Human T-cell Leukemia/lymphotropic Virus Type I Research Articles

Isolation of bioactive 5α,8α-epidioxy sterols from the marine sponge Luffariella cf. variabilis

The lipid extract of the marine sponge Luffariella cf. variabilis from Mayotte (Indian Ocean) has proven to be an extraordinarily rich source of a wide variety of unusual steroids belonging to the 5α,8α-epidioxy sterol family. Ten epidioxy sterols have been identified: 5α,8α-epidioxycholesta-6,22-dien-3β-ol, 5α,8α-epidioxycholest-6-en-3β-ol, 5α,8α-epidioxy-24(ξ)-methylcholesta-6,22-dien-3β-ol, 5α,8α-epidioxy-24(S)-methylcholest-6-en-3β-ol and its epimer (24R), 5α,8α-epidioxy-24(S)-ethylcholesta-6,22-dien-3β-ol and its epimer (24R), 5α,8α-epidioxy-24(S)-ethylcholest-6-en-3β-ol and its epimer (24R), along with 5α,8α-epidioxy-24(ξ)-methylcholesta-6,9(11),22-trien-3β-ol. Their structures were determined by nuclear magnetic resonance (1H, 13C) and electron impact mass spectrometry. The mixture of the 10 steroids showed inhibitive activity against the human T-cell leukemia/lymphotropic virus type I (HTLV-I) and also displayed cytotoxic activity against the human breast cancer cell line (MCF7WT).Key words: Luffariella cf. variabilis, marine sponge, 5α,8α-epidioxy sterols, antitumor and antiviral activities.

Mitochondrial targeting of the p13II protein coded by the x-II ORF of human T-cell leukemia/lymphotropic virus type I (HTLV-I).

The X region of the HTLV-I genome contains four major open reading frames (ORFs), two of which, termed x-I and x-II, are of still undefined biological significance. By indirect immunofluorescence and dual labeling with marker proteins, we demonstrate that p13II, an 87-amino acid protein coded by the x-II ORF, is selectively targeted to mitochondria. Mutational analysis revealed that mitochondrial targeting of p13II is directed by an atypical 10-amino acid signal sequence that is not cleaved upon import and is able to target the Green Fluorescent Protein to mitochondria. Expression of p13II results in specific alterations of mitochondrial morphology and distribution from a typical string-like, dispersed network to round-shaped clusters, suggesting that p13II might interfere with processes relying on an intact mitochondrial architecture. Functional studies of mitochondria with the cationic fluorochrome tetramethylrhodamine revealed that a subpopulation of the cells with p13II-positive mitochondria show a disruption in the mitochondrial inner membrane potential (Apsi), an early event observed in cells committed to apoptosis. Taken together, these results suggest novel virus-cell interactions that might be important in HTLV-I replication and/or pathogenicity.

Analysis of Tax-Expressing Cell Lines Generated from HTLV-ITax-Transgenic Mice: Correlation between c-mycOverexpression and Neoplastic Potential

Human T-cell leukemia/lymphotropic virus type I (HTLV-I) infection causes a variety of human diseases, including adult T-cell leukemia/lymphoma. The viral transactivator Tax has been implicated as a key factor in the HTLV-I-induced transformation pathway. To investigate the components of this pathway, we derived fibroblast-like cell lines, designated T6 and T9, from tail biopsies oftax-transgenic C57BL/6 mice that do not develop tumors. Phenotypic characterization of T6 and T9 cells and T6-derived subclones revealed that they differ in their abilities to form fociin vitroand tumorsin vivo.The observed differences in the levels of Tax expression did not correlate with their degree of neoplastic potential. However, a control cell line derived from a nontransgenic C57BL/6 mouse did not form fociin vitroor tumorsin vivo,indicating that Tax was required for the transformation process. Results of Northern analyses showed that the T9 cells and the highly malignant derivatives of T6 cells expressed elevated levels of c-mycmRNA. These findings suggest that progression of thetax-transgenic cells toward a more malignant phenotype might involve c-mycderegulation.

HTLV-Is in Argentina are phylogenetically similar to those of other South American countries, but different from HTLV-Is in Africa.

To understand the origin and past dissemination of human T-cell leukemia/lymphotropic virus type I (HTLV-I) in Latin America, we conducted a phylogenetic study of five new HTLV-I isolates from Argentina. We sequenced partial fragments of long terminal repeats (LTR) of the new HTLV-Is, and then the sequences were subjected to a phylogenetic analysis for comparison with other HTLV-Is of various geographical origins. Our results indicated that all the isolates were members of the Cosmopolitan group. Furthermore, most (four out of five isolates) of the new HTLV-Is belonged to the Transcontinental (A) subgroup, the most widespread subgroup of the four subgroups in the Cosmopolitan group. In this subgroup, they were closely related to HTLV-Is found in other South American countries including those of Amerindians, and were different from those found in Africa. In contrast, the remaining one HTLV-I (ARGMF) did not show any clear similarity to known HTLV-I isolates belonging to the Cosmopolitan group. The close similarity of South American HTLV-Is strongly suggests a common origin of the virus in this continent. Our results do not support the proposed idea of recent introduction of HTLV-I into South America as a consequence of the slave trade from Africa, where phylogenetically different HTLV-Is predominate.

Proliferation of adult T cell leukemia/lymphoma cells is associated with the constitutive activation of JAK/STAT proteins.

Human T cell leukemia/lymphotropic virus type I (HTLV-I) induces adult T cell leukemia/lymphoma (ATLL). The mechanism of HTLV-I oncogenesis in T cells remains partly elusive. In vitro, HTLV-I induces ligand-independent transformation of human CD4+ T cells, an event that correlates with acquisition of constitutive phosphorylation of Janus kinases (JAK) and signal transducers and activators of transcription (STAT) proteins. However, it is unclear whether the in vitro model of HTLV-I transformation has relevance to viral leukemogenesis in vivo. Here we tested the status of JAK/STAT phosphorylation and DNA-binding activity of STAT proteins in cell extracts of uncultured leukemic cells from 12 patients with ATLL by either DNA-binding assays, using DNA oligonucleotides specific for STAT-1 and STAT-3, STAT-5 and STAT-6 or, more directly, by immunoprecipitation and immunoblotting with anti-phosphotyrosine antibody for JAK and STAT proteins. Leukemic cells from 8 of 12 patients studied displayed constitutive DNA-binding activity of one or more STAT proteins, and the constitutive activation of the JAK/STAT pathway was found to persist over time in the 2 patients followed longitudinally. Furthermore, an association between JAK3 and STAT-1, STAT-3, and STAT-5 activation and cell-cycle progression was demonstrated by both propidium iodide staining and bromodeoxyuridine incorporation in cells of four patients tested. These results imply that JAK/STAT activation is associated with replication of leukemic cells and that therapeutic approaches aimed at JAK/STAT inhibition may be considered to halt neoplastic growth.

Cross-neutralizing antibodies against cosmopolitan and Melanesian strains of human T cell leukemia/lymphotropic virus type I in sera from inhabitants of Africa and the Solomon Islands.

The so-called cosmopolitan strains of human T cell leukemia/lymphotropic virus type I (HTLV-I) from Japan, Africa, the West Indies, and the Americas differ only slightly (< 3%) in their genomic sequence. On the other hand, the Melanesian strains of HTLV-I are somewhat more divergent, exhibiting only 93% sequence similarity with the cosmopolitan strains. Despite this difference, sera from individuals infected with Melanesian strains cross-react with T cells infected with cosmopolitan strains of HTLV-I, indicating an overall conservation of the B cell immunoprevalent epitopes. Neutralizing antibodies against HTLV-I in sera from virus-infected Africans and Melanesians were assayed by determining their ability to block the formation of syncytia in cocultures of 8166 and T cell lines harboring either cosmopolitan or Melanesian HTLV-I isolates. All six African sera blocked the formation of syncytia with cells infected with HTLV-IC91-PL, a viral isolate from the United States. Similarly, all six Melanesian sera inhibited syncytium formation with cells infected with HTLV-IMEL3, a virus isolate from the Solomon Islands. Although most of these sera inhibited syncytium formation in cell cultures carrying the cosmopolitan as well as the Melanesian HTLV-I strains, neutralizing antibody titers tended to be higher against the homologous virus. All sera failed to inhibit syncytium formation when cells were infected with HTLV-II. These data indicate the involvement of one or more epitopes in syncytial formation, some of which are conserved in all strains of HTLV-I.(ABSTRACT TRUNCATED AT 250 WORDS)

The human T-cell leukemia/lymphotropic virus type I p12I protein cooperates with the E5 oncoprotein of bovine papillomavirus in cell transformation and binds the 16-kilodalton subunit of the vacuolar H+ ATPase

The human T-cell leukemia/lymphotropic virus type I (HTLV-I) induces T-cell leukemia and transforms human T cells in vitro. A recently identified protein with a molecular weight of 12,000 (12K) (p12I), encoded by single- and double-spliced mRNAs transcribed from the 3' end of the HTLV-I genome, has been shown to localize in the perinuclear compartment and in the cellular endomembranes. The p12I protein exhibits significant amino acid sequence similarity to the E5 oncoprotein of bovine papillomavirus type 1 (BPV-1). Both proteins are very hydrophobic, contain a glutamine residue in the middle of a potential transmembrane region(s), and are localized in similar cellular compartments. Because of these observations, we investigated whether the p12I resemblance to E5 correlated with a similarity in their biological behavior. We expressed the p12I protein to evaluate its ability to functionally cooperate with the BPV-1 E5 oncoprotein and to bind to a cellular target of the E5 protein, the 16K component of the vacuolar H+ ATPase. Cotransfection of the mouse C127 cell line with the p12I and E5 cDNAs showed that although p12I alone could not induce focus formation, it strongly potentiated the transforming activity of E5. In addition, the p12I protein bound to the 16K protein as efficiently as the E5 protein. These findings might provide new insight for potential mechanisms of HTLV-I transformation and suggest that p12I and E5 represent an example of convergent evolution between RNA and DNA viruses.

Complete nucleotide sequence of a highly divergent human T-cell leukemia (lymphotropic) virus type I (HTLV-I) variant from melanesia: genetic and phylogenetic relationship to HTLV-I strains from other geographical regions

The high prevalences of antibodies against human T-cell leukemia (lymphotropic) virus type I (HTLV-I) reported for remote populations in Papua New Guinea and the Solomon Islands and for some aboriginal populations in Australia have been verified by virus isolation. Limited genetic analysis of the transmembrane portion (gp21) of the envelope gene of these viruses indicates the existence of highly divergent HTLV-I strains in Melanesia. Here, we report the complete nucleotide sequence of an HTLV-I isolate (designated HTLV-IMEL5) from the Solomon Islands. The overall nucleotide divergence of HTLV-IMEL5 from the prototype HTLV-IATK was approximately 8.5%. The degree of variability in the amino acid sequences of structural genes ranged between 3 and 11% and was higher (8.5 to 25%) for the regulatory (tax and rex) genes and the other genes encoded by the pX region. Since HTLV-IMEL5 was as distantly related to HTLV-II as to the other known HTLV-I strains, it could not have arisen from a reocmbinational event involving HTLV-II but rather might be an example of independent viral evolution in this remote population. These data provide important insights and raise new questions about the origin and global dissemination of HTLV-I.

In vitro infection of human macrophages by human T-cell leukemia/lymphotropic virus type I (HTLV-I).

HTLV-I is associated with a neurological syndrome designated Tropical Spastic Paraparesis/HTLV-I associated myelopathy (TSP/HAM). To determine whether HTLV-I can replicate in human primary macrophages and thus contribute to HTLV-I dissemination in the nervous system, elutriated human macrophages were infected cell-free with the HTLV-ICR and HTLV-IBOU isolates from patients with adult T-cell leukemia and TSP/HAM, respectively. Viral production was monitored by measuring the viral p24 gag antigen in the cell culture supernatant, by electron microscopy (EM) and by polymerase chain reaction (PCR) on viral DNA and RNA. The HTLV-I p24 gag antigen was detected 21 days after infection with either isolate, and the presence of mature viral particles was demonstrated by electron microscopy one month after infection. Viral sequences were amplified by PCR analysis of the infected macrophages' DNA. Spliced mRNAs for the p40tax and p27rex proteins, as well as the p12I, and p30II proteins encoded by the pX region were readily identified by reverse transcriptase PCR. Altogether, these data indicate that HTLV-I replication occurs in vitro in primary human macrophages. Whether macrophage infection occurs also in vivo and is a crucial step in the induction of the neurological manifestations observed in TSP/HAM remains an open question.

Protein isoforms encoded by the pX region of human T-cell leukemia/lymphotropic virus type I.

The pX region of the human T-cell leukemia/lymphotropic virus type I (HTLV-I) contains at least four open reading frames (orfI-orfIV). orf III and orf IV encode the regulatory HTLV-I proteins Rex and Tax, which together modulate viral expression, and the p21rex protein of unknown function. By using the reverse transcriptase and polymerase chain reaction techniques on the RNA of an HTLV-I-infected cell culture, we uncovered the existence of alternatively spliced mRNAs generated through the use of three splice acceptor sites. These mRNAs encoded protein isoforms derived from the HTLV-I orf I (p12I) and orf II (p13II and p30II). An additional acceptor splice site, used in the processing of the env and tax/rex mRNAs and a singly spliced mRNA for the p21rex protein, was also identified. All of these HTLV-I mRNAs were also detected in freshly isolated cells from HTLV-I-infected individuals. Thus HTLV-I, like the human immunodeficiency virus type 1, has developed fine posttranscriptional mechanisms to increase the complexity of its genome.

Serological characterization of human T-cell leukemia (lymphotropic) virus, type I (HTLV-I) small envelope protein

Murine monoclonal antibodies were developed against the protein products produced by murine C127 cells which had been transfected with a recombinant plasmid clone containing the human T-cell leukemia (lymphotropic) virus type I (HTLV-I) proviral DNA coding regions for part of env, px, and the 3prime;TR. Four antibodies with different binding patterns were obtained. One of these antibodies, F1.6, reacted against HTLV-I infected cells but not against noninfected cell lines. This antibody also reacted with sucrose-gradient purified HTLV-I and -II particles with preferential binding against the HTLV-I preparation. The F1.6 antibody bound to two proteins of approximately 21 and 43 kDa in gradient purified HTLV-I preparations, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blots, and to a 43-kDa protein in cell lysates of HTLV-I infected cell lines; the F1.6 antibody did not bind significantly to any HTLV-II proteins in western blots. The three other antibodies F1.1, F1.2, and F1.5, recognized the same size proteins, 21 and 43 kDa in the gradient purified viral preparations of HTLV-I and in the case of the F1.2 antibody, the same size proteins in purified virus preparation of HTLV-II and -III. The F1.2 and F1.5 antibodies bound not only to purified HTLV particles but also to a variety of cellular proteins in HTLV-I infected and noninfected cells suggesting that they recognized epitopes which were shared between HTLV proteins and normal cells. The identity of the 21-kDa viral protein is most likely that of the small envelope glycoprotein. The identity of the larger protein is undetermined.

Molecular analysis of a deletion mutant provirus of type I human T-cell lymphotropic virus: evidence for a doubly spliced x-lor mRNA.

The genome of the human T-cell leukemia/lymphotropic virus type I (HTLV-I) contains a functional gene denominated x-lor that may be important in HTLV-I transformation of human T cells. To study the role of x-lor and other HTLV-I genes in cellular transformation, we obtained a transformed nonproducer human T-cell line containing a single defective HTLV-I provirus (HTLV-I 55/PL). This 7-kilobase provirus had undergone a deletion involving the entire envelope gene and the nonconserved region. The point of the deletion corresponded to the junction of a donor splice site, located between the polymerase gene and the envelope gene (nucleotide 5183), and the acceptor site for the mRNA of the x-lor gene (nucleotide 7302). The juxtaposition of nucleotides 5182 and 7302 brings the initiating methionine codon of the envelope gene immediately 5' to the x-lor region, leaving the DNA sequence in frame for expression of a protein product. This finding suggests that a double splicing mechanism is used to express the x-lor gene, and that the defective provirus 55/PL was generated through the reverse transcription of a partially spliced mRNA. Analysis of the x-lor mRNA of other HTLV-I-transformed cell lines revealed that a double splicing process is commonly used. Furthermore, since 55/PL can be faithfully transmitted and is able to immortalize recipient T cells, we can conclude that the envelope gene is not necessary for in vitro transformation by HTLV-I.