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PubMed1992

New human gene encoding a positive modulator of HIV Tat-mediated transactivation.

The human immunodeficiency virus-1 (HIV-1) protein Tat is a potent activator of virus gene expression. Tat functions through a sequence known as TAR, located immediately downstream of the transcription start site in the long terminal repeat. Several observations suggest that cellular factors cooperate with Tat in the overall transactivating process. We have isolated a human complementary DNA from the new gene MSS1, which may encode such a cellular factor, by transcomplementation of a yeast sgv1- mutant. The MSS1 protein shares 42% sequence identity with the human TBP-1 protein, which binds Tat in vitro and suppresses Tat-mediated transactivation in vivo (ref. 6). We report here that the levels of HIV activation by Tat correlate with endogenous levels of MSS1 messenger RNA. Furthermore, we provide evidence that expression of MSS1 enhances the Tat-mediated transactivation. Our results suggest that MSS1 has a key role in activation of HIV genes regulated by Tat.

ATPases Associated with Diverse Cellular Activitie↗
PubMed1990

Transactivation of interleukin 2 and its receptor induces immune activation in human T-cell lymphotropic virus type I-associated myelopathy: pathogenic implications and a rationale for immunotherapy.

A state of T-cell activation, reflected by a marked degree of spontaneous proliferation in vitro, exists among patients with human T-cell lymphotropic virus type I (HTLV-I)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) but not in those with retroviral-induced adult T-cell leukemia (ATL). We wished to define the mechanism by which the immune activation of circulating cells from HAM/TSP is driven, thus gaining insight into the pathogenesis of this HTLV-I-associated disease. By using a modification of the polymerase chain reaction, we compared the levels of interleukin 2 (IL-2) and IL-2 receptor alpha chain (IL-2R alpha) mRNA expression to the transcription of the HTLV-I transactivator gene, pX, in peripheral blood mononuclear cells of HAM/TSP and ATL patients as well as seropositive carriers. Up-regulation of IL-2 and IL-2R alpha transcripts was detected in HAM/TSP and seropositive carriers that paralleled the coordinate mRNA expression of the pX transactivator. In addition, IL-2 and soluble IL-2R alpha serum levels in HAM/TSP and seropositive carriers were elevated. Despite markedly elevated levels of soluble IL-2R alpha in ATL, transcripts for IL-2 and pX were not demonstrable in the circulating cells. Finally, the marked degree of in vitro spontaneous proliferation present in HAM/TSP was profoundly inhibited by specific anti-IL-2R or anti-IL-2 blocking antibodies. Collectively, these results suggest that immune activation in HAM/TSP, in contrast to ATL, is virally driven by the transactivation and coordinate expression of IL-2 and IL-2R alpha. This deregulated autocrine process may contribute to the evolution of inflammatory nervous system damage in HAM/TSP.

Base Sequence↗
PubMed1996

Infectivity of chimeric human T-cell leukemia virus type I molecular clones assessed by naked DNA inoculation.

Two human T-cell leukemia virus type I (HTLV-I) molecular clones, K30p and K34p were derived from HTLV-I-infected rabbit cell lines. K30p and K34p differ by 18 bp with changes in the long terminal repeats (LTRs) as well as in the gag, pol, and rex but not tax or env gene products. Cells transfected with clone K30p were infectious in vitro and injection of the K30p transfectants or naked K30p DNA into rabbits leads to chronic infection. In contrast, K34p did not mediate infection in vitro or in vivo, although the cell line from which it was derived is fully infectious and K34p transfectants produce intact virus particles. To localize differences involved in the ability of the clones to cause infection, six chimeric HTLV-I clones were constructed by shuffling corresponding fragments containing the substitutions in the LTRs, the gag/pol region and the rex region between K30p and K34p. Cells transfected with any of the six chimeras produced virus, but higher levels of virus were produced by cells transfected with those constructs containing the K30p rex region. Virus production was transient except in cells transfected with K30p or with a chimera consisting of the entire protein coding region of K30p flanked by K34p LTRs; only the transfectants showing persistent virus production mediated in vitro infection. In vivo infection in rabbits following intramuscular DNA injection was mediated by K30p as well as by a chimera of K30p containing the K34p rex gene. Comparisons revealed that virus production was greater and appeared earlier in rabbits injected with K30p. These data suggest that several defects in the K34p clone preclude infectivity and furthermore, provide systems to explore functions of HTLV-I genes.

Animals↗
PubMed1997

Reexamination of human T cell lymphotropic virus (HTLV-I/II) prevalence.

In the United States, blood donors are being screened for infection with human T cell lymphotropic viruses I and II (HTLV-I/II) by serologic means, which detect antibodies to the structural proteins of these viruses. Because patients with mycosis fungoides (MF) usually do not have such antibodies even though their cells harbor HTLV-I Tax and/or pol proviral sequences, it was questioned whether the prevalence of HTLV infection among healthy blood donors may also be underestimated by current means of testing. To examine this possibility, a study on specimens of relatives of mycosis fungoides patients (MFR) was begun. In addition, to collect data more expeditiously, a cohort of former injection drug users (IDUs) was tested by routine serologic methods, as well as by PCR/Southern blot analysis for Tax, pol, and gag proviral sequences and Western blot analysis for antibodies to the Tax gene product. To date, 6/8 MFRs and 42/81 (51.8%) of HIV-negative IDUs proved to be positive for HTLV, whereas routine serology identified none of the MFR and only 18/81 (22.2%) of the IDUs. Among the latter test subjects, the incidence of HTLV-I also proved to be 10 times higher than expected. Therefore, it is likely that among healthy blood donors infection with HTLV-I/II is more prevalent than is currently assumed. Since Tax is the transforming sequence of HTLV-I/II, testing for Tax sequences and antibodies to its gene product may be desirable in blood transfusion and tissue donor facilities.

Blood Donors↗
PubMed2004

Enhanced activation of tax-dependent transcription of human T-cell leukemia virus type I (HTLV-I) long terminal repeat by TORC3.

Tax, a protein encoded by the env-pX gene of human T-cell leukemia virus type I (HTLV-I), interacts with various host cell transcription factors. Tax activates transcription from the long terminal repeat (LTR) of HTLV-I through association with cyclic AMP-responsive element-binding protein (CREB). Here, we present evidence that transducer of regulated cyclic AMP-response element-binding protein 3 (TORC3), a co-activator of CREB, is involved in Tax-induced transcriptional activation from the HTLV-I LTR. By using a luciferase assay system, we show that TORC3 alone can enhance transcription from the HTLV-I LTR, as well as from a cellular cyclic AMP-response element (CRE). Interestingly, we find that co-expression of TORC3 and Tax dramatically increased transcriptional activation at the HTLV-I LTR. We also show by glutathione S-transferase pull-down and co-immunoprecipitation experiments that TORC3 interacts with Tax. Using deletion mutant analysis, we identify the Tax interaction domain of TORC3 as a region spanning from amino acid 1 to 103, which contains a coiled-coil domain. These results provide important clues toward understanding the molecular mechanism of Tax-dependent transcriptional activation of the HTLV-I LTR.

Animals↗
PubMed1999

Differential patterns of interaction between HIV type 1 and HTLV type I in monocyte-derived macrophages cultured in vitro: implications for in vivo coinfection with HIV type 1 and HTLV type I.

The interaction between human immunodeficiency virus type 1 (HIV-1) and human T cell leukemia-lymphoma virus type I (HTLV-I) has generated substantial interest. However, there is disagreement on the in vivo consequences of the double infection. We investigated the interactions between HIV-1 and HTLV-I in monocyte-derived macrophages cultured in vitro. For study, the T cell-tropic strain IIIB and the macrophagetropic strain Ada-M of HIV-1 were used. The HTLV-I was prepared from the supernatants of the virus-producing MT-2 cell line. We found that coinfection of macrophages with T cell-tropic HIV-1 and HTLV-I significantly enhanced HIV-1 replication, whereas double infection of the cells with macrophage-tropic HIV-1 and HTLV-I resulted in marked upregulation of HTLV-I production. Stimulatory interactions between HIV-1 and HTLV-I were mediated by their trans-acting proteins. Results of study on nuclear translocation of proviral DNA showed that the tax gene product of HTLV-I was able to facilitate the nuclear import of the reverse-transcribed HIV-1(IIIB) DNA. In contrast, the HIV-1 Tat protein did not increase the intranuclear trafficking of HTLV-I DNA, which suggests another mechanism for HTLV-I enhancement by the tat gene product. In conclusion, this study provides possible mechanisms whereby coinfection of an individual with HIV-1 and HTLV-I may influence the clinical outcome of double infection.

Cell Nucleus↗
PubMed1991

Human immunodeficiency virus type 2 (HIV-2) gene expression: downmodulation by sequence elements downstream of the transcriptional initiation site.

Human immunodeficiency virus type 2 (HIV-2) gene expression is downmodulated by sequence elements downstream of the transcriptional initiation site, corresponding to the U5 region of the long terminal repeat (LTR) and further downstream. This repression appeared to be related more to the length of the sequence intervening the transcriptional initiation site and the coding region than to a particular sequence content. The repressive effect of the downstream segment was not affected by HIV-2 and HIV-1 TAT or by the cytomegalovirus transactivator IE-2 gene. Nor was it affected by T-cell activation signals or by such cytokines as tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), interferon-gamma (IFN gamma), and interferon-alpha (IFN alpha). In contrast to HIV-1, HIV-2 LTR-directed gene expression was not modulated by TNF-alpha. A specific sequence element, located downstream of the TAR element in the R region, seemed to participate in modulation of gene expression. This element interacted with a nuclear protein with a mobility of about 26 kD. The repressive effect of the downstream sequence was to a certain extent cell type dependent, suggesting the involvement of cell type-specific factors. It was more effective in human lymphocytic CEM cells than in Jurkat cells. This may be relevant to the HIV-2 cell tropism (replication), latency, and virulence.

Animals↗
PubMed1993

Large granular lymphocytosis in a patient infected with HTLV-II.

HTLV-II has been associated with a variety of lymphoproliferative disorders, including atypical hairy cell leukemia, chronic T cell leukemia, T prolymphocytic leukemia, and large granular lymphocytic leukemia. However, a direct or indirect role for HTLV-II in these disorders is not yet firmly established. We studied a patient diagnosed as having leukemia of the large granular lymphocyte (LGL) type who was HTLV-II seropositive, to determine if the expanded cell population was infected. Two populations of CD3-CD16+ LGL were identified; one was CD8+, the other CD8-. Populations of cells with these surface markers as well as normal CD3+CD4+ and CD3+CD8+ cells were separated by flow cytometric methods, DNA extracted, and gene regions of HTLV-II pol and tax amplified, using the polymerase chain reaction, and probed after Southern blotting. HTLV-II was detected in the CD3+CD8+ population, and not in the CD3-CD16+ large granular lymphocyte population. This finding indicates that the role of HTLV-II, if any, in LGL proliferation is indirect.

Antigens, CD↗
PubMed1994

Familial transmission and minimal sequence variability of human T-lymphotropic virus type I (HTLV-I) in Zaire.

Our group previously reported a strong familial clustering of HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP) in Zaire, suggesting a familial transmission of the virus together with the presence of cofactors. In the present study among 84 relatives of 16 HTLV-I-positive or HAM/TSP index cases, we found that all 15 seropositive children had a seropositive mother and that all 15 children with a seropositive father but a seronegative mother were seronegative. Lymphocytes of 17 relatives from 2 families with a familial HTLV-I-associated neuropathy were tested in 2 polymerase chain reaction (PCR) assays amplifying pol and tax/rex gene fragments. The 10 seropositive individuals were PCR positive for HTLV-I and the 7 seronegatives were negative in both PCR assays. The PCR results showed no evidence for a long lag period between infection with HTLV-I and seroconversion. The HTLV-I long terminal repeat (LTR) of these 10 individuals, related in the first to the fourth degree, was amplified and sequenced. Identical sequences were found within the families except for one woman infected with two variants, one being the familial strain and the other a mutated one with a single nucleotide substitution in the 755 sequenced nucleotides of the LTR region. The family strain and the mutant were both present in two samples taken 1 year apart. Together, the HTLV-I serology, PCR, and sequencing results point toward mother-to-child transmission as the main mode of HTLV-I infection in this population. Comparison of the LTR sequences of the two families with other HTLV-I strains from different geographical regions shows that the Zairean HTLV-I strains form a separate cluster.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1994

Cutaneous disease resembling mycosis fungoides in HIV-infected patients whose skin and blood cells also harbor proviral HTLV type I.

Two homosexual HIV-infected patients with lymphocyte counts of < 50 presented with intense pruritus, hyperpigmentation, and skin lesions clinically suggestive of the cutaneous T cell lymphoma, mycosis fungoides. On light microscopy, the skin biopsies were difficult to interpret because of the sparseness of the lymphocytic infiltrates. However, electron microscopy revealed typical Sézary cells in the peripheral blood and skin. Cultures of blood mononuclear cells of one of the patients generated HTLV-I-like particles. Although both patients lacked antibodies to HTLV, their blood and skin specimens proved to harbor tax and pol HTLV-I proviral sequences as shown by the polymerase chain reaction and Southern blot analysis. Dual infection with HIV and HTLV should be considered in the diagnostic work-up of patients at risk, even in the absence of demonstrable antibodies. Dual infections could result in clinical manifestations and evolution of disease not anticipated in patients who harbor only one of these retroviruses.

AIDS-Related Opportunistic Infections↗
PubMed1993

DNA inoculation induces neutralizing immune responses against human immunodeficiency virus type 1 in mice and nonhuman primates.

DNA, or genetic, inoculation mimics aspects of attenuated vaccines in that synthesis of specific foreign proteins is accomplished in the host. These proteins can be processed and presented on the relevant major histocompatibility complex (MHC) antigens and ultimately become the subject of immune surveillance. Very recently, we have described the use of the new technology to generate immune responses in mice against the human immunodeficiency virus type 1 (HIV-1) envelope using a gp160 DNA construct. Further analysis of this technology specifically in regard to HIV vaccine design is clearly important. In this report, we describe the analysis of additional HIV constructs as immunogens in both mice and report the use of this genetic immunization technology in nonhuman primates. In these studies, successful seroconversion occurs in more than 70% of the mice following the second immunization with 100 micrograms of construct DNA; three and four immunizations result in routinely 100% seroconversion of the mice. Furthermore, the same strategy has successfully seroconverted primates following their second inoculation, resulting in the generation of both antiviral and neutralizing antibodies in this animal species. These studies are the first report of which we are aware that demonstrate successful immunization of nonhuman primates through genetic vaccination technology and the first to describe genetic immunization of primates against HIV antigens. This technology has relevance for the development of safe and efficacious immunization strategies against HIV because it provides for relevant antigen production in vivo without the use of infectious agents.

AIDS Vaccines↗
PubMed1991

Detection of human T-cell leukemia virus type I proviral sequences from fixed tissues of seropositive patients.

Fixed lymphoma tissues from 11 patients seropositive for human T-cell leukemia virus type I (HTLV-I) antibodies were analyzed for proviral sequences by the polymerase chain reaction. Typical adult T-cell leukemia/lymphomas (ATLLs) from nine patients were positive for HTLV-I sequences. In contrast, one of two lymphomas with immunophenotypes atypical for ATLL was negative for HTLV-I. This HTLV-I DNA-negative lymphoma, although present in an HTLV-I-seropositive patient, was therefore reclassified. The HTLV-I tax gene was always detected in the ATLL tissues, whereas segments of the pol gene were not detected in half the cases. These studies demonstrate that fixed non-ATLL specimens can be distinguished from ATLL specimens.

Adult↗
PubMed2002

Detection of HTLV-I tax-rex and pol gene sequences of thymus gland in a large group of patients with myasthenia gravis.

We report in this study the use of polymerase chain reaction (PCR) for the amplification of the genomic DNA, isolated from thymic tissue, using the primers flanking HTLV-I/II tax-rex genes and the sequence method to analyze the HTLV-I pol sequence of 27 Italian patients with myasthenia gravis. These molecular methods showed that 92.5% of patients tested positive for tax gene and 55% for pol genes; 55.5% samples were positive for both the tax gene of HTLV-I/II, and the pol gene of HTLV-I. Histologic investigation of the thymus showed that 15 samples had thymic hyperplasia, 93% tested positive for the tax gene, and 40% tested positive for both the tax and pol genes of HTLV-I. In contrast, 91.6% of thymoma-positive samples were positive for tax gene I/II and 75% positive for both genes, tax and pol type I. The sequence analysis of PCR product for tax and pol genes confirmed that these amplified products were HTLV-I, with minimal variations. Our date suggested that either HTLV-I or part of the virus genome is involved in the etiopathogenesis of myasthenia gravis.

Adult↗
PubMed1998

Complete nucleotide sequence of an African human T-lymphotropic virus type II subtype b isolate (HTLV-II-Gab): molecular and phylogenetic analysis.

We report the first complete nucleotide sequence of an African human T-cell lymphotropic virus type II. This new strain, called HTLV-II-Gab (Gab), was obtained from the uncultured peripheral blood mononuclear cells of a 44-year-old healthy Gabonese male who lived in a remote rural area, with neither history of blood transfusion nor sexual intercourse with non-Africans. Using nested PCR, 25 overlapping fragments, representing the entire proviral genome, were obtained, cloned and sequenced. The overall nucleotide sequence comparison with the four other available complete HTLV-II genomes indicated that Gab was more closely related to the HTLV-II subtype b prototypes (98.9, 99.3 and 98.2% nucleotide similarity with G12, NRA and GU respectively) than to the subtype a prototype (95.1% nucleotide similarity with Mo). Restriction profiles studies and phylogenetic analyses confirmed that Gab was a subtype b strain. However, this strain represents a newly described restriction fragment length polymorphism subtype, closely related to one of the rare partially sequenced African isolates originating from a pygmy living in Cameroon (PYGCAM). Nevertheless, the very low genetic divergence observed between this new African strain and the American strains raises several questions on the origins and level of genetic variability over time of this human retrovirus.

Adult↗
PubMed1988

Isolation of virus-producing transformants from human gastric cancer cell line, HGC-27, infected with human T-cell leukemia virus type I.

A human anaplastic gastric cancer cell line, HGC-27, showed marked degeneration with formation of multinucleated syncytia and cell detachment of nearly all cells which began 24 hr after and reached a maximum 2 to 3 days after co-cultivation with X-irradiated MT-2 cells, HTLV-I producing human cord leukocytes. Less severe degeneration without formation of syncytia was also observed in the cultures inoculated with cell-free MT-2 culture media. Morphologically altered cells began to proliferate and formed piled up colonies in some of the cultures co-cultivated with X-irradiated MT-2 cells after a long culture period. The two clones designated HGC/MT2 (Cl-1) and HGC/MT2 (Cl-2) were separated by cell cloning. HGC/MT2 (Cl-1) and HGC/MT2 (Cl-2) cells were positive for HTLV-I gag proteins (p19 and p24) and pX gene products, p40x, as demonstrated by immunohistochemistry and immunoblotting analysis, contained HTLV-I provirus DNA, and consistently produced type C virus particles.

Cell Transformation, Viral↗
PubMed2005

Proviral status of HTLV-1 integrated into the host genomic DNA of adult T-cell leukemia cells.

Human T-cell leukemia virus type-1 (HTLV-1) is the etiological agent of adult T-cell leukemia (ATL), and leukemic cells always carry the proviral genome monoclonally integrated into their host genomes at the same sequence site, designated as the monoclonal integration. Using Southern blot hybridization (SBH) and sequenced tagged site polymerase chain reaction assays, we examined the proviral status in 558 clinical specimens from 350 patients who are suspected to have ATL. A total of 321 specimens (57.5%) from 241 patients showed positive results for the monoclonal integration according to SBH, using EcoR1 and Pst1. The 241 patients consisted of 136 patients (56.4%) with the complete provirus (C-type), 62 patients (25.7%) with a defective provirus (D-type), and 43 patients (17.8%) with multibands (M-type). The incidence of the D- and M-types were in the order of smoldering, chronic, and acute subtypes of ATL, suggesting that such an aberrant proviral status is generated on the way to multistep carcinogenesis and is subsequently clinically important for the malignant behavior of the disease. Moreover, our data showed that the partial deletion of the proviral genome is initiated first at the site of the gag region and spreads into the sites of the pol and env regions, whereas the long terminal repeats and pX regions are almost always conserved. These results suggest that analysis of the proviral status provides useful diagnostic and virologic-oncological information about ATL and HTLV-1 pathology, especially the important role of pX gene in tumorigenesis.

Adult↗
PubMed1992

Sensitive and specific polymerase chain reaction assays for diagnosis of human T-cell lymphotropic virus type I (HTLV-I) and HTLV-II infections in HTLV-I/II-seropositive individuals.

To confirm and differentiate between human T-cell lymphotropic virus type I (HTLV-I) and HTLV-II infections, we analyzed by polymerase chain reaction (PCR) samples of peripheral blood lymphocytes from 98 individuals seropositive for HTLV-I/II using pol (SK110/111) and tax (SK43/44) consensus primer pairs. A total of 96 samples (97.9%) were positive by the tax generic probe, while 95 were typed by the HTLV-I and HTLV-II pol probes. The three pol-negative samples were successfully amplified and typed by nested PCR with primers internal to SK110 and SK111. Results of PCR with a lysate of leukocyte nuclei obtained by whole blood lysis were comparable to those obtained with peripheral blood lymphocytes from 16 HTLV-seropositive subjects.

Base Sequence↗