Studies of heterologous promoter trans-activation by the HTLV-II tax protein.

The tax protein of HTLV-II increases the level of steady-state mRNA produced from the HTLV-II long terminal repeat (LTR) and also activates heterologous promoters. We have previously shown that the adenovirus E3 promoter, which is trans-activated by the adenovirus E1a protein, is also trans-activated by the tax protein. To investigate the mechanism of trans-activation by tax, we analyzed E3 promoter deletion mutants to determine nucleotide sequence requirements for activation of this promoter. Our results show that removal of different upstream regions within the promoter does not result in loss of trans-activation, indicating that tax does not appear to interact with a single DNA binding protein to activate the E3 promoter. In addition, tax and E1a together activate the E3 promoter in a greater than additive fashion, suggesting that these proteins function differently. Possible mechanisms of activation by the tax protein are discussed.     

Mutational analysis of the HTLV-I trans-activator, Tax

The gene coding for the trans-activating factor (Tax) of the human T-cell leukemia virus, type I (HTLV-I) was mutagenized in vitro using oligonucleotide-directed mutagenesis and recombinant DNA techniques. All except one of the mutagenized tax constructs failed to trans-activate the HTLV-I LTR in a eukaryotic test system. Moreover, negative Tax mutant Arg-39----Gly was found to be trans-dominant. This observation suggests that Tax contains distinct functional domains mediating different interactions of the protein in the process of trans-activation.     

Synergism between two distinct elements of the HTLV-I enhancer during activation by the trans-activator of HTLV-I

We have conducted functional studies of the enhancer elements of human T-cell leukemia virus type I (HTLV-I) using the human T-cell lines Jurkat and MOLT 4, which are negative for HTLV-I, and MT-2 and TL-Mor, which carry the proviral genome of HTLV-I. Two distinct elements have been implicated in function of the HTLV-I enhancer. One is the 21-base-pair (bp) core element that is responsible for trans-activation by the HTLV-I trans-activator p40tax and that has the ability to bind to cyclic-AMP responsive element binding factor (CREB)-like factor(s). The other is a region interposed between the 21-bp elements. In this study we demonstrate that a subfragment (C26) in the region between the 21-bp elements is involved in trans-activation by p40tax, possibly through binding to an NF-kappa B-like nuclear factor or factors. Formation of the protein-DNA complex with the C26 subfragment was positively affected by p40tax. The C26 element conferred partial responsiveness to p40tax when linked to one copy of the 21-bp element that, by itself, showed little activation in response to p40tax. However, the C26 element alone, even when repeated, could not be activated by p40tax, unlike other NF-kappa B-binding elements. In contrast, the C26 element itself was profoundly activated upon stimulation with 12-O-tetradecanoylphorbol-13-acetate. These findings therefore suggest that the HTLV-I enhancer contains multiple functional elements, including binding sites for at least CREB- and NF-kappa B-like factors, which synergistically cooperate in activation of the HTLV-I enhancer in response to p40tax. Our results also demonstrate that TPA-dependent activation of the HTLV-I enhancer may be mediated through the C26 element.     

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.     

Complete nucleotide sequence of an Amerindian human T-cell lymphotropic virus type II (HTLV-II) isolate: identification of a variant HTLV-II subtype b from a Guaymi Indian.

The complete nucleotide sequence of a human T-cell lymphotropic virus type II (HTLV-II) isolate from a Panamanian Guaymi Indian was determined and analyzed. When this new viral isolate (HTLV-IIG12) was compared with prototypic HTLV-IIMoT, the overall nucleotide sequence similarity was 95.4%, while the predicted amino acid sequence similarity was 97.5%. Although the overall percentage of nucleotide and amino acid identity with prototypic HTLV-IIMoT (subtype a) was high, HTLV-IIG12 displayed several distinctive features that defined it as an HTLV-II subtype b. However, there were several characteristics unique to this isolate, which included a cluster of nucleotide substitutions in the pre-gag region and changes in restriction enzyme sites within the pre-gag region and the gag, pol, env, and pX genes. In addition, two nucleotide changes in the C terminus of the Tax protein coding sequence inserted an Arg residue for a stop codon and appeared to result in a larger tax gene product in HTLV-IIG12. Although the HTLV-IIG12 isolate appears to be a variant of the prototypic HTLV-IIb, this information represents the first complete nucleotide sequence of any HTLV-II subtype b. These data will allow further studies on the evolutionary relationships between the HTLV-II subtypes and between HTLV-I and HTLV-II.     

Delineation of type-specific regions on the envelope glycoproteins of human T cell leukemia viruses.

Two different approaches were used to map the type-specific regions on human T cell leukemia virus (HTLV) envelope glycoproteins. 1) Antibody reactivities of polymerase chain reaction-confirmed HTLV-I or HTLV-II carriers' sera were analyzed by Western blot assay with seven recombinant proteins containing different regions of HTLV-I or HTLV-II envelope proteins. 2) Rabbit antibodies elicited by nine HTLV-I Env synthetic peptides were used to react with the native HTLV envelope glycoproteins in an antibody-dependent cellular cytotoxicity (ADCC) assay. The results of the Western blot analysis showed that RP-B2, which contains amino acid residues 166 to 213 from HTLV-II exterior glycoprotein, was specifically reactive with 90.6% (48 of 53) of the HTLV-II carriers' sera but not with any of the HTLV-I carriers' serum (0 of 71). In contrast, RP-B, which contains amino acid residues 166 to 229 from HTLV-I exterior glycoprotein, was reactive with 85.1% (114 of 134) of the HTLV-I carriers' sera but not with any HTLV-II carrier serum (0 of 62). Furthermore, anti-HTLV-I Env synthetic peptide antibody-mediated ADCC identified several distinguishing HTLV-I ADCC epitopes in the middle region (amino acid residues 177 to 257) of the HTLV-I exterior glycoprotein. Therefore, HTLV type-specific epitopes reside mainly in a 69-amino acid sequence bounded by two cysteine residues (amino acids 157 and 225 for HTLV-I and 153 and 221 for HTLV-II), in the middle region of the exterior envelope glycoproteins.     

No Proof of HTLV-I/II in Intravenous Drug Abusers with a High Rate of HIV-1 Infection in Central Thailand

Serum specimens of 1,074 intravenous drug abusers (IVDA) were examined for infection with HIV-1, HTLV-I and HTLV-II in central Thailand. Three hundred and sixty-two of the specimens were seropositive for HIV-1 (33.7%). The HIV-1 seropositive IVDAs exhibited increased seropositivity with age through group 40–44 and significantly decreased seropositivity over the age of 45. In contrast, no seropositivity to either HTLV-I or -II was detected in the samples tested by a particle-agglutination assay for HTLV followed by type-specific Western blotting for HTLV. Reference to previous reports suggested that the rate of HIV infection in IVDAs has decreased with no HTLV-I or HTLV-II in Thailand when compared with that of the HIV infection in 1992.     

Expression of the x-lor gene of human T-cell leukemia virus I in Escherichia coli.

The 3'-terminal regions of the human T-cell leukemia virus I (HTLV-I) and HTLV-II genomes encode a novel gene product. We showed that expression of this region fused to the beta-galactosidase gene in bacteria produces a protein recognized by adult T-cell leukemia-lymphoma patient sera. Rabbit antibodies raised against this protein specifically precipitated the 42-kilodalton x-lor gene protein from HTLV-I-infected cells.     

Identification of an 80-kilodalton membrane glycoprotein important for human T-cell leukemia virus type I and type II syncytium formation and infection.

Human T-cell leukemia virus type I and type II (HTLV-I and HTLV-II, respectively) infect certain sublines of the BJAB human B-cell line. We observed that the WH subline, but not the CC/84 subline, of BJAB cells were infectible by cell-free HTLV-I or HTLV-II and formed syncytia with cells infected by these retroviruses. This suggests that the BJAB-CC/84 cells possibly lack a membrane molecule(s) important for syncytium formation and infectibility. In order to identify this antigen, we generated polyclonal anti-BJAB-WH antisera which were adsorbed on BJAB-CC/84 cells. The adsorbed antisera bound only BJAB-WH and BJAB-CC/79 cells as demonstrated by complement-dependent cytotoxicity and flow cytometric assays. Furthermore, this adsorbed antisera bound several human T-cell clones, including SupT-1, as determined by flow cytometric assays. The adsorbed antiserum was monospecific as it immunoprecipitated only one 78- to 80-kDa protein from lysates of metabolically labeled BJAB-WH, BJAB-CC/79, and SupT-1, but not BJAB-CC/84, cells. The monospecific antisera detected a glycoprotein composed of a 64- to 66-kDa core protein containing tunicamycin-sensitive N-linked oligosaccharides. This membrane glycoprotein appears to be involved in HTLV-I- and HTLV-II-induced fusion and infection, as the monospecific antisera were capable of inhibiting both of these processes. The monospecific antisera diluted 1:50 and 1:90 inhibited 85 to 90% of syncytium formation induced in BJAB-WH, BJAB-CC/79, and SupT-1 cells cultured with HTLV-I- or HTLV-II-infected MT2, MoT, or FLW human T- or B-cell lines. At the same dilution, antisera inhibited 70 to 80% of infection of BJAB-WH cells by cell-free HTLV-I or HTLV-II. Thus, these studies indicate a role for a 78- to 80-kDa glycoprotein in HTLV-I or HTLV-II infection and syncytium formation.     

HTLV-II-specific antisera raised in rabbits immunized with a synthetic peptide of HTLV-II envelope protein.

In order to discriminate HTLV-II from HTLV-I, HTLV-II-specific polyclonal antibodies against a synthetic peptide of HTLV-II envelope sequence were raised in rabbits. We immunized two adult rabbits with a KLH-conjugated synthetic peptide corresponding to the amino acid sequence 171-196 of the HTLV-II envelope sequence, which is a specific region for HTLV-II as evaluated with an ELISA method. The resulting rabbit antisera to the synthetic peptide reacted with gp46 of HTLV-II lysates in Western blot analysis but not with that of HTLV-I. Flow cytometric analysis and immunohistochemical study revealed that these affinity purified antisera recognized some HTLV-II-producing cell lines examined, but not HTLV-I-producing cell lines or other cell lines uninfected by HTLV. These findings indicate that these antisera specifically recognized the envelope glycoprotein (gp46) of HTLV-II and suggest the specificity of this region in the immune response to HTLV-II. Such antisera are useful in distinguishing between HTLV-I and HTLV-II infection and in determining the presence of individual HTLV-II-infected cells both in vivo and in vitro, including non-lymphoid cells. They may also assist in the elucidation of the pathogenesis of HTLV-II.     

Genetic heterogeneity in human T-cell leukemia/lymphoma virus type II.

DNA from the peripheral blood mononuclear cells of 17 different individuals infected with human T-cell lymphoma/leukemia virus type II (HTLV-II) was successfully amplified by the polymerase chain reaction (PCR) with the primer pair SK110/SK111. This primer pair is conserved among the pol genes of all primate T-cell lymphoma viruses (PTLV) and flanks a 140-bp fragment of DNA which, when used in comparative analyses, reflects the relative degree of diversity among PTLV genomes. Cloning, sequencing, and phylogenetic comparisons of these amplified 140-bp pol fragments indicated that there are at least two distinct genetic substrains of HTLV-II in the Western Hemisphere. These data were confirmed for selected isolates by performing PCR, cloning, and sequencing with to 10 additional primer pair-probe sets specific for different regions throughout the PTLV genome. HTLV-II isolates from Seminole, Guaymi, and Tobas Indians belong in the new substrain of HTLV-II, while the prototype MoT isolate defines the original substrain. There was greater diversity among HTLV-II New World strains than among HTLV-I New World strains. In fact, the heterogeneity among HTLV-II strains from the Western Hemisphere was similar to that observed in HTLV-I and simian T-cell lymphoma/leukemia virus type I isolates from around the world, including Japan, Africa, and Papua New Guinea. Given these geographic and anthropological considerations and assuming similar mutation rates and selective forces among the PTLV, these data suggest either that HTLV-II has existed for a long time in the indigenous Amerindian population or that HTLV-II isolates introduced into the New World were more heterogeneous than the HTLV-I strains introduced into the New World.