Simian T-cell lymphotropic virus type 1 from Mandrillus sphinx as a simian counterpart of human T-cell lymphotropic virus type 1 subtype D

A recent serological and molecular survey of a semifree-ranging colony of mandrills (Mandrillus sphinx) living in Gabon, central Africa, indicated that 6 of 102 animals, all males, were infected with simian T-cell lymphotropic virus type 1 (STLV-1). These animals naturally live in the same forest area as do human inhabitants (mostly Pygmies) who are infected by the recently described human T-cell lymphotropic virus type 1 (HTLV-1) subtype D. We therefore investigated whether these mandrills were infected with an STLV-1 related to HTLV-1 subtype D. Nucleotide and/or amino acid sequence analyses of complete or partial long terminal repeat (LTR), env, and rex regions showed that HTLV-1 subtype D-specific mutations were found in three of four STLV-1-infected mandrills, while the remaining monkey was infected by a different STLV-1 subtype. Phylogenetic studies conducted on the LTR as well as on the env gp21 region showed that these three new STLV-1 strains from mandrills fall in the same monophyletic clade, supported by high bootstrap values, as do the sequences of HTLV-1 subtype D. These data show, for the first time, the presence of the same subtype of primate T-cell lymphotropic virus type 1 in humans and wild-caught monkeys originating from the same geographical area. This strongly supports the hypothesis that mandrills are the natural reservoir of HTLV-1 subtype D, although the possibility that another monkey species living in the same area could be the original reservoir of both human and mandrill viruses cannot be excluded. Due to the quasi-identity of both human and monkey viruses, interspecies transmission episodes leading to such a clade may have occurred recently.     

Induction of tumor necrosis factor alpha in human neuronal cells by extracellular human T-cell lymphotropic virus type 1 Tax

To examine the role of human T-lymphotropic virus type 1 (HTLV-1) Tax1 in the development of neurological disease, we studied the effects of extracellular Tax1 on gene expression in NT2-N cells, postmitotic cells that share morphologic, phenotypic, and functional features with mature human primary neurons. Treatment with soluble HTLV-1 Tax1 resulted in the induction of tumor necrosis factor alpha (TNF-alpha) gene expression, as detected by reverse-transcribed PCR and by enzyme-linked immunosorbent assay. TNF-alpha induction was completely blocked by clearance with anti-Tax1 monoclonal antibodies. Furthermore, cells treated with either a mock bacterial extract or with lipopolysaccharide produced no detectable TNF-alpha. Synthesis of TNF-alpha in response to soluble Tax1 occurred in a dose-dependent fashion between 0.25 and 75 nM and peaked within 6 h of treatment. Interestingly, culturing NT2-N cells in the presence of soluble Tax1 for as little as 5 min was sufficient to result in TNF-alpha production, indicating that the induction of TNF-alpha in NT2-N does not require Tax1 to be continually present in the culture medium. Treatment of the undifferentiated parental embryonal carcinoma cell line NT2 with soluble Tax1 did not result in TNF-alpha synthesis, suggesting that differentiation-dependent, neuron-specific factors may be required. These results provide the first experimental evidence that neuronal cells are sensitive to HTLV-1 Tax1 as an extracellular cytokine, with a potential role in the pathology of HTLV-1-associated/tropical spastic paraparesis.     

Enhanced human T-cell lymphotropic virus type I expression following induction of the cellular stress response

Human T-cell lymphotropic virus type I (HTLV-I) infection is typically associated with long incubation periods between virus exposure and disease manifestation. Although viral protein expression is considered to play an important role in the pathogenesis of HTLV-I-associated diseases, limited information is known regarding host cell mechanisms that control viral gene expression. This study was designed to evaluate modulation of HTLV-I gene expression following induction of the cellular stress response in HTLV-I-infected lymphocytes. The cellular stress response was elicited by treatment with either Na arsenite or thermal stress and was monitored by demonstrating increased expression of the 72-kDa heat shock protein. Induction of the cellular stress response in HTLV-I-infected lymphocytes resulted in significantly increased HTLV-I-mediated syncytia formation due to enhanced HTLV-I envelope (gp46) expression. Intracellular viral proteins and released p24 capsid protein were increased in stressed infected lymphocytes as compared to nonstressed infected lymphocytes. Furthermore, HTLV-I-LTR reporter gene constructs had increased activity (three- to sixfold) in a transiently transfected, uninfected lymphocyte cell line following induction of the cellular stress response. Quantitation of HTLV-I RNA expression by slot blot analysis of infected lymphocytes suggested variable increases in RNA accumulation. Northern blot analysis demonstrated no qualitative changes in expression of RNA species. These data suggest a relationship between modulation of viral replication and a basic cellular response to stress and have important implications for understanding host cell control mechanisms of HTLV-I expression.     

71-kilodalton heat shock cognate protein acts as a cellular receptor for syncytium formation induced by human T-cell lymphotropic virus type 1

We previously reported that the region corresponding to amino acids 197 to 216 of the gp46 surface glycoprotein (gp46-197) served as a binding domain for the interaction between gp46 and trypsin-sensitive membrane components of the target cell, leading to syncytium formation induced by human T-cell lymphotropic virus type 1 (HTLV-1)-bearing cells. Our new evidence shows that the 71-kDa heat shock cognate protein (HSC70) acts as a cellular receptor for syncytium formation. Using affinity chromatography with the peptide gp46-197, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, we isolated three components (bands A, B, and C) from MOLT-4 cell lysate which exhibited specific interactions with gp46 and inhibitory activities for syncytium formation induced by HTLV-1-bearing cells. Band A and B components were identified as HSC70 and beta-actin, respectively, through amino acid sequencing by tandem mass spectrometry and immunostaining with specific monoclonal antibodies. Band C is likely to be a nonprotein component, because full activity for syncytium formation was seen after extensive trypsin digestion. Anti-HSC70 monoclonal antibody clearly blocked syncytium formation in a coculture of HTLV-1-bearing cells and indicator cells, whereas no inhibition was seen with anti-beta-actin monoclonal antibody. Furthermore, flow cytometric analysis indicated that anti-HSC70 antibody reacted with MOLT-4 cells. Thus, we propose that HSC70 expressed on the target cell surface acts as a cellular acceptor to gp46 exposed on the HTLV-1-infected cell for syncytium formation, thereby leading to cell-to-cell transmission of HTLV-1.     

Diagnostics of persistent viruses: human cytomegalovirus as an example

Infections with persistent viruses such as herpesviruses have become of significant clinical importance with the increasing number of immunocompromised patients at risk to suffer from severe disease. As antiviral chemotherapy is available for herpesvirus infections, the diagnostic methods for rapid and sensitive detection of symptomatic infection have been developed and recently refined. In human cytomegalovirus (HCMV), the use of recombinant viral antigens provides a rationale to improve serological assays. This may be of use for the discrimination of primary versus secondary infection. Early diagnosis of symptomatic HCMV infection in immunosuppressed patients can be most effectively achieved by the detection of a viral tegument protein, pp65, in peripheral blood leukocytes. This early diagnosis has been shown to be of major importance for the effective treatment of these patients. HCMV infection in solid organs can be demonstrated by immunohistochemistry using monoclonal antibodies against viral proteins. HCMV involvement in diseases of the central nervous system in AIDS patients can be verified by the detection of very small amounts of HCMV DNA in cerebrospinal fluid by polymerase chain reaction. This method may prove useful for monitoring HCMV encephalitis and neuropathy.     

A source of glycosylated human T-cell lymphotropic virus type 1 envelope protein: expression of gp46 by the vaccinia virus/T7 polymerase system

Heterologous expression of the human T-cell lymphotropic virus type 1 (HTLV-1) envelope surface glycoprotein (gp46) in a vaccinia virus/T7 polymerase system resulted in the production of authentic recombinant gp46. Five differentially glycosylated forms of the surface envelope protein were produced by this mammalian system, as demonstrated by tunicamycin inhibition of N-glycosylation and N-glycan removal with endoglycosidase H and glycopeptidase F. These studies revealed that all four potential N-glycosylation sites in gp46 were used for oligosaccharide modification and that the oligosaccharides were mannose-rich and/or hybrid in composition. Conformational integrity of the recombinant HTLV-1 envelope protein was determined by the ability to bind to various HTLV-1-infected human sera and a panel of conformational-dependent human monoclonal antibodies under nondenaturing conditions. Furthermore, this recombinant gp46 was recognized by a series of HTLV-2-infected human sera and sera from a Pan paniscus chimpanzee infected with the distantly related simian T-cell lymphotropic virus STLVpan-p. Maintenance of highly conserved conformational epitopes in the recombinant HTLV-1 envelope protein structure suggests that it may serve as a useful diagnostic reagent and an effective vaccine candidate.     

Viral pollution in the environment and in shellfish: human adenovirus detection by PCR as an index of human viruses

A study of the presence of human viruses (adenoviruses, enteroviruses, and hepatitis A viruses [HAVs]) in environmental and shellfish samples was carried out by applying DNA and cDNA amplification techniques by PCR. The detection of human adenoviruses by PCR was also examined as a potential molecular test to monitor viral pollution. The samples studied were urban and slaughterhouse sewage, river water, seawater, and shellfish. Enteroviruses were quantified by PFU in Buffalo green monkey kidney cells and fecal coliforms and phages of Bacteroides fragilis HSP40 were also evaluated in some of the samples. The amplification of viral DNA and cDNA has shown a high prevalence of human viruses that would not be detected by the use of classical techniques, such as the quantification of PFU in cell lines. The results of the analysis of slaughterhouse sewage samples together with the test of farm animal feces indicate that the adenoviruses and the HAVs detected in the environment are mostly of human origin. A significative correlation between the detection of human viruses by PCR and the values of bacteriophages of B. fragilis HSP40 in urban raw sewage was observed. Human adenoviruses were the viruses most frequently detected throughout the year, and all the samples that were positive for enteroviruses or HAVs were also positive for human adenoviruses. The results suggest that the detection of adenoviruses by PCR could be used as an index of the presence of human viruses in the environment where a molecular index is acceptable.     

Immunocytochemical localization of the endogenous neuroexcitotoxin quinolinate in human peripheral blood monocytes/macrophages and the effect of human T-cell lymphotropic virus type I infection

Quinolinate (Quin), a metabolite in the kynurenine pathway of tryptophan degradation and a neurotoxin that appears to act through the N-methyl-D-aspartate receptor system, was localized in cultured human peripheral blood monocytes/macrophages (PBMOs) by using a recently developed immunocytochemical method. Quin immunoreactivity (Quin-IR) was increased in gamma interferon (IFN-gamma)-stimulated monocytes/macrophages (MOs). In addition, the precursors, tryptophan and kynurenine, significantly increased Quin-IR. Infection of MOs by human T-cell lymphotropic virus type I (HTLV-I) in vitro substantially increased both the number of Quin-IR cells and the intensity of Quin-IR. At the peak of the Quin-IR response, about 40% of the cells were Quin-IR positive. In contrast, only about 2-5% of the cells were positive for HTLV-I, as detected by both immunofluorescence for the HTLV-I antigens and PCR techniques for the HTLV-I Tax gene. These results suggest that HTLV-I-induced Quin production in MOs occurs by an indirect mechanism, perhaps via cytokines produced by the infection but not directly by the virus infection per se. The significance of these findings to the neuropathology of HTLV-I infection is discussed.     

Vertical transmission human T-cell lymphotropic virus type-I (HTLV-I)--genetic diagnosis and assessment of the probable routes of HTLV-I infection

Human T-cell lymphotropic virus type-I (HTLV-I) provirus DNA from peripheral lymphocyte of 39 infants delivered by 26 pregnant carriers was detected by the nested double polymerase chain reaction (PCR) method to identify vertical transmission (VT) of HTLV-I. The 39 infants included 12 breast-fed and 27 bottle-fed infants. Particle agglutination (PA) assay and indirect immunofluorescence (IF) test with 467 cells were performed to detect anti-HTLV-I antibody. In breast-fed infants, two (16.7%) cases were both seropositive and PCR-positive and others were both negative, so there was perfect agreement between seropositivity and PCR-positivity. In bottle-fed infants, two (7.4%) cases were seropositive but PCR-negative. This seropositivity was supposed to be due to the transplacental maternal anti-HTLV-I antibody. In 25 seronegative bottle-fed infants, 4 (14.8%) cases were PCR-positive. No significant difference was found in the PCR-positivity rate between the breast-fed and bottle-fed groups. Our study showed the usefulness of the PCR method in identifying VT, the existence of silent carriers especially in bottle-fed infants and the possibility of transplacental or birth canal routes of HTLV-I infection.     

Patterns of prehistoric human mobility in polynesia indicated by mtDNA from the Pacific rat

Human settlement of Polynesia was a major event in world prehistory. Despite the vastness of the distances covered, research suggests that prehistoric Polynesian populations maintained spheres of continuing interaction for at least some period of time in some regions. A low level of genetic variation in ancestral Polynesian populations, genetic admixture (both prehistoric and post-European contact), and severe population crashes resulting from introduction of European diseases make it difficult to trace prehistoric human mobility in the region by using only human genetic and morphological markers. We focus instead on an animal that accompanied the ancestral Polynesians on their voyages. DNA phylogenies derived from mitochondrial control-region sequences of Pacific rats (Rattus exulans) from east Polynesia are presented. A range of specific hypotheses regarding the degree of interaction within Polynesia are tested. These include the issues of multiple contacts between central east Polynesia and the geographically distinct archipelagos of New Zealand and Hawaii. Results are inconsistent with models of Pacific settlement involving substantial isolation after colonization and confirm the value of genetic studies on commensal species for elucidating the history of human settlement.     

Nucleotide sequence and restriction fragment-length polymorphism analysis of human T-cell lymphotropic virus type II (HTLV-II) in southern Europe: evidence for the HTLV-IIa and HTLV-IIb subtypes

Human T-cell lymphotropic virus type II (HTLV-II) has been subtyped into two major groups, IIa and IIb, according to molecular studies involving env gene sequencing. Subsequently, this retrovirus was further subclassified by examining the long terminal repeat (LTR), the most divergent genomic region. Sequence analysis and restriction fragment-length polymorphism (RFLP) applied to the LTR region identified either four or five groups within the IIa subtype (depending on the restriction enzyme sets used) and six within the IIb subtype. In this study, we analyzed the LTR sequences of 29 samples obtained from HTLV-II-infected individuals living in Spain and Italy, which included 24 injecting drug users (IDUs), three blood donors, and two subjects at risk for HIV/HTLV infection. Sequence analysis and phylogenetic analysis of 720 base pairs of the LTR performed in 10 Spanish samples showed that all of these samples belonged to IIb subtype, with a divergence of 7.5% and 1.66% compared with MoT (IIa) and NRA/G12 (IIb) isolates, respectively. RFLP analysis demonstrated the presence of the IIb 4-subtype restriction pattern in 26 samples, a IIb5-subtype pattern in one Italian IDU, and a IIa0-subtype pattern in two Italian samples (blood donors), according to W.M. Switzer's nomenclature. This is the first report of the presence of IIb5 in Southern Europe and IIa0 among Italian blood donors. RFLP correlated with nucleotide sequence and phylogenetic data obtained in this study, demonstrating the ability of the RFLP method to predict the phylogroup of HTLV-II-infected samples.     

Breast milk is not a significant source for early Epstein-Barr virus or human herpesvirus 6 infection in infants: a seroepidemiologic study in 2 endemic areas of human T-cell lymphotropic virus type I in Japan

The relationship between energy expenditure and protein metabolism during amino acid (AA) administration was evaluated in normal humans. A balanced AA solution was infused for 180 min at five different rates: 20 (study I), 40 (study II), 80 (study III), 160 (study IV), and 240 mg.m2(-1).min-1 (study V), on separate days, in seven normal, overnight-fasted subjects (age 25 +/- 2 y; height 172 +/- 5 cm; weight 68 +/- 4 kg). Indirect calorimetry and [1-14C] leucine infusion techniques were employed. Basal total plasma AA concentration averaged 1827 +/- 121 mumol/L and increased to 2192 +/- 142, 2576 +/- 158, 3677 +/- 195, 5638 +/- 237, and 7185 +/- 261 mumol/L in studies I-V, respectively. Basal energy expenditure averaged 0.60 +/- 0.02 kcal.m2(-1).min-1 and increased slightly in studies I and II (to 0.62 +/- 0.03, 0.63 +/- 0.02, respectively), and significantly in studies III-V (to 0.65 +/- 0.03, 0.70 +/- 0.04, and 0.77 +/- 0.05 kcal.m2(-1).min-1, respectively, all P < 0.01 versus basal; P < 0.05-0.01 for each study versus preceding study). Basal nonoxidative leucine disposal (NOLD), an index of protein synthesis, averaged 73 +/- 3 mumol.m2(-1).min-1 and increased, albeit not significantly, in studies I and II (to 75 +/- 5, 76 +/- 4, respectively). In contrast, a significant increase in NOLD was observed in studies III-V (to 87 +/- 7, 103 +/- 7, and 127 +/- 9 mumol.m2(-1).min-1, respectively; all P < 0.01 versus basal; P < 0.05-0.01 for each study versus preceding study). Basal respiratory quotient averaged 0.81 +/- 0.02 and did not change significantly in studies I-V (0.80 +/- 0.02, 0.79 +/- 0.02, 0.80 +/- 0.03, 0.82 +/- 0.02 and 0.82 +/- 0.03, respectively). The thermic effect of AA administration, calculated as percent of the AA energy infused, was constant and averaged 24 +/- 4, 19 +/- 3, 17 +/- 4, 17 +/- 3, and 18 +/- 3% in studies I-V, respectively. When AA-induced increase in protein synthesis was plotted with the increment in energy expenditure, a positive correlation was obtained (r = 0.792, P < 0.001). In summary, during AA administration (1) the absolute rise in energy expenditure is dose-dependent and does not show evidence of achieving a plateau; (2) it is positively correlated with AA-induced protein synthesis; and (3) the thermic effect is not dependent upon the AA dose administered. The data provide a quantitative assessment of AA-induced thermogenesis in normal humans and the energy needs associated with an acute stimulation of protein synthesis.     

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.     

Modification of human T-cell responses by altered peptide ligands: a new approach to antigen-specific modification

Human CD4+ T-cells recognize antigenic peptides in the context of human leukocyte antigen (HLA) class II molecules and produce various lymphokines to proliferate and activate other cells. It was once considered that the T-cell response is an all or nothing type event, but recent studies have clearly indicated that T-cells show many different types of activation in recognition of altered ligands for T-cell receptors (TCR). In this review, we summarize our recent findings on the human CD4+ T-cell response to altered peptide ligands (APL); peptides carrying single residue substitutions in antigenic peptides. We observed the following: 1) TCR antagonism for T-cell clones reactive to non-self or autoantigenic peptides, 2) partial activation (agonism) without cell proliferation, including production of lymphokines and increases in cell size, and in expression levels of several cell surface proteins or survival time in the absence of antigenic stimulus, 3) augmentation in cell proliferation and production of interferon-gamma (IFN-gamma) and granulocyte monocyte colony stimulating factor (GM-CSF), 4) augmentation of interleukin (IL)-12 production by antigen presenting cell (APC) and the subsequent augmented production of IFN-gamma by T-cells. This information provides basic knowledge regarding the characteristics of T-cell recognition of antigens and the subsequent activation, and a novel method for modification of human T-cell responses by altered peptide ligands (APLs), as a possible candidate for antigen-specific immunopotentiating or immunosuppressive therapy against autoimmune diseases, allergies, infectious diseases and malignant tumors.     

Structure-function relationships of human liver cytochromes P450 3A: aflatoxin B1 metabolism as a probe

Cytochromes P450 3A4 and 3A5, the dominant drug-metabolizing enzymes in the human liver, share >85% primary amino acid sequence identity yet exhibit different regioselectivity toward aflatoxin B1 (AFB1) biotransformation [Gillam et al., (1995) Arch. Biochem. Biophys. 317, 374-384]. P450 3A4 apparently prefers AFB1 3alpha-hydroxylation, which results in detoxification and subsequent elimination of the hepatotoxin, over AFB1 exo-8,9-oxidation. In contrast, P450 3A5 is incapable of appreciable AFB1 3alpha-hydroxylation and converts it predominantly to the exo-8,9-oxide which is genotoxic. To elucidate the structural features that govern the regioselectivity of the human liver 3A enzymes in AFB1 metabolism and bioactivation, a combination of approaches including sequence alignment, homology modeling, and site-directed mutagenesis was employed. Specifically, the switch in AFB1 regioselectivity was examined after individual substitution of the divergent amino acids in each of the six putative substrate recognition sites (SRSs) of P450 3A4 with the corresponding amino acid of P450 3A5. Of the P450 3A4 mutants examined, P107S, F108L, N206S, L210F, V376T, S478D, and L479T mutations resulted in a significant switch of P450 3A4 regioselectivity toward that of P450 3A5. The results confirmed the importance of some of these residues in substrate contact in the active site, with residue N206 (SRS-2) being critical for AFB1 detoxification via 3alpha-hydroxylation. Moreover, the P450 3A4 mutant N206S most closely mimicked P450 3A5, not only in its regioselectivity of AFB1 metabolism but also in its overall functional capacity. Furthermore, the other SRS-2 mutant, L210F, also resembled P450 3A5 in its overall AFB1 metabolism and regioselectivity. These findings reveal that a single P450 3A5 SRS domain (SRS-2) is capable of conferring the P450 3A5 phenotype on P450 3A4. In addition, some of these P450 3A4 mutations that affected AFB1 regioselectivity had little influence on testosterone 6beta-hydroxylation, thereby confirming that each substrate-P450 active site fit is indeed unique.