Clonality of HTLV-2 in natural infection.

Human T-lymphotropic virus type 1 (HTLV-1) and type 2 (HTLV-2) both cause lifelong persistent infections, but differ in their clinical outcomes. HTLV-1 infection causes a chronic or acute T-lymphocytic malignancy in up to 5% of infected individuals whereas HTLV-2 has not been unequivocally linked to a T-cell malignancy. Virus-driven clonal proliferation of infected cells both in vitro and in vivo has been demonstrated in HTLV-1 infection. However, T-cell clonality in HTLV-2 infection has not been rigorously characterized. In this study we used a high-throughput approach in conjunction with flow cytometric sorting to identify and quantify HTLV-2-infected T-cell clones in 28 individuals with natural infection. We show that while genome-wide integration site preferences in vivo were similar to those found in HTLV-1 infection, expansion of HTLV-2-infected clones did not demonstrate the same significant association with the genomic environment of the integrated provirus. The proviral load in HTLV-2 is almost confined to CD8+ T-cells and is composed of a small number of often highly expanded clones. The HTLV-2 load correlated significantly with the degree of dispersion of the clone frequency distribution, which was highly stable over ∼8 years. These results suggest that there are significant differences in the selection forces that control the clonal expansion of virus-infected cells in HTLV-1 and HTLV-2 infection. In addition, our data demonstrate that strong virus-driven proliferation per se does not predispose to malignant transformation in oncoretroviral infections.

Alpha interferon restricts human T-lymphotropic virus type 1 and 2 de novo infection through PKR activation.

Type I interferon (IFN-I) inhibits the replication of different viruses. However, the effect of IFN-I on the human T-lymphotropic virus type 1 (HTLV-1) viral cycle is controversial. Here, we investigated the consequences of IFN-α addition for different steps of HTLV-1 and HTLV-2 infection. We first show that alpha interferon (IFN-α) efficiently impairs HTLV-1 and HTLV-2 de novo infection in a T cell line and in primary lymphocytes. Using pseudotyped viruses expressing HTLV-1 envelope, we then show that cell-free infection is insensitive to IFN-α, demonstrating that the cytokine does not affect the early stages of the viral cycle. In contrast, intracellular levels of Gag, Env, or Tax protein are affected by IFN-α treatment in T cells, primary lymphocytes, or 293T cells transfected with HTLV-1 or HTLV-2 molecular clones, demonstrating that IFN-α acts during the late stages of infection. We show that IFN-α does not affect Tax-mediated transcription and acts at a posttranscriptional level. Using either small interfering RNA (siRNA) directed against PKR or a PKR inhibitor, we demonstrate that PKR, whose expression is induced by interferon, plays a major role in IFN-α-induced HTLV-1/2 inhibition. These results indicate that IFN-α has a strong repressive effect on the HTLV-1 and HTLV-2 viral cycle during de novo infection of cells that are natural targets of the viruses.

HTLV-2 Tax immortalizes human CD4+ memory T lymphocytes by oncogenic activation and dysregulation of autophagy.

Human T cell leukemia virus type 1 and type 2 (HTLV-1 and -2) are two closely related retroviruses with the former causing adult T cell leukemia. HTLV-2 infection is prevalent among intravenous drug users, and the viral genome encodes the viral transactivator Tax, which is highly homologous to the transforming protein Tax from HTLV-1. However, the link between HTLV-2 infection and leukemia has not been established. In the present study, we evaluated the activity of HTLV-2 Tax in promoting aberrant proliferation of human CD4 T lymphocytes. Tax2 efficiently immortalized CD4(+) memory T lymphocytes with a CD3/TCRαß/CD4/CD25/CD45RO/CD69 immunophenotype, promoted constitutive activation of PI3K/Akt, IκB kinase/NF-κB, mitogen-activated protein kinase, and STAT3, and it also increased the level of Mcl-1. Disruption of these oncogenic pathways led to growth retardation and apoptotic cell death of the Tax2-established T cell lines. We further found that Tax2 induced autophagy by interacting with the autophagy molecule complex containing Beclin1 and PI3K class III to form the LC3(+) autophagosome. Tax2-mediated autophagy promoted survival and proliferation of the immortalized T cells. The present study demonstrated the oncogenic properties of Tax2 in human T cells and also implicated Tax2 in serving as a molecular tool to generate distinct T cell subtype lines.

Human T-cell leukemia viruses: epidemiology, biology, and pathogenesis.

The human T-cell lymphotropic viruses type I and type II are closely related human retroviruses that have similar biological properties, genetic organization and tropism for T lymphocytes. Along with the simian T-cell lymphoma virus type I, they define the group of retroviruses known as the primate T-cell leukemia/lymphoma viruses. Initially identified in 1980, the human T-cell lymphotropic virus type I has been implicated as the etiologic agent of adult T-cell leukemia/lymphoma and of a degenerative neurologic disorder known as tropical spastic paraparesis or human T-cell lymphotropic virus type I-associated myelopathy. The intriguing link between human T-cell lymphotropic virus type, T-cell malignancy, and a totally unrelated and non-overlapping neurological disorder suggests divergent and unique pathogenetic mechanisms. This review will address the epidemiology, molecular biology, and pathogenesis of human T-cell leukemia viruses.

Serological and molecular evidence of infection by human T-cell lymphotropic virus type II in Italian drug addicts by use of synthetic peptides and polymerase chain reaction.

Infection with human T lymphotropic virus type I (HTLV-I) is associated with specific forms of tumours and neurological disorders, but the pathogenic activity of HTLV-II is not yet established. Moreover, due to high crossreactivity between the two viruses, differential diagnosis is not readily achieved. To discriminate between HTLV-I and HTLV-II infections, we employed synthetic peptides specific for HTLV-I and HTLV-II env regions, and the polymerase chain reaction (PCR). In a series of 962 intravenous drug addicts (IVDAs) and 50 patients with haematological malignancies, 51 and 2 samples, respectively, were reactive against HTLV-I proteins; among these, HTLV-I infection was confirmed only in 1 patient with adult T-cell lymphoma, while HTLV-II infections were identified in 6 out of 14 PCR-tested IVDAs. These findings provide evidence of HTLV-II infection among Italian IVDAs. The differentiation between HTLV-I and HTLV-II infections may contribute to a better understanding of HTLV-II pathogenicity in man.

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.

Unintegrated two-long terminal repeat circular human T lymphotropic virus DNA accumulation during chronic HTLV infection.

Accumulation of unintegrated human T lymphotropic virus (HTLV) DNA was analyzed in long-term T cell lines infected with HTLV type I (HTLV-I) or type II (HTLV-II). By using a polymerase chain reaction-based assay, amplified products of expected size were obtained in all of the HTLV-I-infected (n = 7) and HTLV-II-infected (n = 8) cell lines. The signal intensities of the hybridizing band varied greatly among the cell lines and did not correlate with HTLV p24gag antigen production. Further analysis of HTLV-I-infected clones demonstrated considerable variability in the unintegrated DNA accumulation, suggesting that either the epigenetic status of the host cell or some environmental factor determines the occurrence of unintegrated DNA. The presence of lower levels of unintegrated DNA in most of the HTLV-infected, long-term cell lines presumably results in persistent noncytopathic infection.

Molecular analysis of HTLV-I and HTLV-II isolates from Italian blood donors, intravenous drug users and prisoners.

Using molecular methods three or five major variants of HTLV-I have been identified; moreover two subtypes of HTLV-II defined as HTLV-IIa and HTLV-IIb with six variants within each of these groups have been described. In the present study we analysed proviral DNA obtained from the peripheral blood mononuclear cells (PBMCs) of a significant group of Italian intravenous drug users (IVDUs), prison inmates and blood donors (BDs) who were HTLV antibody positive. Restriction fragment length polymorphism (RFLP) of amplified LTR region with ApaI, NdeI, DraI, SacI and MaeIII endonucleases was used to define the HTLV-I subtypes, while the different variants of HTLV-IIa and -IIb were defined by RFLP of the LTR region with the AvaII, BglI, SauI, XhoI and BanII endonucleases. The four HTLV-I isolated from BDs were characterized as C type. All the 11 HTLV-II detected in the IVDUs were HTLV-IIb4, while among the prisoners one HTLV-IIb5 and five HTLV-IIb4 were found. Interestingly, in the BDs group two HTLV-IIa0 and one HTLV-IIb4 were detected. It should also be noted that 82% of the IVDUs and 50% of the prisoners were coinfected with HIV, while all the BDs were HIV negative. These data indicate that HTLV-IIb4 is the predominant genotype in Italian IVDUs and prisoners, while the significant variability observed in the BD HTLV-II isolates could be due to the different source of infection among this group.

Intracellular localization and cellular factors interaction of HTLV-1 and HTLV-2 Tax proteins: similarities and functional differences.

Human T-lymphotropic viruses type 1 (HTLV-1) and type 2 (HTLV-2) present very similar genomic structures but HTLV-1 is more pathogenic than HTLV-2. Is this difference due to their transactivating Tax proteins, Tax-1 and Tax-2, which are responsible for viral and cellular gene activation? Do Tax-1 and Tax-2 differ in their cellular localization and in their interaction pattern with cellular factors? In this review, we summarize Tax-1 and Tax-2 structural and phenotypic properties, their interaction with factors involved in signal transduction and their localization-related behavior within the cell. Special attention will be given to the distinctions between Tax-1 and Tax-2 that likely play an important role in their transactivation activity.

Postgenomic up-regulation of CCL3L1 expression in HTLV-2-infected persons curtails HIV-1 replication.

Leukocytes of persons coinfected with HTLV-2 and HIV-1 secrete chemokines that prevent CCR5-dependent (R5) HIV-1 infection of CD4+ T cells and macrophages, with HTLV-2-induced MIP-1alpha as dominant HIV-1 inhibitory molecule. Two nonallelic genes code for CCL3 and CCL3L1 isoforms of MIP-1alpha, and the population-specific copy number of CCL3L1 exerts a profound effect on HIV-1 susceptibility and disease progression. Here, we demonstrate that CCL3L1 is secreted spontaneously by leukocytes of HTLV-2-infected persons and superinduced when cells of HTLV-2/HIV-1 multiply exposed-uninfected seronegative (MEU) persons were stimulated with HIV-1 Env peptides. The CCL3L1 median copy number in MEU, HTLV-2/HIV-1-coinfected long-term nonprogressors (LTNPs) and HIV-1-monoinfected LTNPs were 1, 2, and 3, respectively. An increased CCL3L1/CCL3 mRNA ratio versus PHA-activated healthy leukocytes was observed in both HIV-1-monoinfected LTNPs and in HTLV-2/HIV-1(MEU) subjects. An additional potential correlate of HTLV-2 infection was a rapid and persistent leukocyte secretion of GM-CSF and IFN-gamma, 2 cytokines endowed with CCR5 down-regulation capacity. This study confirms a crucial protective role of CCL3L1 from both HIV infection and disease progression, highlighting a previously not described functional up-regulation of this chemokine variant in both HIV-positive and -negative persons infected with HTLV-2.

Neuropilin-1 is involved in human T-cell lymphotropic virus type 1 entry.

Human T-cell lymphotropic virus type 1 (HTLV-1) is transmitted through a viral synapse and enters target cells via interaction with the glucose transporter GLUT1. Here, we show that Neuropilin-1 (NRP1), the receptor for semaphorin-3A and VEGF-A165 and a member of the immune synapse, is also a physical and functional partner of HTLV-1 envelope (Env) proteins. HTLV-1 Env and NRP1 complexes are formed in cotransfected cells, and endogenous NRP1 contributes to the binding of HTLV-1 Env to target cells. NRP1 overexpression increases HTLV-1 Env-dependent syncytium formation. Moreover, overexpression of NRP1 increases both HTLV-1 and HTLV-2 Env-dependent infection, whereas down-regulation of endogenous NRP1 has the opposite effect. Finally, overexpressed GLUT1, NRP1, and Env form ternary complexes in transfected cells, and endogenous NRP1 and GLUT1 colocalize in membrane junctions formed between uninfected and HTLV-1-infected T cells. These data show that NRP1 is involved in HTLV-1 and HTLV-2 entry, suggesting that the HTLV receptor has a multicomponent nature.

Comparative performances of an HTLV-I/II EIA and other serologic and PCR assays on samples from persons at risk for HTLV-II infection.

BACKGROUND: HTLV-I and HTLV-II are related exogenous pathogenic human retroviruses. Until recently, ELISAs based on HTLV-I antigens have been used to screen for the presence of HTLV-I or -II antibodies. The HTLV-I-based assays have not been as sensitive in detecting antibodies to HTLV-II as in detecting antibodies to HTLV-I. The Abbott HTLV-I/HTLV-II ELISA uses a combination of HTLV-I and HTLV-II antigens to detect antibodies to the whole HTLV group. The performance of this ELISA was compared to that of several HTLV-I-based serologic assays and an HTLV-II PCR assay in cohorts of South American Indians and New York City IV drug users (IVDUs) in whom HTLV-II is endemic. STUDY DESIGN AND METHODS: Sera from 429 South American Indians and New York City IVDUs were evaluated for HTLV antibodies by the use of three ELISAs (rgp21-enhanced HTLV-I/II, Cambridge Biotech; Vironostika HTLV-I/II, Organon Teknika; and HTLV-I/HTLV-II, Abbott), and a Western blot (WB) assay. Peripheral blood leukocyte DNA from each person was analyzed for HTLV-I and HTLV-II pol DNA via PCR. The HTLV-II PCR-positive samples were further subtyped via cloning and sequencing and/or oligomer restriction. RESULTS: Two hundred four samples (48%) were positive for HTLV-II by serologic and/or PCR assays. All of the positive samples from the Indians and approximately one-third of the positive samples from the IVDUs were of the HTLV-IIB subtype. Comparative analyses indicate that the sensitivity and specificity of the various assays were: PCR, 98 and 100 percent; Abbott HTLV-I/HTLV-II, 78 and 95 percent; Cambridge Biotech HTLV-I/II, 76 and 96 percent; Vironostika HTLV-I/II, 71 and 98 percent; and WB, 73 and 100 percent, respectively. CONCLUSION: There were no significant differences among the sensitivities and specificities of the HTLV-I/II ELISAs (p values, 0.056-0.438). The WB and PCR assays were much more specific than the other serologic assays (p

Role of the genetic background of rats in infection by HTLV-I and HTLV-II and in the development of associated diseases.

Three aspects of the rat model of HTLV-I/II infection were investigated. (i) The efficacy of HTLV-I-transformed rat cell lines in infecting different strains of rats: WKY and Lewis HTLV-I-transformed cell lines were injected into adult WKY, Lewis and Brown Norway rats, representing syngeneic and allogeneic combinations. The HTLV-I provirus was not detected in peripheral-blood mononuclear cells (PBMC) from these rats 18 weeks after inoculation, showing that HTLV-I-transformed rat cells are not suitable for virus challenge in vaccination experiments. Rats inoculated with Lewis HTLV-I-transformed cells produced an antibody response to HTLV-I, which was higher in allogeneic (WKY and Brown Norway) than in syngeneic rats. (ii) The susceptibility of rats to HTLV-II infection: After human HTLV-II-producing cells (MO) were injected into adult WKY rats, the HTLV-II provirus was detected in PBMC 12 weeks later. Sequencing of a portion of this provirus confirmed its identity with the HTLV-II from MO cells. (iii) The role of MHC haplotype in susceptibility to neurological disease in rats inoculated as newborns with HTLV-I: The hypothesis that the RT-Ik haplotype confers susceptibility was tested by inoculating newborn OKA (RT-Ik), WKY (RT-Il), Lewis (RT-Il) and Fischer 344 (RT-I lvl) rats with human HTLV-I-producing cells (MT-2). Eighteen months later, only the WKY rats showed histological abnormality of the spinal cord, without clinical paralysis. Fischer 344 rats developed cutaneous tumors and OKA rats mammary tumors. The HTLV-I provirus was not detected in these tumors.

HTLV-II risk factors in Native Americans in Florida.

Earlier virologic studies established that human T-cell lymphotropic virus type II (HTLV-II) is the predominant retrovirus type found among Seminole Indians in southern Florida. We studied 46 members of the Seminole tribe living on 3 reservations to determine the risk factors for HTLV-II and to investigate disease association with the virus. The donors' plasma samples were evaluated with the enzyme-linked immunosorbent and Western blot assays. DNA extracted from their peripheral blood mononuclear cells were analyzed by type-specific polymerase chain reaction amplification and detection of the HTLV pol gene using the primer pair SK110/SK111, and the probes SK112 or SK188. One of 46 (2%) subjects was identified as HTLV-I positive and 11 (24%) were identified as HTLV-II positive. Restriction fragment length polymorphism and sequence analyses indicated that all of the HTLV-II strains were subtype b. Mitochondrial DNA analyses indicated that all of the HTLV-II-positive subjects had an Amerindian haplotype. HTLV-II was more prevalent in Indians who were >45 years of age or female, had multiple sex partners or had received a blood transfusion. However, only the latter risk factor was statistically significant. Three of the HTLV-II-positive Indians demonstrated signs and symptoms of an ataxic neuropathy. The data support that HTLV-IIb is endemic among the Seminoles and that they will be a key population for further virologic studies.

Type-specific antigens for serological discrimination of HTLV-I and HTLV-II infection.

55 HTLV-I (human T-cell lymphotropic virus) and 45 HTLV-II carriers, confirmed by HTLV-type specific polymerase chain reaction (PCR), were distinguished by western blot assays with recombinant HTLV I or II envelope glycoproteins. Recombinant protein (RP) B1 contains aminoacids 166-201 from HTLV-I exterior glycoprotein gp46 and was reactive with HTLV-I samples only. RP-IIB, which contains aminoacids 96-235 from HTLV-II exterior glycoprotein gp52, was reactive with all HTLV-II samples. 39 patients (86.6%) had high reactivity by densitometry. Of 55 HTLV-I samples, 35 (65.5%) had antibody reactivity to RP-IIB, but only 1 (1.8%) had high reactivity by densitometry. RP B1 and IIB western blot assays may replace the PCR test in diagnosis of HTLV infection.

High rate of infection with the human T-cell leukemia retrovirus type II in four Indian populations of Argentina.

Sera from 215 non-drug-injecting Toba and Mataco-Mataguayo pure Indians belonging to four communities in northern Argentina were examined using assays that allow differentiation between reactivities due to type-specific antigens of the human T-cell leukemia/lymphoma virus (HTLV). Three of these populations have very little contact with non-Indian groups and reside in remote, isolated areas. HTLV-II type-specific seroreactivity was present in 24 (13.7%) of the 175 Indians older than 13 years of age and in none of the 40 who were of younger ages. None of the Indians had antibodies reacting with HTLV-I type-specific antigen. Seroreactivity was more prevalent and appeared at younger ages in females than in males. The majority of the HTLV-II-seropositive Indians belonged to the more isolated communities. The seroprevalences among the Tobas and Mataco-Mataguayo Indians were comparable. With the exception of a Toba who was positive in a test for Treponema pallidum, no serological evidence of sexually transmitted infections with this spirochete, hepatitis B virus, hepatitis C virus, and human immunodeficiency virus was found among the Indians tested. None of the 55 non-Indian people tested in the region showed HTLV-II type-specific seroreactivity. PCR analysis of DNA isolated from peripheral blood lymphocytes of seropositive Indians confirmed that the virus present in these populations is HTLV-II. Sequence analysis of PCR-amplified genomic segments showed that the virus belongs to the HTLV-II subtype which has been found to be endemic in other Paleo-American Indians.

Detection of human T lymphotropic virus type II/b in human immunodeficiency virus type 1-coinfected persons in southeastern Italy.

A preliminary screening of 511 persons at risk for AIDS living in southeastern Italy disclosed 20 cases of seroreactivity to human T lymphotropic viruses (HTLV). To verify and type the HTLV infection among these subjects, confirmatory serologic tests, polymerase chain reaction (PCR), and virus culture were done. No evidence of HTLV-I infection was found. HTLV-II infection was confirmed in 8 cases by HTLV-specific, synthetic peptide EIAs and PCR on uncultured cells; restriction analysis of the PCR-amplified env regions revealed the presence of HTLV-II/b strains in all 8 cases. Four sera were nontypeable by EIA. The finding of such indeterminate reactivities in a geographic area in which HTLV variants were previously described indicates the need for more extensive surveys among the healthy population. HTLV-II was isolated in 5 cases, and virus isolation was mostly dependent on the presence of an actively replicating human immunodeficiency virus type 1 in culture.

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.