repeated interspecies transmissions between primates and is now present in many species of Old World monkeys and apes and in humans. The human subtypes of PTLV-I arose from at least three acquisitions from separate simian reservoirs.

Stochastic events in the amplification of HTLV-I integration sites by linker-mediated PCR.

Human T-cell leukaemia virus type I (HTLV-I) proviral integration sites from an asymptomatic carrier and from the MT4 cell line were analysed by linker-mediated PCR (LMPCR) and inverse PCR (IPCR). LMPCR was more sensitive, allowing detection of a greater number of integrated proviruses. Reconstruction experiments using a cloned integrated HTLV-1 provirus indicated that > 100 copies were necessary to be detected frequently by LMPCR. To circumvent this problem, the LMPCR analysis was performed approximately 20 times per sample. Thus, for the MT4 cell line, the seven major integration sites were accompanied by approximately 20 clones of lesser frequency. For an asymptomatic HTLV-I carrier, nine integration sites were identified in a single amplfication, while a further 9 followed from 14 additional reactions. These findings show that there is a stochastic element to sampling HTLV-I integration sites by LMPCR, which tends to underestimate the actual number of HTLV-I bearing clones. Accordingly, those detected in at least two reactions represent the most abundant clones.

Hypercalcemia, parathyroid hormone-related protein expression and human T-cell leukemia virus infection.

Adult T-cell leukemia (ATL) associated with HTLV-1 infection is characterized by the development of hypercalcemia in over two thirds of patients. Dysregulation of cellular gene transcription by viral proteins is an emerging paradigm for molecular pathogenesis of disease. A recent example is the parathyroid hormone-related protein (PTHrP) gene, which has been implicated in the hypercalcemia of ATL, and is transactivated by the HTLV-1 tax and HTLV-11 tax proteins. PTHrP is expressed at high levels in leukemia cells derived from ATL patients, as well as in asymptomatic HTLV-1 positive carriers. This article reviews the interaction of the HTLV-1 transcriptional regulator tax with the PTHrP promoter. Tax mediates its effects on PTHrP via cellular transcription factors AP-2 and AP-1, and transactivation via an AP-2 motif represents a novel interaction of tax with a cellular transcription factor.