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.

HTLV-II down-regulates HIV-1 replication in IL-2-stimulated primary PBMC of coinfected individuals through expression of MIP-1alpha.

The influence of human T-cell leukemia/lymphoma virus type II (HTLV-II) in individuals also infected with HIV-1 is poorly understood. To evaluate the reciprocal influence of HTLV-II and HIV-1 infection, primary peripheral blood mononuclear cell (PBMC) cultures from coinfected individuals were established in the presence of interleukin 2 (IL-2). In these cultures, the kinetics of HTLV-II replication always preceded those of HIV-1. Noteworthy, the kinetics of HIV-1 production were inversely correlated to the HTLV-II proviral load in vivo and its replication ex vivo. These observations suggested a potential interaction between the 2 retroviruses. In this regard, the levels of IL-2, IL-6, and tumor necrosis factor-alpha (TNF-alpha) were measured in the same coinfected PBMC cultures. Endogenous IL-2 was not produced, whereas IL-6 and TNF-alpha were secreted at levels compatible with their known ability to up-regulate HIV-1 expression. The HIV-suppressive CC-chemokines RANTES, macrophage inflammatory protein-1alpha (MIP-1alpha), and MIP-1beta were also determined in IL-2-stimulated PBMC cultures. Of interest, their kinetics and concentrations were inversely related to those of HIV-1 replication. Experiments were performed in which CD8(+) T cells or PBMCs from HTLV-II monoinfected individuals were cocultivated with CD4(+) T cells from HIV-1 monoinfected individuals separated by a semipermeable membrane in the presence or absence of antichemokine neutralizing antibodies. The results indicate that HTLV-II can interfere with the replicative potential of HIV-1 by up-regulating viral suppressive CC-chemokines and, in particular, MIP-1alpha. This study is the first report indicating that HTLV-II can influence HIV replication, at least in vitro, via up-regulation of HIV-suppressive chemokines. (Blood. 2000;95:2760-2769)

Evolutionary strategies of human T-cell lymphotropic virus type II.

Human T-cell lymphotropic virus type II (HTLV-II) primarily infects two different populations in which the virus is transmitted in very diverse ways. In endemically infected populations, the virus is propagated through sexual contact, and by mother to child transmission via breast-feeding, among intravenous drug users (IDUs), spread is mainly due to blood-borne transmission via needle sharing. The phylogeny of HTLV-II strains isolated from American Indian and Pygmy tribes and strains from IDUs, reveal that the virus originated on the African continent as a result of a simian to human transmission at least 400,000 years ago. HTLV-II was very likely introduced into the American continent during one or more migrations of HTLV-II infected Asian populations over the Bering land bridge, some 15,000-35,000 years ago. During the last few decades, HTLV-II has been transmitted from native American Indians to IDUs at least twice, followed by a rapid spread of the virus in the drug users population world-wide due to the practice of needle sharing. Molecular clock analysis showed that HTLV-II has two different evolutionary rates, with the molecular clock for the virus in IDUs ticking 150-350 times faster than the one in endemically infected tribes: 2.7x10(-4) compared to 1.7/7.3x10(-7) nucleotide substitutions per site per year in the LTR region. Although many of the HTLV-II infected drug users are co-infected with HIV, the dramatic acceleration of the evolutionary rate seems to be mainly related to the different modes of transmission in the two populations. These contrasting evolutionary rates correlate with an endemic spread of HTLV-II in infected tribes compared to an epidemic spread in IDUs.

The origin and evolution of human T-cell lymphotropic virus type II (HTLV-II) and the relationship with its replication strategy.

In this review, the origin and evolution of the human T-cell lymphotropic virus type II (HTLV-II) are discussed, with particular emphasis on its high genomic stability. In particular, it appears that the virus originated in the African continent and has been infecting human populations for several thousands of years. The very low divergence accumulated on average between different viral strains during such a long period could be explained by considering that in infected individuals the viral amplification could be due mainly to the clonal expansion of the infected cells, via cellular mitosis, rather than to reverse transcription. HTLV-II was introduced into the American continent during one or more migrations of HTLV-II-infected Asian populations over the Bering land bridge, some 15,000-35,000 years ago. Finally, during the last few decades, HTLV-II has been transmitted from native Amerindians to injecting drug users (IDUs). It might be speculated that at least two separate introductions of HTLV-II in European IDUs from US IDUs have occurred, due to the practice of needle-sharing among IDUs.

Evolutionary rate and genetic heterogeneity of human T-cell lymphotropic virus type II (HTLV-II) using isolates from European injecting drug users.

Seven new Italian and two new British HTLV-II isolates were obtained from injecting drug users and the entire long terminal repeat (LTR) region was sequenced. Restriction analysis showed that all the Italian isolates are of the IIb subtype, whereas the British isolates are of the IIa subtype. To understand whether the further differentiation of each two principal HTLV-II subtypes in several subgroups could be statistically supported by phylogenetic analysis, the neighbor-joining, parsimony, and maximum likelihood methods were used. The separation between IIa and IIb is very well supported by all three methods. At least two phylogenetic subgroups exist within the HTLV-IIa and at least three within the HTLV-IIb subtype. In the present analysis, no statistical support was obtained for additional phylogroups. Two particular subgroups seem interesting because they include all European and North American injecting drug user strains within the IIa and IIb subtypes, respectively. These data confirm that European HTLV-II infection among drug users is probably derived from North America. They also suggest that though a certain differentiation by restriction analysis in different subgroups is possible, carefully interpreted phylogenetic analyses remain necessary. Using the likelihood ratio test, a molecular clock for the drug user strains was calibrated. A fixation rate between 1.08 x 10(-4) and 2.7 x 10(-5) nucleotide substitutions per site per year was calculated for the IIa and IIb injecting drug user strains. This is the lowest fixation rate so far reported for RNA viruses, including for HIV, which typically range between 10(-2) and 10(-4).

Use of a generic polymerase chain reaction assay detecting human T-lymphotropic virus (HTLV) types I, II and divergent simian strains in the evaluation of individuals with indeterminate HTLV serology.

In countries with a low prevalence of human T-lymphotropic virus (HTLV) infection, indeterminate HTLV serologies are a major problem in blood bank screening because of the uncertainties about infection in these cases. The recent discovery of two new types of simian T-lymphotropic viruses (STLV), which give an HTLV-indeterminate serology, raises the question whether indeterminate serologies in humans may be linked to new types of HTLV. Starting from a Tax sequence alignment of all available primate T-cell lymphotropic virus strains (PTLV), including the two new types STLV-PH969 and STLV-PP1664, we developed generic and type-specific nested polymerase chain reactions (PCRs). The generic PCR proved to be highly sensitive and cross-reactive for all four types of PTLV, while the discriminatory PCRs had a high sensitivity and a specificity of 100%. There was no cross-reactivity with human immunodeficiency virus (HIV), ensuring correct interpretation of results from coinfected patients. Among the 77 serologically indeterminate samples tested, 6 were found to be HTLV-1 PCR positive and 1 was HTLV-II PCR positive. Sequencing of one of the HTLV-I PCR positives excluded PCR contamination, and revealed a divergent type of HTLV-I. The majority of the seroindeterminate samples (91%) were however HTLV-PCR negative, and no new types of HTLV were found. This new assay can identify otherwise undetected HTLV-I or HTLV-II infections and is a useful tool of screening for new types of HTLV among seroindeterminate samples.

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.

Complete nucleotide sequence of the Italian human T-cell lymphotropic virus type II isolate Gu and phylogenetic identification of a possible origin of South European epidemics.

The complete nucleotide sequence of a human T-cell lymphotropic virus type II isolate (HTLV-II-Gu) from an Italian injecting drug user was obtained, representing the first entire sequence of a European HTLV-II isolate. The HTLV-II-Gu genome was more similar to the HTLV-IIb-NRA isolate (98.4%) and HTLV-IIb-G12 (98.2%) than to HTLV-IIa-Mo (95.2%). The classification of HTLV-II-Gu as subtype IIb was confirmed by restriction analysis. Just as for HTLV-IIa strain Mo, HTLV-IIb-Gu cultured lymphocytes produce two additional mRNAs generated through alternative splicing in the pX region. A phylogenetic analysis was performed by using the methods of neighbour-joining and parsimony with bootstrapping, and maximum likelihood. The different gene regions were analysed separately, comparing Gu with all other HTLV-II strains presently available. In the LTR, as well as in other genome regions, a clear separation between IIa and IIb was evident, and within the IIb subtype three clusters were present of which two were well supported; one contained exclusively Amerindian strains and the other included all Italian and Spanish strains together with two strains obtained from New York drug users. All data clearly showed that HTLV-IIa and IIb subtypes are closely related and are equidistant from HTLV-I, suggesting that both groups evolved simultaneously. The results suggest that HTLV-II-Gu and other IIb South European isolates were probably derived from North American IIb isolates. The data also indicate that sequence analysis is necessary to further classify IIa and IIb subtypes.

Langerhans cells and HIV infection.

Epidermal Langerhans cells (LCs) isolated from individuals infected with human immunodeficiency virus (HIV-1) harbour HIV-1 proviral DNA and RNA, indicating productive infection by the virus in vivo. Furthermore, normal LCs can be infected in vitro by HIV and can present HIV antigens to helper T cells. Here, Giovanna Zambruno and colleagues discuss the possibility that LCs of genital mucosae are among the first targets of HIV infection following sexual contact, and can be involved both in the transmission of the infection to T cells and in T-cell priming to HIV antigens. In addition, epidermal LCs might acquire HIV infection from dermal T cells during transit from blood vessels through the dermis and may, in turn, represent a reservoir of the virus for continued T-cell infection.

Quantification of HTLV-II proviral copies by competitive polymerase chain reaction in peripheral blood mononuclear cells of Italian injecting drug users, central Africans, and Amerindians.

To better correlate the burden of human T cell leukemia virus type I (HTLV-I) and type II (HTLV-II) infection with diagnostic and prognostic markers, we developed a new competitive polymerase chain reaction (PCR) assay for the quantitative determination of proviral copy numbers in infected cells. A competitive plasmid was constructed that carried a 112-bp fragment from a highly conserved region of the HTLV tax gene and that was further modified by inserting a sequence of 24 bp. This competitive PCR assay system can be used for the quantification of HTLV-I and HTLV-II proviral DNA as demonstrated by using HTLV-I- and HTLV-II-infected cell lines and/or patient material. We determined the HTLV-II proviral load in peripheral blood mononuclear cells (PBMCs) of 11 Italian injecting drug users (IDUs) infected by this virus and in PBMCs of 10 seropositive Amerindian and Central African individuals from endemically infected ethnic groups. A great variation was observed in the number of HTLV-II proviral sequences in the PBMCs of Italian drug abusers, ranging from 5-10 to 16,239 copies/10(5) cells. There was no clear-cut correlation between proviral load, CD8 count, stage of HIV-1 infection, and therapy. A considerable variation in HTLV-II proviral load was also observed in PBMCs of Amerindians and Central Africans with no correlation between the amount of HTLV-II provirus and the geographic origin of the infected individuals.

Identification of IIa and IIb molecular subtypes of human T-cell lymphotropic virus type II among Italian injecting drug users.

The molecular characterization of two human T-cell lymphotropic virus type II (HTLV-II) isolates, Gu and Va, obtained from Italian injecting drug users (IDUs) has indicated that these isolates belong to the HTLV-IIb subtype. To establish whether Italian IDUs are also infected by the HTLV-IIa variant, sequencing of the gp21 env gene of proviral DNA from further patients was carried out. Two new isolates, Bo and Md, were found, which presented a divergence of 0.4-0.7% from the IIa prototype HTLV-II-Mo, thus indicating that they belong to the HTLV-IIa subtype. The results strongly support the existence of two distinct molecular subtypes of HTLV-II infecting Italian IDUs and demonstrate that, although the IIb subtype appears to be prevalent, there is the same variability for this virus in Europe as found in the United States. The Italian IIb isolates were also seen to encode an additional 25 amino acids at the C-terminal end of tax protein, as already shown for other IIb isolates. The identification of two HTLV-II molecular subtypes among Italian drug addicts will be useful in tracing the worldwide distribution of this virus and in further understanding its molecular structure and biology.

Prognostic value of adenosine deaminase compared to other markers for progression to acquired immunodeficiency syndrome among intravenous drug users.

This study was designed to determine the prognostic value of erythrocyte adenosine deaminase (ADA) as a possible indicator of progression to AIDS, and compare this with other known cellular and serological markers. At the end of a 3-year study, a cohort of 114 human immunodeficiency virus-1 (HIV-1) seropositive intravenous drug users (IVDUs) from the five different Center for Disease Control (CDC) groups was examined in order to estimate the prognostic relevance with respect to the progression to acquired immunodeficiency syndrome (AIDS) of each of the following markers at baseline value: number and percentage of CD4+ T cells, number of CD8+ T cells, CD4+/CD8+ ratio, IgA and beta 2 microglobulin and ADA levels, and the presence of HIV antigens. Moreover, 57 IVDUs belonging to II and III CDC groups were analyzed in a follow-up study at 6-month intervals, in order to evaluate and compare the behavior of each marker over time. The prognostic significance of each marker was assessed by computing the survival distribution and the Cox analysis in a multivariate model providing the set of markers with greatest predictive value. The levels of ADA and the CD4+/CD8+ ratio showed a linear association with disease staging, whereas beta 2 microglobulin and CD4+/CD8+ ratio were the best predictors for AIDS progression. A highly significant increase in ADA and beta 2 microglobulin was observed during follow-up. The results obtained among HIV-positive IVDUs clearly indicate that the erythrocyte ADA may be considered a reliable marker of the development of HIV infection from the intermediate stages of the disease onwards.(ABSTRACT TRUNCATED AT 250 WORDS)

Utilization of a DNA enzyme immunoassay for the detection of proviral DNA of human immunodeficiency virus type 1 by polymerase chain reaction.

BACKGROUND: The detection of proviral DNA by Polymerase Chain Reaction (PCR) is regarded as an important tool in the diagnosis of HIV-1 infection, specially among adults at risk of AIDS and children born to seropositive mothers. However, application of PCR in routine testing is hampered by the need to use radioactive probes. OBJECTIVES: In this study, a non-radioactive test based on a microtiter plate (DNA Enzyme ImmunoAssay, DEIA) was used for the detection of proviral sequences of HIV-1 in peripheral blood cells of different patients. The results of the PCR-DEIA assay were compared to those obtained by liquid hybridization (PCR-LH), virus isolation (VI) and Western blot (WB). STUDY DESIGN: The study population included 92 patients belonging to three different groups: seropositive subjects with a well-defined clinical status and WB profile; adults at risk of infection with negative or indeterminate WB; children born to seropositive mothers with still unestablished HIV-1 infection. RESULTS: In the seropositive subjects, both PCR-LH and PCR-DEIA confirmed infection and gave the same results as WB. In adults at risk of infection, PCR with both methods anticipated the seroconversion in one patient with indeterminate WB and confirmed the absence of infection among seronegative and other indeterminate patients. In children born to seropositive mothers, both PCR systems as well as VI permitted an early diagnosis of infection, as confirmed by the clinical follow-up. CONCLUSION: This study has shown that in subjects at risk of AIDS and in children born to seropositive mothers, the non-isotopic DEIA method presents the same sensitivity and specificity for the detection of HIV-1 infection as the radioactive procedure. The DEIA method appears to be particularly useful for the detection of PCR products in routine diagnostic analyses.

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 expression is associated with an increase in proviral load.

Spatial discontinuities in human immunodeficiency virus type 1 quasispecies derived from epidermal Langerhans cells of a patient with AIDS and evidence for double infection.

A nonhomogeneous spatial distribution of human immunodeficiency virus type 1 quasispecies was observed for epidermal Langerhans cells purified from skin patches taken from a patient with AIDS soon after death. Each patch presented a unique collection of sequences, distinct from those of juxtaposed patches or those derived from the other leg. Infection of Langerhans cells by virus from underlying T cells in the dermis might explain this partition. The analysis revealed the presence of two distinct cocirculating viral strains, indicating double infection.

Quantitation by competitive PCR of HIV-1 proviral DNA in epidermal Langerhans cells of HIV-infected patients.

Langerhans cells (LC) belong to the dendritic cell family and represent the principal antigen presenting cells populating squamous epithelia. We have reported the presence of human immunodeficiency virus Type 1 (HIV-1) proviral DNA and RNA in purified LC from the epidermis of seropositive patients. The aim of this study was to quantify HIV-1 proviral DNA in LC of infected patients using a competitive polymerase chain reaction (PCR) assay. Bulk epidermal cell (EC) suspensions were obtained from the skin of nine AIDS patients and six seronegative subjects. Purified LC and LC-depleted EC were prepared by immunomagnetic separation using an anti-CD1a monoclonal antibody. LC preparations did not contain T cells, as assessed by reverse transcription PCR analysis of the T cell receptor beta-chain gene (C region). In addition, no CD14+ cells could be detected in LC fractions by immunostaining of cytospin preparations. To quantify HIV-1 DNA, a new competitive PCR system was devised using SK145/150 as primers (gag) and a competitor plasmid DNA with a modified sequence (209 instead of 142 bp). The number of HIV-1 DNA copies found in the LC of AIDS patients ranged from 107 to 3,645/10(5) LC. In contrast, LC-depleted EC from the same subjects were all negative. The results indicate that in AIDS patients the frequency of infected LC is comparable to that reported for peripheral blood CD4+ T cells.

Differential expression and stability of poly(ADP-ribose)polymerase mRNA in human cells.

The regulation of the expression of the poly(ADP-ribose)polymerase gene was studied in HeLa cells and in quiescent and mitogen-stimulated human lymphocytes by quantitating the mRNA molecules with a new technique based on the polymerase chain reaction. Using plasmid constructs containing defined sequences of the poly(ADP-ribose)polymerase cDNA as internal standards in a competitive PCR reaction, precise measurements of reverse transcribed mRNA copies per microgram of total RNA were obtained. The value found for asynchronously growing HeLa cells (8.6 x 10(5) copies) was very close to that observed for proliferating lymphocytes (8.7 x 10(5)) whereas a 20-fold lower value (0.4 x 10(5)) was obtained for quiescent lymphocytes. The determination of the stability of the mRNA of the enzyme in G0 and stimulated lymphocytes, and in HeLa cells was performed by devising a new PCR amplification system, using non-competitive conditions and plasmid target sequences as internal standards. The half-life of mRNA for poly(ADP-ribose)polymerase was approx. 1 h in G0 lymphocytes and 4-5 h in stimulated lymphocytes and in HeLa cells. This observed difference in stability of the transcripts can partially account for the observed difference in mRNA levels between G0 and stimulated human lymphocytes.

ADP-ribosylation of nonhistone proteins in HeLa cells: modification of DNA topoisomerase II.

We have studied the nonhistone proteins which are modified by ADP-ribosylation in HeLa cells. When isolated nuclei were incubated with 32P-NAD, the main labeled proteins presented sizes of 170, 116, 70, and 45 kDa. To provide evidence for the identification of the 170-kDa band as DNA topoisomerase II, the enzyme was immunoprecipitated from isolated nuclei incubated with 32P-NAD and a labeled peptide of 170 kDa was observed. The label was sensitive to the action of venom phosphodiesterase which specifically degrades ADP-ribose. ADP-ribosylated proteins were also isolated from HeLa cells by affinity chromatography on boronate-agarose gel. Using a monoclonal antibody against the 170-kDa isoform of topoisomerase II, a single 170-kDa immunoreactive peptide was recognized by Western blot among the retained protein acceptors. When ADP-ribosylation was blocked by treating HeLa cells with 3-aminobenzamide, topoisomerase II was no longer retained on the boronate column. These results provide experimental evidence indicating that DNA topoisomerase II is ADP-ribosylated in HeLa cells. To possibly correlate ADP-ribosylation of nuclear proteins with the extent of DNA damage, permeabilized HeLa cells were incubated with 32P-NAD after treatment with the alkylating agent dimethylsulfate. ADP-ribosylated proteins were isolated by boronate chromatography. A strong increase in the ADP-ribosylation of the poly(ADP-ribose)polymerase was observed, whereas no further modification of topoisomerase II was noted.

Direct detection of HIV-1 RNA in epidermal Langerhans cells of HIV-infected patients.

Human Langerhans cells (LC) are bone marrow-derived, HLA-DR+, CD1a+, and CD4+ dendritic antigen-presenting cells found in stratified squamous epithelia. As other members of the dendritic leukocyte family, to which they belong, LC have been reported as targets for HIV-1 infection. The aim of the present study was to investigate whether HIV-1 RNA is expressed in epidermal LC of HIV-1-infected patients. Bulk epidermal cell (EC) suspensions were prepared from skin of nine recently deceased AIDS patients and 11 seronegative controls. Purified LC (94 +/- 4% HLA-DR+ cells with no CD3+ cells, as assessed by flow microfluorimetry analysis) and LC-depleted EC were obtained by immunomagnetic separation using an anti-CD1a monoclonal antibody. Samples were analyzed for the presence of HIV-1 RNA by reverse transcription of a spliced mRNA region of the tat gene, followed by polymerase chain reaction amplification. HIV-1-spliced RNA was detected in LC from 6 of 9 patients examined, whereas LC-depleted EC fractions from the same patients were all negative. The results indicate that epidermal LC from HIV-seropositive patients actively transcribe HIV-1 proviral DNA, further supporting the hypothesis that HIV productively infected LC could serve as a reservoir of the virus in the epidermis and as a source for the infection of T lymphocytes.