Human T-cell Leukemia Virus Type II Research Articles

Human T cell leukemia virus type II increases telomerase activity in uninfected CD34+ hematopoietic progenitor cells.

The aging process of long-term self-renewing hematopoietic stem/progenitor cells is not yet completely understood and recent studies on antiapoptotic cell pathways have demonstrated a close linkage between telomerase activation and Bcl-2 deregulation in human cancer cells. The present work shows that human T cell leukemia virus type II (HTLV-II) Mo virions that have originated from the T cell line (C344), but not from the B cell line (BJAB), are critically involved in mediating survival and growth effects on hematopoietic precursors (represented by both the TF-1 CD34+ cell line and by peripheral blood-derived CD34+ cells) through the maintenance or enhancement of telomerase activity and the induction of bcl-2 expression. In addition, using an interleukin-3-dependent TF-1 cell line, it was demonstrated that IL-3 deprivation was sufficient to influence the levels of telomerase activity and Bcl-2 expression in CD34+ cells. Taken together, these findings suggest that, in appropriate conditions, extended hematopoietic progenitor cell survival and proliferation following HTLV-II exposure depends on a synergistic interaction between up-regulation of Bcl-2 and activation of telomerase activity.

Human T-Cell Leukemia Virus Type II Directly Acts on CD34+ Hematopoietic Precursors by Increasing Their Survival Potential. Envelope-Associated HLA Class II Molecules Reverse This Effect

The role of human T-cell leukemia virus type II (HTLV-II) in human lymphoproliferative and hematopoietic abnormalities in which the retrovirus can be isolated is still elusive. Here we show that the C344 T-cell–derived lymphotropic HTLV-II type IIa Mo strain acts directly on CD34+ hematopoietic precursors by rescuing them from apoptosis induced by interleukin-3 (IL-3) deprivation. This effect is viral strain-specific, as it is not observed with the B-lymphotropic HTLV-II type IIb Gu strain, it does not require infection of the hematopoietic precursors, and, interestingly, it is strongly dependent on the infected cellular host from which the virus was derived. Indeed, growth adaptation of the Mo strain to the permissive B-cell line, BJAB, renders the virus no longer capable of mediating the antiapoptotic effect. However, pretreatment of the BJAB-adapted Mo strain with antibodies specific for HLA class II, but not class I, histocompatibility antigens restores the antiapoptotic potential of the virus. These results constitute the first evidence that HTLV-II retrovirus can directly influence the homeostasis of human progenitors, without infecting them, and that this crucial activity is strongly inhibited by the presence of host-derived envelope-associated HLA class II antigens.

Human T-Cell Leukemia Virus Type II Directly Acts on CD34+ Hematopoietic Precursors by Increasing Their Survival Potential. Envelope-Associated HLA Class II Molecules Reverse This Effect

AbstractThe role of human T-cell leukemia virus type II (HTLV-II) in human lymphoproliferative and hematopoietic abnormalities in which the retrovirus can be isolated is still elusive. Here we show that the C344 T-cell–derived lymphotropic HTLV-II type IIa Mo strain acts directly on CD34+ hematopoietic precursors by rescuing them from apoptosis induced by interleukin-3 (IL-3) deprivation. This effect is viral strain-specific, as it is not observed with the B-lymphotropic HTLV-II type IIb Gu strain, it does not require infection of the hematopoietic precursors, and, interestingly, it is strongly dependent on the infected cellular host from which the virus was derived. Indeed, growth adaptation of the Mo strain to the permissive B-cell line, BJAB, renders the virus no longer capable of mediating the antiapoptotic effect. However, pretreatment of the BJAB-adapted Mo strain with antibodies specific for HLA class II, but not class I, histocompatibility antigens restores the antiapoptotic potential of the virus. These results constitute the first evidence that HTLV-II retrovirus can directly influence the homeostasis of human progenitors, without infecting them, and that this crucial activity is strongly inhibited by the presence of host-derived envelope-associated HLA class II antigens.

Molecular phylogeny of human T-cell leukemia virus type I and II of Amerindians in Colombia and Chile.

Six human T-cell leukemia virus type I (HTLV-I) and eight human T-cell leukemia virus type II (HTLV-II) cases newly isolated from the South American countries of Colombia and Chile were analyzed together with the two Amerindian HTLV-I isolates previously reported. All of the HTLV-I isolates belonged to the transcontinental subgroup of the "cosmopolitan" group, and Colombian isolates, including those from native Amerindians and Negroes, formed a single tight cluster within this subgroup. The transcontinental subgroup consisted of isolates from various regions such as the Caribbean basin, India, Iran, South Africa, Sakhalin, and Japan, and included isolates from the "Ainu" and "Okinawa" people, regarded as relatively pure Japanese descended from the prehistoric "Jomon" period which began more than 10,000 years ago. This implied a dissemination of the subgroup associated with the movement of human beings in ancient times. On the other hand, all of the HTLV-II isolates from native Amerindians in Colombia and Chile belonged to the HTLV-IIb subtype which has previously been reported to be mainly endemic in certain populations of native Amerindians. The southernmost isolate from Chile, showing wide distribution of the IIb subtype in native South Amerindians and largest heterogeneity of the subtype in Colombian isolates, supported the idea that the HTLV-IIb subtype has been endemic for a long time in native Indians of South America.

Three-Dimensional Structure of the HTLV-II Matrix Protein and Comparative Analysis of Matrix Proteins from the Different Classes of Pathogenic Human Retroviruses

The matrix protein performs similar roles in all retroviruses, initially directing membrane localization of the assembling viral particle and subsequently forming a stable structural shell associated with the inner surface of the mature viral membrane. Although conserved structural elements are likely to perform these functions in all retroviral matrix proteins, invariant motifs are not evident at the primary sequence level and three-dimensional structures have been available for only the primate lentiviral matrix proteins. We have therefore used NMR spectroscopy to determine the structure of the matrix protein from human T-cell leukemia virus type II (HTLV-II), a member of the human oncovirus subclass of retroviruses. A total of 577 distance restraints were used to build 20 refined models that superimpose with an rmsd of 0.71 Å for the backbone atoms of the structured regions. The globular HTLV-II matrix structure is composed of four α-helices and a 3 10helix. Exposed basic residues near the C terminus of helix II form a putative membrane binding surface which could act in concert with the N-terminal myristoyl group to anchor the protein on the viral membrane surface. Clear structural similarities between the HTLV-II and HIV-1 matrix proteins suggest that the topology and exposed cationic membrane binding surface are likely to be conserved features of retroviral matrix proteins.

Clonal Expansion of Human T-Cell Leukemia Virus Type II in Patients with High Proviral Load

In previous studies we demonstrated that individuals infected by human T-cell leukemia virus type II (HTLV-II) presented a high degree of variation in proviral load and that the cellular tropism of this virus was expanded in some patients to B-lymphocytes. To understand whether the observed high proviral load could be associated with the clonal expansion of the infected cells, we have studied the mode of integration of HTLV-II in six infected individuals with proviral load higher than 1% of total peripheral blood mononuclear cells (PBMCs). An inverse polymerase chain reaction (PCR) analysis, which allowed the amplification of the region flanking the 5′ end of the provirus, was developed for HTLV-II. A single band, corresponding to a monoclonal expansion, was found in four of six patients analyzed, while in the other two patients an oligoclonal type of integration was observed. The results for inverse PCR analysis were confirmed by sequencing the PCR products and showing that the 5′ LTR flanking sequences of proviral DNA obtained from the different subjects presented no homology, thus suggesting that no specific site or sequence is required for the integration process of HTLV-II. The results indicate that the HTLV-II high proviral load observed in PBMCs from infected patients is associated with a clonal expansion of HTLV-II-infected cells. This study also suggests that the very high genetic stability of HTLV-II could be explained by viral amplification via clonal expansion rather than by reverse transcription.

Negative Regulation of Gene Expression from the HTLV Type II Long Terminal Repeat by Rex: Functional and Structural Dissociation from Positive Posttranscriptional Regulation

Regulation of human T cell leukemia virus type II (HTLV-II) gene expression by Rex is mediated by cis-acting elements in the 5' viral long terminal repeat (LTR). Rex acts posttranscriptionally to enhance cytoplasmic accumulation of incompletely spliced viral mRNAs encoding structural proteins. We report a distinct negative regulatory function mediated by Rex affecting expression from the viral 5' LTR. Using both LTR-driven CAT reporters and a full-length HTLV-II proviral construct, we demonstrate that Rex decreases total cellular levels of LTR-containing mRNA in a dose-dependent manner. Negative regulation is an independent function as demonstrated by structural and functional dissociation from Rex positive posttranscriptional regulation. This negative regulatory action was dependent on nuclear localization sequences, but did not require the previously defined Rex-responsive element (RxRE). Negative regulation was observed in T cell lines but not in B cell lines, suggesting the involvement of cell type-specific factors distinct from those involved in posttranscriptional regulation. An internal deletion mutant of Rex removing aa 38-80 retained the ability to repress, but did not posttranscriptionally increase expression, while negative regulation requires a previously uncharacterized carboxy-terminal region (aa 154-170). These findings suggest that Rex may serve two simultaneous functions: to decrease overall levels of transcribed viral mRNA, and to facilitate nuclear to cytoplasmic export of mRNAs encoding structural proteins. The negative regulatory function of Rex may play a role in viral latency.

Specific binding of polypyrimidine tract binding protein and hnRNP A1 to HIV-1 CRS elements.

The human immunodeficiency virus (HIV) Rev and human T-cell leukemia virus (HTLV) Rex proteins regulate viral RNA processing. Both proteins act to overcome the block to viral structural gene expression, at least in part, by reversing the inhibitory effect of intronic RNA sequences, termed cis-acting repressive (CRS) sequences. Using HTLV type II (HTLV-II) as a model, we recently showed that the function of a 5' long terminal repeat (LTR) CRS correlates with in vitro binding by both polypyrimidine tract binding (PTB) protein (also known as hnRNP I) and hnRNP A1 to CRS RNA (1,2). Using radioimmunoprecipitation of proteins ultraviolet (UV) crosslinked to each HIV CRS RNA with monoclonal anti-hnRNP antibodies, we now demonstrate that hnRNP I and hnRNP A1 bind to two different HIV-1 CRS RNAs. In addition, we show that hnRNP I and hnRNP A1 binding to HIV-1 CRS RNAs can be specifically competed by HTLV-II CRS RNAs using electrophoretic mobility shift assay (EMSA)/UV crosslinking assays. Binding by both hnRNP I and hnRNP A1 to HIV-1 and HTLV-II CRS RNAs suggests a role for these proteins in CRS function that may be influenced by the Rev and Rex proteins, respectively.

Rapid Generation of Antibodies against the HTLV-II External Envelope Protein by Growth of Mouse Plasmacytomas in SCID Mice

Recombinant human T-cell leukemia virus type II (HTLV-II) envelope external glycoprotein, gp46-II, was expressed using a vaccinia virus vector. A recombinant gp46-II fused to an epitope of the influenza virus hemagglutinin, YPYDVPDYA, was purified by immunoaffinity chromatography. The purified glycoprotein was used to immunize Balb/c mice, and antibodies against gp46-II were detected by Western blot analysis and syncytium inhibition assays. We transformed spleen cells from the immunized mice by retroviral infection with ABL-MYC (Ψ2) and intraperitoneally transplanted the infected cells into syngeneic Balb/c and severe combined immunodeficient (SCID) mice. The plasmacytomas established ascitic tumors that produced antibodies directed against HTLV-II gp46-II. Ascites developed more rapidly in SCID mice than in normal syngeneic mice. This procedure provides a general means to generate antibodies rapidly.

Cellular Tropism of Human T-Cell Leukemia Virus Type II Is Enlarged to B Lymphocytes in Patients with High Proviral Load

To establish the in vivo cellular tropism of human T-cell leukemia virus type II (HTLV-II) in peripheral blood, subpopulations of mononuclear cells isolated from patients with a history of drug abuse and with high proviral load were analyzed by polymerase chain reaction for the presence of the proviral sequences. After purification of cellular subsets by immunomagnetic fractionation of blood cells of an infected patient, HTLV-II DNA was detected in CD4 + and CD8 + T-cells as well as in CD19 + B-cells. A positive PCR signal was obtained for purified B-cells also at limiting dilutions. This observation was confirmed by purifying the B-cell fraction by a two-step immunomagnetic procedure from the peripheral blood of another patient with very high HTLV-II copy number and quantifying the B-cell proviral load by means of competitive PCR. A proviral copy number of 90/100 B-cells was found, demonstrating that the great majority of these cells were infected by HTLV-II in this subject. The results indicate that HTLV-II has a broad host range in some infected individuals, showing an enlargement of cellular tropism to B lymphocytes and suggesting that this expansion is associated with an increase in proviral load.

Identification of a novel neutralization epitope on envelope gp46 antigen of human T-cell-leukemia virus-type-II (HTLV-II).

Having observed that multiple neutralization epitopes of human T-cell-leukemia-virus-type-I (HTLV-I) envelope gp46 clustered in a region between amino acids 187 and 199, we speculated that a region of HTLV-II gp46 corresponding to the HTLV-I-neutralization region may contain HTLV-II-specific neutralization epitopes. To test this, we immunized a NZW rabbit and BALB/c mice with a synthetic peptide containing the HTLV-II gp46 amino acids 182 to 199 (pep182-199) conjugated to OVA. The serum from the rabbit reacted to the HTLV-II gp46 and neutralized HTLV-II-mediated syncytium formation. One monoclonal antibody (MAb), M2E186N1, generated from the BALB/c mice, stained specifically the surface of HTLV-II-positive cells and reacted with HTLV-II gp46 by Western blot. This MAb was of the IgA isotype and inhibited HTLV-II- but not HTLV-I-mediated syncytium formation at a final antibody concentration of 200 micrograms/ml; it also neutralized an HTLV-II-VSV pseudotype virus specifically. Epitope mapping by ELISA using overlapping synthetic peptides indicated that the neutralization epitope recognized by the M2E186N1 MAb was located between HTLV-II gp46 amino acids 186 and 192, corresponding to the sequence Leu-Gln-His-Val-Ile-Leu-Gln-Pro. These new observations will be helpful in developing vaccines against HTLV-II infection.

In vivo cellular tropism of human T cell leukemia virus type II (HTLV-II).

To investigate the in vivo cellular tropism of human T cell leukemia virus type II (HTLV-II), subpopulations of fresh peripheral blood mononuclear cells from infected individuals were isolated and analyzed by polymerase chain reaction for the presence of provirus. In eight of nine patients, HTLV-II was detected exclusively in the CD8+ T lymphocyte population. In the remaining patient, provirus was also detected in CD4+ T lymphocytes. Provirus was not detected in B lymphocytes or monocytes of any patient. These results suggest that in vivo HTLV-II has a preferential, and perhaps in some cases, an exclusive tropism for CD8+ T lymphocytes. The findings contrast sharply with those on HTLV-I where there is a preferential tropism for CD4+ T lymphocytes. Although HTLV-II infection has not been consistently associated with any lymphoproliferative disorders, the results suggest that if these occur, they may be different from those known to be associated with HTLV-I.

Human T-cell leukemia virus type II Rex binding and activity require an intact splice donor site and a specific RNA secondary structure

The human T-cell leukemia virus type II (HTLV-II) regulatory protein Rex augments cytoplasmic levels of unspliced gag-pol mRNA by acting through a Rex-responsive element (RxRE) in the long terminal repeat. Purified Rex protein binds to long terminal repeat mRNA. Here, using an immunobinding assay to measure the binding of Rex protein to mutated RxRE RNAs, we show that efficient Rex binding requires a stem-bulge-loop RNA secondary structure (nucleotides [nt] 465 to 500) and specific sequences both within the stem-bulge (nt 470 to 476) and within a conserved upstream splice donor site (nt 449 to 455). Rex function in a transient transfection expression system correlates with Rex protein-RxRE RNA binding. The ability of HTLV-II Rex to interact directly with the HTLV-II splice donor site suggests that HTLV-II Rex may increase expression of unspliced gag-pol mRNA, in part, by inhibiting splicing.

Transformation of CD8 + T‐Cells Producing a Strong Cytopathic Effect on CD4+ T‐Cells through Syncytium Formation by HTLV‐II

Human T‐cell leukemia virus type II (HTLV‐II) is thought to play an important role in the development of CD8 + T‐cell malignancies resembling hairy cell leukemia. In this study, dramatic cytopathic effects characterized by syncytium formation in various CD4+ T‐cell lines were observed upon their cocultivation with HTLV‐II infected T‐cells. The HTLV‐II infected T‐cells, however, did not die as a result of syncytium formation. HTLV‐II also transformed CD4 + T‐cells and CD8 + T‐cells at various coculture ratios. Furthermore, sera from antiHTLV‐II antibody‐positive specific carriers inhibited syncytium formation in the CD4+ T‐cells. These results suggest that HTLV‐II infection may contribute to the pathogenesis of associated CD8 + T‐cell malignancies.

Syndrome of severe skin disease, eosinophilia, and dermatopathic lymphadenopathy in patients with HTLV-II complicating human immunodeficiency virus infection

Two intravenous drug users dually infected with human immunodeficiency virus type 1 (HIV-1) and human T-cell leukemia virus type II (HTLV-II) developed an unusual severe dermatitis characterized by progressive brawny induration, fissuring, and ulceration of the skin, with an associated CD8 cell infiltration in one patient. Both patients had persistent eosinophilia. Lymph node biopsy revealed dermatopathic lymphadenopathy, an unusual pathologic finding in HIV-1 infection but one seen in association with mycosis fungoides and other skin disorders. Two new isolates of HTLV-II virus were established from these patients and were identified as HTLV-II by Southern blotting. This type of skin disease and lymph node pathology has not been found in other intravenous drug users who have been infected with HIV-1 alone or in patients in other risk groups for HIV-1 infection. HTLV-II may play a role in this unique new disease pattern in patients infected with HIV-1.

Identification of a Recombinant HTLV-II Envelope Protein for Serological Detection of HTLV-II Carriers

To understand the pathogenic potential and the true extent of human T-cell leukemia virus type II (HTLV-II) infection, it is important to develop a specific HTLV-II antigen-based serological test. Plasmid pIIB was constructed and induced in Escherichia coli to express a recombinant protein (RP) containing 140 amino acids (amino acid residues 96 to 235) from the middle region of the HTLV-II exterior envelope glycoprotein gp52. Serum samples from polymerase chain reaction-confirmed HTLV-II-infected people, HTLV-I carriers, and adult T-cell leukemia (ATL) patients were tested for antibody reactivity to RP-IIB by Western blot assay. The results showed all 27 HTLV-II carriers, 10 of 20 HTLV-I carriers, and 4 of 17 (23.5%) ATL patients had antibody reactivities to RP-IIB. The difference in rates of seropositivity to RP-IIB between HTLV-II carriers (100%) and HTLV-I-infected people (carriers plus ATL patients) (37.8%) is statistically significant (Fisher's exact test, p = 4.30E-08).

Human T-cell leukemia virus (HTLV) type II Rex protein binds specifically to RNA sequences of the HTLV long terminal repeat but poorly to the human immunodeficiency virus type 1 Rev-responsive element

The human T-cell leukemia viruses (HTLVs) encode a trans-regulatory protein, Rex, which differentially regulates viral gene expression by controlling the cytoplasmic accumulation of viral mRNAs. Because of insufficient amounts of purified protein, biochemical characterization of Rex activity has not previously been performed. Here, utilizing the baculovirus expression system, we purified HTLV type II (HTLV-II) Rex from the cytoplasmic fraction of recombinant baculovirus-infected insect cells by heparin-agarose chromatography. We directly demonstrated that Rex specifically bound HTLV-II 5' long terminal repeat RNA in both gel mobility shift and immunobinding assays. Sequences sufficient for Rex binding were localized to the R-U5 region of the HTLV-II 5' long terminal repeat and correlate with the region required for Rex function. The human immunodeficiency virus type 1 (HIV-1), has an analogous regulatory protein, Rev, which directly binds to and mediates its action through the Rev-responsive element located within the HIV-1 env gene. We demonstrated that HTLV-II Rex rescued an HIV-1JR-CSF Rev-deficient mutant, although inefficiently. This result is consistent with a weak binding activity to the HIV-1 Rev-responsive element under conditions in which it efficiently bound the HTLV-II long terminal repeat RNA.

Regulation of HTLV-II gene expression by rex involves positive and negative cis-acting elements in the 5′ long terminal repeat

Regulation of human T-cell leukemia virus type II (HTLV-II) gene expression by the trans-acting viral protein, Rex, is mediated through specific cis-acting sequences in the HTLV-II long terminal repeat (LTR). Augmentation of 5′ LTR-linked gene expression by Rex requires two distinct cis-acting elements: one termed the “Rex-responsive element” (RxRE), which allows Rex to overcome the inhibitory effect of a second, termed the “ cis-acting repressive sequences” (CRS). The HTLV-II RxRE is located between nt +91 and +317 relative to the cap site in the R/U5 region of the 5′ LTR, and the HTLV-II CRS is contained within the RxRE from nt +208 to +317, which is downstream of the splice donor site, in the R/U5 region of the 5′ LTR. Deletion of the CRS results in significantly increased cytoplasmic levels of LTR-linked mRNA independent of the presence of Rex. Our results show that Rex acts post-transcriptionally and induces a shift from nucleus to cytoplasm of gag/ pol mRNA, the only HTLV-II mRNA that contains both the RxRE and the CRS in the 5′ LTR-derived leader sequence.

Rex transregulation of human T-cell leukemia virus type II gene expression

The Rex protein of the human T-cell leukemia virus type II (HTLV-II), Rex-II, plays a central role in regulating the expression of the structural genes of this retrovirus. Rex-II acts posttranscriptionally by inducing the cytoplasmic expression of the incompletely spliced viral mRNAs that encode the Gag and Env structural proteins and the enzymes derived from the pol gene. We now define a 295-nucleotide cis-acting regulatory element within the 3' long terminal repeat of HTLV-II that is required for the effects of Rex-II. This Rex-II response element (RexIIRE) corresponds to a predicted, highly stable RNA secondary structure and functions when present in the sense but not in the antisense orientation. The RexIIRE confers responsiveness not only to Rex-II but also to the Rex protein of HTLV-I. Deletion and substitution mutagenesis of the RexIIRE permitted identification of a small subregion within the larger element critically required for Rex-II responsiveness and further suggested that the structurally distinct RexIIREs generated from the 5' and 3' long terminal repeats of HTLV-II may differentially regulate the cytoplasmic expression of unspliced gag-pol and singly spliced env mRNAs. While the Rev protein of human immunodeficiency virus type 1 fails to function via the RexIIRE, the Rex-II protein, like Rex-I, can functionally replace the Rev protein of human immunodeficiency virus type 1 via its interaction with the Rev response element (RevRE).

Transformation of Animal Cells with Human T‐Cell Leukemia Virus Type II

Human T‐cell leukemia virus type II (HTLV‐II) was tested for its ability to transform normal animal cells. The HTLV‐II‐infected human T‐cell line, HTLV‐IIA was lethally X‐irradiated and cocultivated with normal leukocytes of rabbit and crab‐eating monkey and spleen cells of hamster. The transformed cell lines, designated Ra‐IIA, Si‐IIA and Ham‐IIA, were established. These cell lines were shown to be infected with HTLV‐II by the polymerase chain reaction method combined with the digoxigenin‐enzyme‐linked immunosorbent assay method. These cell lines were examined for viral antigens by the indirect immunofluorescence method. Although the cytoplasma of over 90% of the cells of Si‐IIA cell line was brilliantly stained, Ra‐IIA and Ham‐IIA cells were not stained. Electron microscopy of cells of the Si‐IIA line revealed C‐type virus particles in the extracellular spaces.