Characterization of human T-cell leukemia virus type I integrase expressed in Escherichia coli

The C-terminal part of the pol gene of the human T-cell leukemia virus type I (HTLV-I) is predicted to encode the integrase (IN) of the virus; however, this protein has not yet been detected in virions or infected cells. We expressed the putative IN from an infectious molecular clone of HTLV-I in Escherichia coli. Comparison with protein resulting from coexpression of HTLV-I protease (PR) and Pol in insect cells indicated that the bacterially expressed protein is identical with or very similar to IN released from a PR-Pol precursor by proteolytic cleavage. HTLV-I IN was purified from E. coli under native conditions. The protein behaved like a dimer in size-exclusion chromatography. It carried out activities characteristic of retroviral IN with high efficiency, displaying a strong preference for U5-derived vs. U3-derived sequences in the processing and strand-transfer reactions. In the disintegration reaction, HTLV-I IN not only accepted the double-stranded branched substrate corresponding to the product of a strand-transfer reaction, but was also able to carry out a phosphoryl transfer on a branched molecule with a single-stranded or a single adenosine overhang.     

Risk factors for adult T-cell leukemia among carriers of human T-lymphotropic virus type I

The presence of circulating "flower cells" and a low prevalence of antibody to Tax regulatory protein of human T-lymphotropic virus type I (HTLV-I) are characteristics of adult T-cell leukemia (ATL). To examine the predictability of levels of HTLV-I antibodies and of flower cell-like abnormal lymphocytes (Ably) for the risk of ATL among asymptomatic HTLV-I carriers, we prospectively evaluated the levels of viral markers of five HTLV-I carriers who developed ATL and 38 age-, sex-, and screen-matched HTLV-I-positive controls in the Miyazaki Cohort Study. After accounting for matching factors, Ably level was slightly, but not significantly, higher among cases than among controls (P =.13). Anti-HTLV-I (odds ratio [OR] = 1.6 per twofold dilution; 95% confidence interval [CI] 0.94, 3.8) was associated with ATL diagnosis, but antibody to Tax regulatory protein (anti-Tax) was not (OR = 0.78; 95% CI 0.26, 1.7). Anti-Tax level was low for all ATL cases for up to 10 years preceding their diagnosis, independent of the level of anti-HTLV-I titer. HTLV-I carriers with a higher anti-HTLV-I titer and a lower anti-Tax reactivity may be at greatest risk of ATL.     

Transmission of human T-cell leukemia virus type 1 to mice

Human T-cell leukemia virus type 1 (HTLV-1) is associated with adult T-cell leukemia/lymphoma, HTLV-1-associated myelopathy/tropical spastic paraparesis, and other diseases. For prevention of the transmission of HTLV-1 and manifestation of these diseases, a small-animal model, especially a mouse model, would be useful. We injected HTLV-1-producing T cells (MT-2) intraperitoneally into neonatal C3H/HeJ mice. While the antibody against HTLV-1 antigens was not detectable in C3H/HeJ mice, HTLV-1 provirus was frequently detected in the spleen, lymph nodes, and thymus by PCR. HTLV-1 provirus was present at the level of 0 to 30 molecules in 10(5) spleen cells at the age of 15 weeks. In addition, a 59-bp flanking sequence of the HTLV-1 integration site was amplified from the spleen DNA by linker-mediated PCR and was confirmed to be derived from the mouse genome. HTLV-1 provirus was found in the T-cell fraction of the mouse spleen. These results indicate that mice can be infected by HTLV-1 and could serve as an animal model for the study of HTLV-1 infection and its pathogenesis in vivo.     

Antinuclear autoantibodies in human T-cell leukemia/lymphoma virus type I carriers in French Guiana

To compare the rate of antinuclear antibodies (ANA) in HTLV-I carriers and negative individuals in French Guiana, 350 sera (175 HTLV-I carriers, either symptomatic or not, and 175 controls) were screened for ANA, using an immunofluorescence assay. All positive sera were tested for autoantibodies against extractable nuclear antigens, histones and double stranded DNA. ANA were detected in 9.71% of the HTLV-I carriers and 3.43% of the control group (p < 0.05). There was no difference in ANA distribution by age, sex, or ethnic group. Neither was there any difference between asymptomatic and symptomatic HTLV-I individuals. However, ANA of medical interest were significantly higher (p < 0.04) in HTLV-I seropositive Creoles than in seropositive Noir-Marrons.     

In vitro infection of human macrophages with human T-cell leukemia virus type 1

The tropism of the human T-cell leukemia virus type 1 (HTLV-1) for the cells of monocyte-macrophage lineage was evaluated by the coculture of blood monocyte-derived macrophages, with irradiated cells of HTLV-1 producing cell lines MT2 or C91/PL. The susceptibility to HTLV-1 was assessed by the detection of viral DNA using the polymerase chain reaction method. HTLV-1 gene expression in the cells was detected using in situ hybridization and by immunofluorescent staining of viral antigen. The presence of type C virus-like particles detected by electron microscopy and the ability to infect normal cord blood lymphocytes demonstrated that the infected macrophages produced infectious virus. These results indicate that human macrophages are susceptible in vitro to productive HTLV-1 infection, and thus might be involved in the pathogenesis of HTLV-1-related diseases.     

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.     

Human immature thymocytes as target cells of the leukemogenic activity of human T-cell leukemia virus type I

The risk of developing adult T-cell leukemia (ATL) associated with neonatal infection by human T-cell leukemia virus type I (HTLV-I) suggests that early events triggered by HTLV-I might be of crucial importance in initiating the multistep lymphoproliferative process leading several decades later to the development of leukemic disease. Thus, infection of thymocytes early in life might be directly correlated with the development of ATL. In the present study, we show that in vitro infection of mature (CD2+CD3+) or immature (CD2+CD3-) thymocytes resulted in the exogenous interleukin (IL)-2-dependent proliferation of HTLV-I-positive thymocytes, most of them displaying a CD2+CD3-CD4+ phenotype and expressing the CD25 molecule, the alpha chain of the IL-2 receptor. Furthermore, the CD80 and CD54 antigens, normally expressed by thymic stromal cells, were detected on these transformed thymocytes, indicating that HTLV-I infection may disturb the cooperation between thymocytes and their thymic environment. These HTLV-I-positive thymocytes were producing significant amounts of IL-6, which was found to be implicated in their proliferation and in the expression of CD25, as demonstrated by blocking experiments using a monoclonal antibody to IL-6. The present study suggests that immature thymocytes may provide an environment favorable to the unfolding of events leading to leukemia.     

Constrained evolution of human immunodeficiency virus type 1 protease during sequential therapy with two distinct protease inhibitors

Human immunodeficiency virus type 1 (HIV-1) variants that have developed protease (PR) inhibitor resistance most often display cross-resistance to several molecules within this class of antiretroviral agents. The clinical benefit of the switch to a second PR inhibitor in the presence of such resistant viruses may be questionable. We have examined the evolution of HIV-1 PR genotypes and phenotypes in individuals having failed sequential treatment with two distinct PR inhibitors: saquinavir (SQV) followed by indinavir (IDV). In viruses where typical SQV resistance mutations were detected before the change to IDV, the corresponding mutations were maintained under IDV, while few additional mutations emerged. In viruses where no SQV resistance mutations were detected before the switch to IDV, typical SQV resistance profiles emerged following the introduction of IDV. We conclude that following suboptimal exposure to a first PR inhibitor, the introduction of a second molecule of this class can lead to rapid selection of cross-resistant virus variants that may not be detectable by current genotyping methods at the time of the inhibitor switch. Viruses committed to resistance to the first inhibitor appear to bear the "imprint" of this initial selection and can further adapt to the selective pressure exerted by the second inhibitor following a pathway that preserves most of the initially selected mutations.     

Cytokine expression and tumorigenicity of large granular lymphocytic leukemia cells from mice transgenic for the tax gene of human T-cell leukemia virus type I

The human T-cell leukemia virus type I (HTLV-I) regulatory protein, Tax, has been speculated to play a major role in HTLV-I leukemogenesis. Indeed, several studies have suggested that upregulation of various cellular oncogenes and cytokines by Tax may explain the pathogenesis observed in HTLV-I-infected individuals, as well as several Tax-transgenic animal models. We report here the analysis of cytokine expression in a Tax-transgenic animal model with large granular lymphocytic (LGL) leukemia. Two different transgenic mice showed identical expression of interleukin-1alpha (IL-1alpha), IL-1beta, interferon gamma (IFNgamma), and granulocyte-macrophage colony-stimulating factor (GM-CSF) in peripheral tail tumors. Interestingly, LGL cell lines derived from these same tumors expressed high levels of both IFNgamma and GM-CSF, which correlated with the level of Tax expression. These same LGL cell lines also expressed high levels of lymphocyte function-associated antigen-1 (LFA-1) and intracellular adhesion molecule-1 (ICAM-1). Engraftment of these LGL cell lines into severe combined immunodeficient (SCID) mice led to the development of leukemia and lymphomas. Examination of these SCID mice showed that their pathology was nearly identical to that observed in the original Tax-transgenic mouse model. Both the Tax-transgenic and engrafted SCID mouse models allow for the analysis of cellular events that are required for tumor development associated with HTLV infection and suggest that Tax expression may be responsible for the upregulation of certain cytokines and adhesion molecules that affect the infiltrating capabilities of HTLV-I-infected cells.     

Seroepidemiology of human T-cell lymphotropic virus type I/II in northeastern Brazil

We investigated the prevalence of human T-lymphotropic virus I/II (HTLV-I/II) infection in Bahia, a state in Northeastern Brazil. Healthy individuals (n = 327) and patients (n = 337) with a variety of diseases were screened for antibodies to HTLV-I/II using an enzyme immunoassay and Western blot. The overall prevalence among healthy subjects was 1.8% (six of 327); among patients it was 18.4% (62 of 337). Patients with AIDS had the highest prevalence of HTLV-I/II infection, 22.7% (20/88), followed by randomly selected patients from an infectious disease hospital, 19.4% (25 of 129), and tuberculosis patients, 11.1% (10 of 90). Four of 14 patients with myelopathy and three of 16 patients with lymphoid leukemia or lymphoma were seropositive for HTLV-I/II. Sixty-three of 68 HTLV-I/II-positive specimens were then typed: 53 patients were HTLV-I positive, three patients were HTLV-II positive, and in seven patients the assay could not distinguish infection by HTLV-I or II. The finding among HIV-seropositive intravenous drug users in Bahia of coinfection with HTLV-I is contrasted with reports from other areas in which dual infection occurs with HTLV-II. Although high prevalence of HTLV-I infection was found in Bahia, the extent and clinical manifestations of HTLV-I/II infection in Brazil remains imprecisely defined, and further studies are needed.     

Development of cervical fat pads following therapy with human immunodeficiency virus type 1 protease inhibitors

Several lines of evidence have suggested a possible involvement of neurotrophic factors in the pathogenesis of neurodegenerative disease. We examined whether a missense mutation (Gly[-63]Glu) of the neurotrophin-3 (NT-3) gene is associated with Alzheimer's disease (AD) in a Japanese sample of 123 patients and 215 controls. We found that homozygotes or heterozygotes for the mutated type (Glu[-63]) were significantly more common among the patients than the controls (P = 0.013, odds ratio 1.77, 95% CI 1.12-2.79). The mutated type was more frequent among the patients than the controls (P = 0.011, odds ratio 1.63, 95%CI 1.11-2.38). This association between NT-3 and AD was more prominent among those who did not carry the epsilon4 allele of the apolipoprotein E gene than those who carried the epsilon4 allele. Our results suggest that the Glu(-63) allele of the NT-3 gene by itself or another mutation nearby which would be in linkage disequilibrium to the mutation is a risk factor for AD.     

Impaired fitness of human immunodeficiency virus type 1 variants with high-level resistance to protease inhibitors

One hope to maintain the benefits of antiviral therapy against the human immunodeficiency virus type 1 (HIV-1), despite the development of resistance, is the possibility that resistant variants will show decreased viral fitness. To study this possibility, HIV-1 variants showing high-level resistance (up to 1,500-fold) to the substrate analog protease inhibitors BILA 1906 BS and BILA 2185 BS have been characterized. Active-site mutations V32I and I84V/A were consistently observed in the protease of highly resistant viruses, along with up to six other mutations. In vitro studies with recombinant mutant proteases demonstrated that these mutations resulted in up to 10(4)-fold increases in the Ki values toward BILA 1906 BS and BILA 2185 BS and a concomitant 2,200-fold decrease in catalytic efficiency of the enzymes toward a synthetic substrate. When introduced into viral molecular clones, the protease mutations impaired polyprotein processing, consistent with a decrease in enzyme activity in virions. Despite these observations, however, most mutations had little effect on viral replication except when the active-site mutations V32I and I84V/A were coexpressed in the protease. The latter combinations not only conferred a significant growth reduction of viral clones on peripheral blood mononuclear cells but also caused the complete disappearance of mutated clones when cocultured with wild-type virus on T-cell lines. Furthermore, the double nucleotide mutation I84A rapidly reverted to I84V upon drug removal, confirming its impact on viral fitness. Therefore, high-level resistance to protease inhibitors can be associated with impaired viral fitness, suggesting that antiviral therapies with such inhibitors may maintain some clinical benefits.     

Inhibition of plating of human T cell leukemia virus type I and syncytium-inducing types of human immunodeficiency virus type 1 by polycations

We examined the effects of polycations, namely, diethylaminoethyl-dextran (DEAE-dextran) and hexadimethrine bromide (Polybrene), on infection with the retroviruses human T cell leukemia virus types I and II (HTLV-I and HTLV-II) and human immunodeficiency virus type 1 (HIV-1). The plating of vesicular stomatitis virus (VSV) pseudotype bearing envelope antigens of HTLV-I [VSV(HTLV-I)] was inhibited about 2- and 10-fold by treatment with DEAE-dextran and Polybrene, respectively. The formation of HTLV-I viral DNA detected 1 day after infection was also inhibited by these polycations. In contrast, polycations enhanced the plating of the VSV (HTLV-II) pseudotype two- to threefold. The polycations did not affect the plating efficiency of HTLV-I or HTLV-II when added after virus adsorption. Infection of human T cell lines, peripheral blood lymphocytes (PBLs), or brain-derived cells with syncytium-inducing (SI) types of HIV-1 strains (GUN1 and IIIB) was inhibited 3- to 20-fold by polycations. However, infection of PBLs or monocyte-derived macrophages with the macrophage-tropic Ba-L or SF162 strain was enhanced 1.5- to twofold by polycations. On the other hand, syncytium formation in coculture induced by HTLV-I, HTLV-II, or HIV-1 was enhanced two- to threefold unanimously by DEAE-dextran or Polybrene. Although polycations have been used to potentiate human retrovirus adsorption, they inhibited infection of cell-free HTLV-I or SI-type HIV-1 strains.     

Novel Gag-Pol frameshift site in human immunodeficiency virus type 1 variants resistant to protease inhibitors

Human immunodeficiency virus type 1 (HIV-1) variants resistant to protease inhibitors have been shown to contain a mutation in the p1/p6 Gag precursor cleavage site. At the messenger RNA level, this mutation generates a U UUU UUU sequence that is reminiscent of the U UUU UUA sequence required for ribosomal frameshifting and Gag-Pol synthesis. To test whether the p1/p6 cleavage site mutation was generating a novel frameshift site, HIV sequences were inserted in translation vectors containing a chloramphenicol acetyltransferase (CAT) reporter gene requiring -1 frameshifting for expression. All sequences containing the original HIV frameshift site supported the synthesis of CAT but expression was increased 3- to 11-fold in the presence of the mutant p1/p6 sequence. When the original frameshift site was abolished by mutation, expression remained unchanged when using constructs containing the mutant p1/p6 sequence, whereas it was decreased 2- to 4.5-fold when using wild-type p1/p6 constructs. Similarly, when introduced into HIV molecular clones, the p1/p6 mutant sequence supported Gag-Pol synthesis and protease activity in the absence of the original frameshift site, indicating that this sequence could also promote ribosomal frameshifting in virus-expressing cells.