NK cells control HIV-1 infection of macrophages through soluble factors and cellular contacts in the human decidua.

BACKGROUND: During the first trimester of pregnancy, HIV-1 in utero transmission is rare despite the permissivity of the placenta and the decidua (the uterine mucosa during pregnancy) to infection. In the decidua from the first trimester of pregnancy, macrophages (dMs) are the HIV-1 main target cells. Decidual natural killer (dNK) cells account for 70 % of decidual leukocytes. They display distinct phenotype and functions compared to peripheral NK cells. At the periphery, NK cells are involved in the control of HIV-1 infection. In this study, we investigate whether human decidual natural killer (dNK) cells control dM HIV-1 infection. RESULTS: Autologous cocultures of infected dMs with dNK cells reveal that dNK cells strongly inhibit dM HIV-1 infection. The addition of dNK cells to dMs at different times after infection suggests that the control occurs before the complete establishment of the infection. Double chamber cocultures show that cellular contacts are necessary for an optimal control of infection. Nevertheless, soluble factors secreted by dMs and dNK cells in double chamber cocultures partially inhibit dM HIV-1 infection, indicating that soluble factors have also a role in the control of infection. IFN-γ secretion is increased in infected and uninfected cocultures. We show that IFN-γ is involved in the control of dM HIV-1 infection by dNK cells. CONCLUSIONS: These results demonstrate that human dNK cells inhibit efficiently HIV-1 infection in dMs in vitro, and highlight the role of innate immune determinants in the control of HIV-1 transmission.

The local environment orchestrates mucosal decidual macrophage differentiation and substantially inhibits HIV-1 replication.

Macrophages from the decidua basalis (dM), the main uterine mucosa during pregnancy, are weakly permissive to HIV-1 infection. Here, we investigated the mechanisms underlying this natural control. We show, by using freshly purified decidual macrophages and ex vivo human decidual explants, that the local decidual environment influences dM differentiation and naturally protects these cells from HIV-1 infection. Interferon (IFN)-γ, present in the decidual tissue, contributes to maintenance of the dM phenotype and restricts HIV-1 infection by mechanisms involving the cyclin-dependent kinase inhibitor p21Cip1/Waf1. We also found that activation of Toll-like receptors 7 and 8 expressed by dM reinforces the low permissivity of dM to HIV-1 by restricting viral replication and inducing secretion of cytokines in the decidual environment, including IFN-γ, that shape dM plasticity. A major challenge for HIV-1 eradication is to control infection of tissue-resident macrophages in the female reproductive tract. Our findings provide clues to the development of novel strategies to prevent HIV-1 macrophage infection.

Characterization of the main placental cytokine profiles from HIV-1-infected pregnant women treated with anti-retroviral drugs in France.

Cytokines are involved in regulating HIV-1 infection. They are also placental environment major components. We assessed the potential impact of HIV-1 infection and/or anti-retroviral drugs on the placental cytokine profiles that may be involved in controlling HIV-1 placental dissemination. Placental explants were obtained after elective caesarean section from anti-retroviral-treated HIV-1-infected pregnant women and from HIV-1 non-infected pregnant women. The main placental cytokines were assessed for protein secretion in the supernatants of 24-h placental culture explants and/or in uncultured placental explants for mRNA expression levels. The cytokine profiles were different between the HIV-1-infected and the non-infected groups. Higher medians of leukaemia inhibiting factor (LIF), tumour necrosis factor (TNF)-alpha and interleukin (IL)-8 secretion were found in the 24-h culture supernatant of term placenta from HIV-1-infected women. High median levels of IL-16 and regulated upon activation normal T cell expressed and secreted (RANTES) levels were found in both groups. The mRNA expression medians were lower for TNF-alpha and IL-8 and higher for stromal cell-derived factor-1 (SDF-1) in uncultured placental explants from HIV-1-infected women. In the HIV-1-infected group, but not in the non-infected group, the secretion levels of TNF-alpha and IL-8, as well as their mRNA expression levels, were highly positively correlated; furthermore, their secretion levels were correlated positively with LIF and IL-10 secretion levels. We found no correlation between the cytokine levels and the immunovirological status of the HIV-1-infected mothers or the type or duration of treatment. These results highlight the potential impact of HIV-1 and of the anti-retroviral treatments on the placental cytokines pattern, independently of their anti-viral activity.

Down modulation of TNF-alpha mRNA placental expression by AZT used for the prevention of HIV-1 mother-to-child transmission.

Mechanisms of HIV-1 in utero mother-to-child transmission (MTCT) protection provided by AZT are not completely understood. The placental cytokine network is involved in the control of HIV-1 in utero transmission but the effect of AZT on this network is unknown. To evaluate the effects of AZT on placental cytokine expression, the chorionic villi from HIV-1 uninfected women term placentae were cultured with 0, 100, and 2,000 ng/ml AZT. Tissue fragments were harvested at days 1, 4, and 7 to determine the level of cytokine mRNA by real-time RT-PCR. The viability and morphology of the placental histocultures were monitored by the expression of beta-human chorionic gonadotropin (beta-hCG) gene, lipopolysaccharide (LPS) activation, and microscopic examination. AZT at 2,000 ng/ml significantly down-regulated TNF-alpha mRNA expression at day 1 and day 4, but had no effect on beta-hCG, stromal cell-derived factor 1 (SDF-1), and IL-10 gene expression. AZT did not induce any deleterious impact on placental tissue structure. Furthermore, activation of chorionic villi by LPS for 24 h up-regulated IL-10 and TNF-alpha mRNA expression. Down-regulation of TNF-alpha mRNA could represent a mechanism through which AZT can decrease the risk of HIV-1 MTCT, in addition to its direct effect on HIV-1 replication.

Cell-free HIV type 1 infection is restricted in the human trophoblast choriocarcinoma BeWo cell line, even with expression of CD4, CXCR4 and CCR5.

The restriction of cell-free HIV-1 infection has been demonstrated in placental trophoblast choriocarcinoma BeWo cells. We tried to determine the level of the viral replication cycle at which this restriction occurs. BeWo cells produce infectious viruses after transfection with HIV-1 plasmids, independently of viral tropism. CCR5 and CXCR4, but not the CD4 molecule, were detected at the cell surface. We therefore derived CD4-expressing clones from transfected BeWo cells. Cell-free virus infection of these clones resulted in neither virus production nor viral sequence integration, indicating that the restriction occurs before integration of the virus. If we used luciferase reporter viruses pseudotyped with HIV-1 Env R5 and X4 for infection, no luciferase activity was detected, even in the BeWo-CD4+ clone, in contrast to what was observed in VSV-G pseudotyped virus infection. Our results show that infection of trophoblast-derived cells with cell-free virus is at least restricted at the level of entry. Thus, BeWo is an interesting human placental cell line that is resistant to HIV-1, even if CD4, CXCR4, and CCR5 are expressed.

Sequence analysis of env C2/V3, gag p17/p24, and pol protease regions of 25 HIV type 1 isolates from Ho Chi Minh City, Vietnam.

Env C2/V3, gag p17/p24, pol protease, and RT regions of HIV-1 isolates recently obtained from 25 HIV-1 seropositive individuals from Ho Chi Minh City (Vietnam) were studied, and genes subtypes were determined by DNA sequence analyses. Twenty-three isolates out of 25 were identified as belonging to subtype E, now recognized as circulating recombinant form 1 (CRF01_AE). The motif at the top of the V3 loop (generally GPGQ) was then preceded by an isoleucine or a methionine (M) residue; the M residue might be a local signature of Vietnamese E isolates compared to Thai E viruses. Two isolates (8%) were shown to be intersubtype recombinants: one E/B and one CRF02_AG(IBNG)/D. The polymorphism of pol protease was considered only for CRF01_AE isolates and is clearly different from that recorded for B viruses with substitutions at positions 13, 35, 36, 41, 69, and 89.

Interleukin-7 and infection itself by human immunodeficiency virus 1 favor virus persistence in mature CD4(+)CD8(-)CD3(+) thymocytes through sustained induction of Bcl-2.

The sequence of events and the mechanisms leading to the destruction of the thymus during human immunodeficiency virus (HIV) infection are still poorly characterized. Investigated here are the survival capacity on HIV-1 infection of the mature single-positive CD4(+)CD8(-)CD3(+) (SP CD4(+)) and the intermediate CD4(+) CD8(-)CD3(-) thymocytes previously shown to be able to replicate the virus in the thymic microenvironment. It is demonstrated that the mature SP CD4(+) thymocytes exhibit a high survival capacity despite the production of a high yield of viruses. Interleukin-7, reported to be a crucial cofactor of tumor necrosis factor (TNF) to promote HIV replication, is shown here to counteract the apoptotic activity of TNF. Resistance to apoptosis of SP CD4(+) cells is conferred by a high expression of the IL-7 receptor (IL-7R) associated with the capacity of IL-7 to permanently up-regulate Bcl-2. In addition, this high Bcl-2 level is further enhanced by infection itself. In contrast, intermediate thymocytes, which replicate the virus at a lower level, are more sensitive to apoptosis, and their differentiation into double-positive CD4(+)CD8(+)CD3(-) (DP CD3(-)) cells strongly increases their death rate on infection. This sensitivity is related to a lower expression of IL-7R and Bcl-2 in intermediate thymocytes, which further decreases at the DP CD3(-) stage. In addition, a decreased level of Bcl-2 is observed in this subset during infection. Altogether these data suggest that in vivo, HIV infection might create a persistent virus reservoir within the SP CD4(+) thymocytes, whereas the later infection of intermediate cells might lead to thymopoiesis failure.

Synthetic peptide strategy for the detection of and discrimination among highly divergent primate lentiviruses.

We developed a simple, rapid, inexpensive, and highly sensitive and specific strategy for the detection and lineage differentiation of primate lentiviruses (PIV-ELISA). It is based on the use of two indirect ELISA methods using synthetic peptides mapping the gp41/36 region (detection component) and the V3 region (differentiation component) of four lentivirus lineages, namely SIVcpz/HIV-1 (groups M, O, N, and SIVcpz-gab), SIVmnd, SIVagm, and SIVsm/SIVmac/HIV-2. This strategy was evaluated with panels of sera originating from both humans and nonhuman primates. The human reference panel consisted of 144 HIV Western blot (WB)-positive sera in which the corresponding virus had been genotyped (HIV-1: 72 group M, 28 group O, and 6 group N; HIV-2: 21 subtype A and 10 subtype B; and 7 HIV-1+2) and 105 HIV WB-negative samples. The nonhuman primate reference panel consisted of 24 sera from monkeys infected by viruses belonging to the four lineages included in the PIV-ELISA strategy (5 chimpanzees, 5 macaques, 8 mandrills, and 6 vervets) and 42 samples from seronegative animals. Additional field evaluation panels consisted of 815 human sera from Gabon, Cameroon, and France and 537 samples from 25 nonhuman primate species. All the samples from the two reference panels were correctly detected and discriminated by PIV-ELISA. In the human field evaluation panel, the gp41/36 component correctly identified all the test samples, with 98% specificity. The V3 component discriminated 206 HIV-1 group M, 98 group O, 12 group M+O, and 128 HIV-2 sera. In the primate field evaluation panel, both gp41/36 and V3 detected and discriminated all the WB-positive samples originating from monkeys infected with SIVcpz, SIVagm-ver, SIVmnd-1, SIVmnd-2, SIVdrl, or SIVsun. These results were confirmed by genotyping in every case. Four SIV-infected red-capped mangabeys (confirmed by PCR) were correctly identified by gp41/36, but only two reacted with the V3 peptides in the absence of a specific SIVrcm V3 peptide. Addition of a V3 SIVrcm peptide discriminated all the SIVrcm-positive samples. Fourteen Papio papio samples were positive for SIVsm gp 36 and by WB, but negative by PCR, whereas three Papio cynocephalus samples were positive by gp41/36 but indeterminate by WB and negative by PCR. This combined ELISA system is thus highly sensitive and specific for antibodies directed against HIV and SIV. In addition, the V3-based serotyping results always agreed with genotyping results. This method should prove useful for studies of lentivirus prevalence and diversity in human and nonhuman primates, and may also have the potential to detect previously undescribed SIVs.

Wild Mandrillus sphinx are carriers of two types of lentivirus.

Mandrillus sphinx, a large primate living in Cameroon and Gabon and belonging to the Papionini tribe, was reported to be infected by a simian immunodeficiency virus (SIV) (SIVmndGB1) as early as 1988. Here, we have identified a second, highly divergent SIVmnd (designated SIVmnd-2). Genomic organization differs between the two viral types; SIVmnd-2 has the additional vpx gene, like other SIVs naturally infecting the Papionini tribe (SIVsm and SIVrcm) and in contrast to the other SIVmnd type (here designated SIVmnd-1), which is more closely related to SIVs infecting l'hoest (Cercopithecus lhoesti lhoesti) and sun-tailed (Cercopithecus lhoesti solatus) monkeys. Importantly, our epidemiological studies indicate a high prevalence of both types of SIVmnd; all 10 sexually mature wild-living monkeys and 3 out of 17 wild-born juveniles tested were infected. The geographic distribution of SIVmnd seems to be distinct for the two types: SIVmnd-1 viruses were exclusively identified in mandrills from central and southern Gabon, whereas SIVmnd-2 viruses were identified in monkeys from northern and western Gabon, as well as in Cameroon. SIVmnd-2 full-length sequence analysis, together with analysis of partial sequences from SIVmnd-1 and SIVmnd-2 from wild-born or wild-living mandrills, shows that the gag and pol regions of SIVmnd-2 are closest to those of SIVrcm, isolated from red-capped mangabeys (Cercocebus torquatus), while the env gene is closest to that of SIVmnd-1. pol and env sequence analyses of SIV from a related Papionini species, the drill (Mandrillus leucophaeus), shows a closer relationship of SIVdrl to SIVmnd-2 than to SIVmnd-1. Epidemiological surveys of human immunodeficiency virus revealed a case in Cameroon of a human infected by a virus serologically related to SIVmnd, raising the possibility that mandrills represent a viral reservoir for humans similar to sooty mangabeys in Western Africa and chimpanzees in Central Africa.

Cell-to-cell contact results in a selective translocation of maternal human immunodeficiency virus type 1 quasispecies across a trophoblastic barrier by both transcytosis and infection.

Mother-to-child transmission can occur in utero, mainly intrapartum and postpartum in case of breastfeeding. In utero transmission is highly restricted and results in selection of viral variant from the mother to the child. We have developed an in vitro system that mimics the interaction between viruses, infected cells present in maternal blood, and the trophoblast, the first barrier protecting the fetus. Trophoblastic BeWo cells were grown as a tight polarized monolayer in a two-chamber system. Cell-free virions applied to the apical pole neither crossed the barrier nor productively infected BeWo cells. In contrast, apical contact with human immunodeficiency virus (HIV)-infected peripheral blood mononuclear cells (PBMCs) resulted in transcytosis of infectious virus across the trophoblastic monolayer and in productive infection correlating with the fusion of HIV-infected PBMCs with trophoblasts. We showed that viral variants are selected during these two steps and that in one case of in utero transmission, the predominant maternal viral variant characterized after transcytosis was phylogenetically indistinguishable from the predominant child's virus. Hence, the first steps of transmission of HIV-1 in utero appear to involve the interaction between HIV type 1-infected cells and the trophoblastic layer, resulting in the passage of infectious HIV by transcytosis and by fusion/infection, both leading to a selection of virus quasispecies.

SIVcpz from a naturally infected Cameroonian chimpanzee: biological and genetic comparison with HIV-1 N.

Thus far, simian immunodeficiency virus from chimpanzees (SIVcpz) genomes have been characterized as Pan troglodytes troglodytes and show a strong relation with human immunodeficiency virus (HIV)-1 N in their env genes. We fully characterized another SIVcpz from P. t. troglodytes. This chimpanzee (Cam5) was, as was also the host of SIVcpz-cam3, wild born in Cameroon, a region where all three groups of HIV-1 (M, N and O) co-occur. In contrast to other SIVcpz, SIVcpz-cam5 was isolated immediately after the rescue of the animal. Our data demonstrate that SIVcpz-cam5, like SIVcpz-cam3, grows easily on human peripheral blood mononuclear cells (PBMCs) and uses CCR5 as a co-receptor similar to HIV-1 N YBF30. Phylogenetic analysis based on the entire env gene shows that SIVcpz-cam5 falls into the same unique subcluster as HIV-1 N YBF30, SIVcpz-cam3 and SIVcpz-US. A phylogenetic relationship was also found with the vif gene of HIV-1 N. This study provides proof that HIV-1 N related viruses circulate in wild P. t. troglodytes.

Evidence of the existence of a new circulating recombinant form of HIV type 1 subtype A/J in Cameroon. The European Network on the Study of In Utero Transmission of HIV-1.

Several genetic subtypes and circulating recombinant forms (CRFs) of HIV-1 have been identified. The greatest degree of genetic diversity is displayed by variants from Central and West Africa. HIV-1 env C2-V5 and protease sequences were obtained from 15 HIV-1-infected pregnant women, who were selected from a larger cohort study in Yaoundé, Cameroon. Fourteen of 15 virus variants were shown to be recombinant, whereas a single variant appeared to be nonrecombinant subtype A. Five viruses were subtype A/J recombinants, with env genes derived from subtype A and protease genes derived from subtype J. Seven viruses clustered with reference sequences for CRF02 AG(IbNG) in both the env and protease gene fragments, and were thus subtype A/G recombinants. Two variants displayed even more complex recombination patterns. Phylogenetic analyses indicated that the five subtype A/J recombinants might be the first representatives of a previously unrecognized CRF.

Generation of CD8+ T cell-generated suppressor factor and beta-chemokines by targeted iliac lymph node immunization in rhesus monkeys challenged with SHIV-89.6P by the rectal route.

The targeted lymph node (TLN) immunization strategy was investigated in macaques, in order to determine the efficacy in generating secretory, systemic, and cellular immune responses, CD8+ T cell-generated suppressor factors, and beta-chemokines. TLN immunization of the rectal and genital mucosa-associated iliac lymph nodes (TILNs) was compared with axillary TLN immunization (TAxLN) using HIV-1 MN/LAI gp140env and SIV p27gag in alum. Significantly higher immune responses, as well as CD8+ T cell-generated anti-SIV factors and the beta-chemokines RANTES, MIP-1alpha, and MIP-1beta, were elicited by iliac as compared with axillary TLN immunization. The immune responses induced by TLN immunization were examined for their capacity to prevent rectal mucosal infection by the pathogenic dual-tropic SHIV-89.6P. Despite significant secretory, serum, cellular, and beta-chemokine responses, the macaques were infected by SHIV-89.6P. Whether the lack of protection was associated with the antigenic unrelatedness of SHIV-89.6P to the immunizing HIV-1 MN/LAI gp140 or to the virus utilizing CXCR4 to a much greater extent than CCR5, remains to be determined.

Clinical and biological characteristics of human immunodeficiency virus-infected and uninfected intravascular drug users in Ho Chi Minh City, Vietnam.

To define the medical characteristics of intravascular drug users in Ho Chi Minh City, Vietnam, we examined 280 men, of whom 235 were infected with human immunodeficiency virus (HIV), being treated in a rehabilitation center. The patients used mainly opium, often in shooting galleries (50%). The prevalence of oral candidiasis (58%) and zoster infection (20%) was high in HIV-seropositive patients, whereas oral hairy leukoplasia and Kaposi's sarcoma were absent. The prevalence of acquired immunodeficiency syndrome was 24%. More than 80% of the patients had infections with hepatitis C virus, hepatitis B virus, cytomegalovirus, or human T cell lymphotropic virus type-1. The CD4+ cell counts correlated well with viral load. Only HIV-1 subtype E was detected in the 30 patients tested. A cohort study of HIV-infected subjects in this population seems feasible, and would permit introduction of anti-retroviral therapy The large number of HIV-seronegative subjects sharing the same at-risk practices as the HIV-infected subjects raises the possibility of natural protection in this population.

Thymocyte-thymic epithelial cell interaction leads to high-level replication of human immunodeficiency virus exclusively in mature CD4(+) CD8(-) CD3(+) thymocytes: a critical role for tumor necrosis factor and interleukin-7.

This work aims at identifying the thymocyte subpopulation able to support human immunodeficiency virus (HIV) replication under the biological stimuli of the thymic microenvironment. In this report we demonstrate that interaction with thymic epithelial cells (TEC) induces a high-level replication of the T-tropic primary isolate HIV-1(B-LAIp) exclusively in the mature CD4(+) CD8(-) CD3(+) thymocytes. Tumor necrosis factor (TNF) and interleukin-7 (IL-7), secreted during this interaction, are critical cytokines for HIV long terminal repeat transactivation through NF-kappaB-dependent activation. TNF is the major inducer of NF-kappaB and particularly of the p50-p65 complex, whereas IL-7 acts as a cofactor by sustaining the expression of the p75 TNF receptor. The requirement for TNF is further confirmed by the observation that the inability of the intermediate CD4(+) CD8(-) CD3(-) thymocytes to replicate the virus is associated with a defect in TNF production during their interaction with TEC and correlates with the absence of nuclear NF-kappaB activity in these freshly isolated thymocytes. Addition of exogenous TNF to the intermediate thymocyte cultures induces NF-kappaB activity and is sufficient to promote HIV replication in the cocultures with TEC. The other major subpopulation expressing the CD4 receptor, namely, the double-positive (DP) CD4(+) CD8(+) CD3(+/-) thymocytes, despite the entry of the virus, do not produce a significant level of virus, presumably because they are unresponsive to TNF and IL-7. Together, these data suggest that in vivo, despite an efficient entry of the virus in all the CD4(+) subpopulations, a high viral load may be generated exclusively within the mature CD4(+) CD8(-) CD3(+) subset of thymocytes. However, under conditions of inflammatory response after infection, TNF might also be present in the intermediate thymocyte compartment, leading to efficient HIV replication in these cells.

Increase of HIV-1 subtype A in Central African Republic.

The concomitant presence of five distinct HIV-1 subtypes and of unclassified HIV-1 was reported in Bangui, Central African Republic (C.A.R.) between 1990 and 1991. This previous study was conducted in individuals belonging to the C.A.R. Armed Forces (FACA) Cohort and in patients from the University Hospital of Baugui. To follow the HIV-1 subtype distribution in Bangui over time, we conducted a cross-sectional surveillance of HIV-1 subtypes between 1987 and 1997 in three groups of individuals in Bangui: 47 men belonging to the FACA Cohort, 38 patients from the CNHUB hospital, and 51 individuals consulting the sexually transmitted diseases (STD) clinic. One hundred and ten HIV-1 were subtyped by heteroduplex mobility assay (HMA) and/or sequencing of env regions encompassing the V3 domain. By comparing the HIV-1 distribution in two time periods (1987-1991 and 1991-1996) in the FACA cohort, we observed a significant increase of subtype A from 43.7% to 83.9%. This subtype distribution does not seem specific to the FACA cohort, in that subtype A accounted for 46.7% of the HIV-1 infections in CNHUB patients in the first time period studied and for 69.6% in the second time period. In STD patients, subtype A infections were predominant in 1995 (72.7%) and 1997 (89.7%). Subtype E viruses could be identified in the second time period, but represented only between 6.5% and 21.8% of the infections in the three groups of individuals studied. Other subtypes (B, C, H) and non-classified HIV-1 in C2-V3 were detected with only a 3.2% to 9.1% frequency for each in the second time period. Phylogenetic analysis excluded infection by a single source for the individuals included in the study. Our data demonstrate an increase in the proportion of HIV-1 subtype A infections in Bangui that raises the question of a preferential transmissibility of specific HIV-1 variants.

High-level replication of human immunodeficiency virus in thymocytes requires NF-kappaB activation through interaction with thymic epithelial cells.

We have previously demonstrated that interaction of infected thymocytes with autologous thymic epithelial cells (TEC) is a prerequisite for a high level of human immunodeficiency virus type 1 (HIV-1) replication in thymocytes (M. Rothe, L. Chêne, M. Nugeyre, F. Barré-Sinoussi, and N. Israël, J. Virol. 72:5852-5861, 1998). We report here that this activation of HIV replication takes place at the transcriptional level through activation of the Rel/NF-kappaB transcription factors. We first demonstrate that an HIV-1 provirus (SF-2 strain) very effectively replicates in thymocytes cocultured with TEC whereas this provirus, with kappaB sites deleted, fails to replicate. We provide evidence that several NF-kappaB complexes are constitutively found in the nuclei of thymocytes either freshly isolated from the thymus or maintained in coculture with autologous or heterologous TEC. The prevalent complex is the heterodimer p50-p65. NF-kappaB activity is tightly correlated with the transcriptional activity of a long terminal repeat (LTR) of HIV-1 transfected in thymocytes. The cotransfection of this LTR with a mutated IkappaBalpha molecule formally demonstrates that LTR transactivation is regulated by members of the Rel/NF-kappaB family in thymocytes. We also showed that tumor necrosis factor (TNF) and to a lesser extent interleukin-1 (IL-1), secreted within the coculture, induce NF-kappaB activity and a correlative LTR transactivation. However IL-7, a crucial factor for thymopoiesis that is secreted mainly by TEC, is a necessary cofactor for NF-kappaB activation elicited by TNF or IL-1. Together, these data indicate that NF-kappaB activation, required for a high level of HIV replication in thymocytes, is regulated in a specific manner in the thymic microenvironment which provides the necessary cytokines: TNF, IL-1, and IL-7.

Human immunodeficiency virus induces a dual regulation of Bcl-2, resulting in persistent infection of CD4(+) T- or monocytic cell lines.

This work aims at characterizing the interplay between human immunodeficiency virus type 1 (HIV-1) and the antiapoptotic cellular protein Bcl-2 responsible for a persistent infection in lymphoblastoid T (J.Jhan) or monocytic (U937) cells. We report that the kinetics of Bcl-2 protein level during the establishment of a chronic infection is biphasic, characterized by a transient decrease followed by restoration to the initial level. The extent and duration of this transient decrease were inversely correlated with the basal level of Bcl-2 as shown by kinetics of Bcl-2 levels in J. Jhan or U937 clones exhibiting different levels of Bcl-2. Using these clones, we also showed that Bcl-2 downregulates HIV-1 replication. Therefore, the cells overexpressing Bcl-2 are characterized by a low viral burden which, in turn, has little effect on the level of this protein. The observed bipasic kinetics is the result of a dual regulation of Bcl-2 induced by HIV-1 infection itself: an upregulation at the transcriptional level of the bcl-2 gene concomitant with a downregulation at the protein level. Convergent data suggest that this downregulation is caused by the oxidative stress induced by the infection itself as shown by the associated modulations of glutathione and thioredoxin levels and by the prevention of these dysregulations by N-acetylcysteine. Altogether, these data indicate that infection first results in a decrease of Bcl-2, permitting an initial boost of replication. Then, as the synthesis at the transcriptional level proceeds, the replication is negatively controlled by Bcl-2 to reach a balance characterized by low virus production and a level of Bcl-2 compatible with cell survival. We suggest that the basal level of Bcl-2, together with infection-inducible transcription factors able to activate bcl-2 gene transcription, is a critical cellular determinant in the tendency toward an acute or a persistent infection.

Contact with thymic epithelial cells as a prerequisite for cytokine-enhanced human immunodeficiency virus type 1 replication in thymocytes.

We report here that human immunodeficiency virus type 1 (HIV-1)-infected human thymocytes, in the absence of any exogenous stimulus but cocultivated with autologous thymic epithelial cells (TEC), obtained shortly (3 days) after thymus excision produce a high and sustained level of HIV-1 particles. The levels and kinetics of HIV-1 replication were similar for seven distinct viral strains irrespective of their phenotypes and genotypes. Contact of thymocytes with TEC is a critical requirement for optimal viral replication. Rather than an inductive signal resulting from the contact itself, soluble factors produced in the mixed culture are responsible for this effect. Specifically, the synergistic effects of tumor necrosis factor, interleukin-1 (IL-1), IL-6, and granulocyte-macrophage colony-stimulating factor may account by themselves for the high level of HIV-1 replication in thymocytes observed in mixed cultures. In conclusion, the microenvironment generated by TEC-thymocyte interaction might greatly favor optimal HIV-1 replication in the thymus.