Exogenous interleukin-2 administration corrects the cell cycle perturbation of lymphocytes from human immunodeficiency virus-infected individuals.

Human immunodeficiency virus (HIV)-induced immunodeficiency is characterized by progressive loss of CD4(+) T cells associated with functional abnormalities of the surviving lymphocytes. Increased susceptibility to apoptosis and loss of proper cell cycle control can be observed in lymphocytes from HIV-infected individuals and may contribute to the lymphocyte dysfunction of AIDS patients. To better understand the relation between T-cell activation, apoptosis, and cell cycle perturbation, we studied the effect of exogenous interleukin-2 (IL-2) administration on the intracellular turnover of phase-dependent proteins. Circulating T cells from HIV-infected patients display a marked discrepancy between a metabolic profile typical of G(0) and a pattern of expression of phase-dependent proteins that indicates a more-advanced position within the cell cycle. This discrepancy is enhanced by in vitro activation with ConA and ultimately results in a marked increase of apoptotic events. Conversely, treatment of lymphocytes with IL-2 alone restores the phase-specific pattern of expression of cell cycle-dependent proteins and is associated with low levels of apoptosis. Interestingly, exogenous IL-2 administration normalizes the overall intracellular protein turnover, as measured by protein synthesis, half-life of newly synthesised proteins, and total protein ubiquitination, thus providing a possible explanation for the effect of IL-2 on the intracellular kinetics of cell cycle-dependent proteins. The beneficial effect of IL-2 administration is consistent with the possibility of defective IL-2 function in vivo, which is confirmed by the observation that lymphocytes from HIV-infected patients show abnormal endogenous IL-2 paracrine/autocrine function upon in vitro mitogen stimulation. Overall these results confirm that perturbation of cell cycle control contributes to HIV-related lymphocyte dysfunction and, by showing that IL-2 administration can revert this perturbation, suggest a new mechanism of action of IL-2 therapy in HIV-infected patients.

Lentivirus-mediated gene transfer in primary T cells is enhanced by a central DNA flap.

Retroviral vectors have become the primary tool for gene delivery into hematopoietic cells, including T lymphocytes. Lentiviral vectors offer an advantage over Moloney murine leukemia virus (MuLV) vectors because of their ability to translocate across an intact nuclear membrane and integrate into the genome of nonproliferating cells. We have recently demonstrated that a central strand displacement event, controlled by the central polypurine tract (cPPT) and the central termination sequence (CTS), results in the formation of a central DNA flap which acts as a cis-determinant of HIV-1 genome nuclear import. Here, we show that insertion of this DNA determinant in a classical lentiviral vector resulted in a significantly higher level of transduction in activated T cells (51 +/- 12.7% versus 15 +/- 1.4%). CD4(+) and CD8(+) T cells were transduced at equivalent levels. Importantly, freshly isolated T cells stimulated only during the 12-h transduction period could be efficiently transduced with this new flap-containing lentiviral vector, but not with the parental lentiviral vector nor an MuLV vector. Transgene expression in the flap-containing lentiviral vector, under the control of either an internal cytomegalovirus or the elongation factor-1 alpha (EF1 alpha) promoter, was significant and expression remained elevated in resting T cells. Thus, this system allows stable expression of transgenes in T lymphocytes following a short ex vivo transduction protocol.

Enhanced transgene expression in cord blood CD34(+)-derived hematopoietic cells, including developing T cells and NOD/SCID mouse repopulating cells, following transduction with modified trip lentiviral vectors.

The recent development of lentivirus-derived vectors is an important breakthrough in gene transfer technology because these vectors allow transduction of nondividing cells such as hematopoietic stem cells (HSC), due to an active nuclear import of reverse-transcribed vector DNA. We recently demonstrated that addition of the central DNA flap of HIV-1 to an HIV-derived lentiviral vector strikingly increases transduction of CD34(+) cells. We now describe improvements of the transduction protocol designed to preserve HSC properties and two modifications of the previously described TRIP-CMV vector. First, deletion of the enhancer/promoter of the 3' LTR in the TRIP-CMV vector resulted in a safer vector (TRIPDeltaU3-CMV) with conserved transduction efficiency and increased EGFP transgene expression. Second, the original internal CMV promoter was replaced with the promoter for the ubiquitously expressed elongation factor 1alpha (EF1alpha). This promoter substitution resulted in a significantly more homogeneous expression of the EGFP transgene in all hematopoietic cell types, including CD34(+)-derived T lymphocytes, in which the CMV promoter was inactive, and NOD/SCID mouse repopulating cells. We thus present here an HIV-derived lentiviral vector, TRIPDeltaU3-EF1alpha, which can very efficiently transduce human cord blood HSC and results in high long-term transgene expression in CD34(+)-derived T, B, NK, and myeloid hematopoietic cells.

Abnormal intracellular kinetics of cell-cycle-dependent proteins in lymphocytes from patients infected with human immunodeficiency virus: a novel biologic link between immune activation, accelerated T-cell turnover, and high levels of apoptosis.

Human immunodeficiency virus (HIV)-infection is characterized by loss of CD4+ T cells associated with high levels of immune activation, T-cell proliferation, and lymphocyte apoptosis. To investigate the role of intrinsic perturbations of cell-cycle control in the immunopathogenesis of acquired immunodeficiency syndrome (AIDS), we studied the expression of cell-cycle-dependent proteins in lymphocytes from HIV-infected patients. Cyclin B1 expression, Nucleolar Organizer Regions (NORs) number, and NORs area of distribution were all consistently increased in HIV-infected patients, but returned to normal after effective antiretroviral therapy, suggesting that viral replication is directly implicated in the genesis of the observed changes. Analysis of cyclin B1 intracellular turnover showed that the increased cyclin B1 expression is (1) caused by defective degradation in the presence of normal rates of synthesis, and (2) is temporally associated with decreased levels of ubiquitination. After in vitro activation of lymphocytes from healthy individuals, cyclin B1 and cdc25 expression and ubiquitination, p34 cdc2 activity, NORs morphology, and C23/nucleolin localization showed a 72- to 96-hour cyclic pattern that led to a biologic state similar to baseline. On the contrary, complex but consistent changes of the same indices followed activation of T lymphocytes from HIV-infected patients, resulting in a 5-fold increase in apoptosis. Overall, our data indicate that a profound dysregulation of cell-cycle control is present in lymphocytes from HIV-infected patients. This finding may provide a novel biologic link between immune activation, accelerated lymphocyte turnover, and increased apoptosis during HIV infection.

Effect of alpha-1 antitrypsin Portland variant (alpha 1-PDX) on HIV-1 replication.

The present work investigated the potential role of alpha-1 antitrypsin Portland variant (alpha 1-PDX), a bioengineered serine proteinase inhibitor (serpin), in the interference with the viral replication of HIV-1, induction of syncytia and maturation of envelope glycoprotein gp160 to gp120 and gp41. A Jurkat lymphoid cell line transfected with a plasmid containing the alpha 1-PDX cDNA (J-PDX) and expressing the protein in a stable manner was infected with HIV-1(Lai). Controls were Jurkat cells transfected with the same vector pcDNA3 without the cDNA insert (J-pcDNA3). The results showed that viral replication of HIV-1 was significantly inhibited with a delay in replication kinetics in J-PDX cells as compared with J-pcDNA3 cells. In addition, a comparison of the infectious capacity of viruses produced in the presence and absence of alpha 1-PDX revealed that this capacity differed. It was found that alpha 1-PDX exerts its effect by interfering with the formation of syncytia between J-PDX cells infected with gp160 recombinant vaccinia virus, or after infection by HIV-1 and co-culture with uninfected Molt-4 cells. In contrast, when the same experiments were performed with J-pcDNA3 cells, a large number of syncytia was obtained. Analysis of viral proteins by Western blotting and densitometry showed that the inhibition of the cytopathic effect of HIV-1 and viral replication was correlated with the capacity of alpha 1-PDX to interfere with the maturation of gp160 to gp120 and gp41.

HIV-1 genome nuclear import is mediated by a central DNA flap.

HIV-1 and other lentiviruses have the unique property among retroviruses to replicate in nondividing cells. This property relies on the use of a nuclear import pathway enabling the viral DNA to cross the nuclear membrane of the host cell. In HIV-1 reverse transcription, a central strand displacement event consecutive to central initiation and termination of plus strand synthesis creates a plus strand overlap: the central DNA flap. We show here that the central DNA flap acts as a cis-determinant of HIV-1 DNA nuclear import. Wild-type viral linear DNA is almost entirely imported into the nucleus where it integrates or circularizes. In contrast, mutant viral DNA, which lacks the DNA flap, accumulates in infected cells as unintegrated linear DNA, at the vicinity of the nuclear membrane. Consistently, HIV-1 vectors devoid of DNA flap exhibit a strong defect of nuclear import, which can be corrected to wild-type levels by reinsertion of the DNA flap sequence.

Molecular evidence for nosocomial transmission of human immunodeficiency virus from a surgeon to one of his patients.

We have investigated the molecular evidence in favor of the transmission of human immunodeficiency virus (HIV) from an HIV-infected surgeon to one of his patients. After PCR amplification, the env and gag sequences from the viral genome were cloned and sequenced. Phylogenetic analysis revealed that the viral sequences derived from the surgeon and his patient are closely related, which strongly suggests that nosocomial transmission occurred. In addition, these viral sequences belong to group M of HIV type 1 but are divergent from the reference sequences of the known subtypes.

Oxidative protein damage and degradation in lymphocytes from patients infected with human immunodeficiency virus.

It has been proposed that oxidative stress is the common mediator of apoptotic cell death in AIDS. However, mechanistic relationships between oxidative damage and cell death are far from clear. It is reported here that the mitogenic activation of T lymphocytes from human immunodeficiency virus-positive subjects involves perturbation of redox balance, as indicated by the increase in hydroethydine intracellular oxidation and manganese superoxide dismutase adaptive induction. Principal molecular targets of oxidative injury are cellular proteins whose content in carbonyl groups increases together with a dramatic increase in degradation of newly synthesized proteins catalyzed by the ATP- and ubiquitin-dependent proteolytic system. The major consequence of this metabolic anomaly is the decrease in protein cell mass leading to cells that are smaller than normal at lethal mitosis.

Human immunodeficiency virus infection and AIDS in a person with negative serology.

A 38-year-old woman resident of Ivory Coast died of AIDS, while remaining human immunodeficiency virus (HIV)-seronegative. She had been regularly tested because her husband was HIV-seropositive. The subject's lack of specific antibodies was assessed using commercial tests and confirmed by a radioimmunoprecipitation assay of the patient's virus. She was unquestionably HIV-1-infected, with a high plasma virus load, and her virus could be isolated. Molecular analyses indicated this retrovirus was clade A, which is common in Africa, and it was highly homologous to the virus isolated from her husband. The subject's seronegative status was thought to be due to rapid depletion of specific CD4+ helper T cells, resulting from accelerated disease progression, and was host-related rather than due to a specific HIV strain.

Inhibition of in vitro HIV infection by trinitrophenyl-protein conjugates.

Levels of natural antibodies (NAb) with high anti-trinitrophenyl (TNP) activity are increased during human immunodeficiency virus (HIV) infection. The aim of the present study was to examine the anti-HIV effect of natural anti-TNP antibodies, as well as that of their internal image, TNP antigen, on HIV infection in vitro. The results obtained with anti-TNP antibodies, as assessed by syncytia formation, were variable, although they demonstrated an inhibitory effect. In contrast, using RT activity assay plus evaluation of syncytia formation and the viral cytopathic effect, we found that bovine serum albumin (BSA) bearing different TNP groups was able to inhibit HIV infection of peripheral mononuclear cells and T4 cell lines without affecting cell metabolism or proliferation. BSA alone was devoid of activity; the antiviral effect depended on TNP substitution of the BSA molecule, and passage through an anti-TNP immunoadsorbent abolished this effect. The mechanism by which TNP exerts this antiviral effect is unclear. Antigenic epitopes may be shared by HIV and TNP, since monoclonal antibodies directed against various HIV proteins reacted with TNP in an enzyme immunoassay. TNP-BSA, however, did not bind to the CD4 receptor.

Isolation and envelope sequence of a highly divergent HIV-1 isolate: definition of a new HIV-1 group.

We report here the isolation and envelope sequence of a divergent HIV-1 isolate from a French woman with AIDS. This virus, HIV-1VAU, is closely related to the recently described Cameroonian viral isolates HIV-1ANT70 and HIV-1MVP5180, until now designated HIV-1 subtype O. Phylogenetic analysis reveals that the three viruses are equidistant from one another and that their mutual divergence is similar to what has been reported between the more conventional HIV-1 subtypes. Therefore, these three viruses could be included in a new viral group, HIV-1 group O (outgroup), distinct from the cluster of other HIV-1 isolates, which we will refer to as group M (Major group). The HIV-1 group O is currently emerging in western central Africa but its spread in Europe has already started.

Specificity of antipeptide antibodies produced against V2 and V3 regions of the external envelope of human immunodeficiency virus type 2.

The V2 region of simian immunodeficiency virus (SIV) and V3 region of human immunodeficiency virus type 1 (HIV-1) have been reported to be neutralization epitopes. We analysed the corresponding regions in HIV-2. Synthetic peptides modeling the V2 (aa 149-168) and V3 (CV3: aa 298-315 and NV3: aa 306-324) regions of the HIV-2 external envelope glycoprotein were coupled to KLH and used as immunogens in rabbits. We characterized the resulting antiV2 and antiV3 antibodies for their ability to recognize native and deglycosylated HIV-2 envelope glycoprotein, to block gp-CD4 interaction and to inhibit syncytium formation in vitro. The three synthetic peptides induced antibodies able to recognize specifically the native HIV-2 envelope glycoprotein with a significant avidity (K0.5 between 6 x 10(-7) and 8 x 10(-9) M). Interestingly, the reactivity of antibodies produced against the V2 peptide, which contains two potential sites of N-glycosylation, was higher against the fully deglycosylated than glycosylated HIV-2 external envelope glycoprotein (gp105). The antipeptide antibodies were used to investigate the topography of these regions in the preformed gp-CD4 complex in indirect immunofluorescence assays. The V2 and V3 regions in the complex remained accessible to their respective antibodies. Moreover, preincubation of gp105 with anti V2 or anti V3 antibodies did not prevent gp-CD4 interaction. Thus the V2 and V3 regions are not directly involved in the gp105 binding site for the CD4 receptor. Finally, in contrast with results obtained with antibodies produced against the V3 region of HIV-1 gp120 and monoclonal antibodies produced against the V3 of SIV, antibodies produced against V2 and V3 of HIV-2 were unable to inhibit syncytium formation induced by HIV-2 in vitro.

Spermatozoa as potential carriers of HIV.

In order to investigate the role of germ cells in the sexual transmission of immunodeficiency virus (HIV), spermatozoa from healthy HIV-seronegative men were incubated in vitro with HIV1. After washing, they were cocultured with peripheral blood leukocytes from seronegative blood donors. Reverse transcriptase assays and p24 antigen tests were performed in culture supernatants. Electron microscopy examination of these HIV-incubated spermatozoa was carried out, as well as the search for CD4 molecules on their surface. Although virus bound to and seemed to enter spermatozoa despite the absence of detectable CD4 epitopes on their surface, no replication of HIV was apparent. However, HIV particles on the surface of spermatozoa were capable of infecting CD4 T lymphocytes. Present results would seem to preclude artificial insemination between an HIV-seropositive man and an HIV-seronegative woman.

Programmed cell death in AIDS-related HIV and SIV infections.

One of the difficulties in understanding the complex pathology of human immunodeficiency virus (HIV) infection is to explain the progressive depletion of the CD4 helper T cell population and consequently the destruction of the immune system. Although cytopathic effects of HIV are observed in vitro, they cannot in vivo account for CD4 T cell depletion because relatively few cells are productively infected. Thus immunological mechanisms must be envisaged. We have found that peripheral blood lymphocytes (PBLs) from asymptomatic HIV-infected individuals are primed for a suicide process known as apoptosis or programmed cell death (PCD). DNA fragmentation characteristic of apoptosis was enhanced by stimulation of lymphocytes with ionomycin, a known inducer of apoptosis in suitably primed cells. Identification of the T cell subpopulations programmed for apoptosis indicated that both CD4+ and CD8+ cells died when cultured without stimulation or when polyclonally stimulated with ionomycin. Activation-induced cell death was also observed after stimulation with self-MHC class II-dependent superantigens, namely bacterial toxins from Staphylococcus (SEB), Streptococcus (ETA), and Myocoplasma (MAM) and under these conditions the CD4+ T cells were preferentially affected. To explore whether new macromolecular synthesis were required for apoptosis, various known inhibitors of apoptosis such as cycloheximide, cyclosporin A, Zn2+, or EGTA were tested. Activation-induced apoptosis was found sensitive to these inhibitors, indicating an active mechanism, but apoptosis observed in nonstimulated cultures was not, suggesting that these cells already contained the complete machinery for death. Prevention of apoptosis could be obtained in the presence of a mixture of cytokines and the minimal signal necessary for this prevention was IL-1 alpha and IL-2. Finally, a correlation between PCD and AIDS-pathogenesis was suggested by the comparison of lymphocytes from lentivirus-infected primates suceptible (SIV-infected macaques) and resistant (HIV-infected chimpanzees) to AIDS. Altogether our results suggest that, during HIV or SIV infection, PCD may contribute in vivo to the deletion of reactive T cells after antigenic stimulation.

In vitro non-productive infection of purified natural killer cells by the BRU isolate of the human immunodeficiency virus type 1.

Highly purified natural killer (NK) cell lines and clones, displaying the typical phenotype, morphology and function and obtained from healthy blood donors, were infected in vitro with the BRU isolate of human immunodeficiency virus type 1 (HIV-1). There was no significant increase in reverse transcriptase activity and levels of p24 antigen in the supernatants, but positive staining was observed using an immunogold technique with polyclonal anti-HIV-1 antibodies. When infected NK cells were co-cultivated with autologous non-infected CD4+ mitogen-activated cells, significant levels of reverse transcriptase activity and p24 antigen in supernatants were detected. Giant syncytial cells and a high number of mature virion particles were also evident. When NK cell lines or clones from HIV-1-infected patients were studied, neither the presence of p24 antigen nor reverse transcriptase activity was detected in the supernatants after stimulation with mitogens, cytokines or co-culture with allogeneic CD4+ mitogen-activated cells. PCR studies did not detect HIV-1 genes in freshly purified NK cells, cell lines or clones from infected patients. Taken together these results suggest that (i) normal NK cells can be infected in vitro by the HIV-1 BRU isolate in a non-productive fashion, (ii) PCR with NK cell DNA of HIV-1-infected patients indicates that in vivo few of these cells, if any, are infected by HIV-1 and (iii) the mechanisms responsible for the impairment of NK cell function during HIV-1 infection remain to be determined and are probably not related to a direct cytopathic effect of the virus.

Study of the interaction of HIV-1 and HIV-2 envelope glycoproteins with the CD4 receptor and role of N-glycans.

In order to further characterize the interaction of human immunodeficiency viruses (HIV) with the CD4 receptor at the molecular level, a binding test was performed using iodine-labeled glycoproteins, 125I-gp160 from HIV-1 and 125I-gp140 from HIV-2, to bind to lymphoid cells expressing the CD4 receptor. The inhibition of binding of the radiolabeled glycoproteins to CD4+ cells by increasing concentrations of nonradiolabeled gp160 or gp140 was used to determine the affinity of the interaction between the glycoproteins and CD4. The gp-CD4 association occurs with a high affinity: K0.5 gpHIV-1 = 9 x 10(-9) M and K0.5 gpHIV-2 = 7 x 10(-8) M, indicating that the affinity of the interaction between HIV-2 gp140 and CD4 is 10 times lower than that observed with HIV-1 gp160. The N-linked glycans of the HIV-1 and HIV-2 glycoproteins account for a high proportion of their molecular mass (about 50%). Total deglycosylation of gp160 and gp140 by enzymatic treatment with Endo F-N glycanase occurred under nondenaturing conditions, indicating the high accessibility of the N-linked glycan chains in the three-dimensional structure of the molecule. Moreover, the deglycosylated proteins retained a significant binding capacity to CD4. These results show that the carbohydrate chains of HIV-2 gp140, as those of HIV-1 gp160, do not play a major role in the gp-CD4 interaction.

Modulation of cell growth and host protein synthesis during HIV infection in vitro.

During HIV infection of CEM cells cultured in vitro, significant differences in growth rate and protein turnover were observed with different viral preparations. There was a significant inhibition of proliferation after infection with crude HIV supernatants. On the other hand, infection with purified HIV particles obtained by filtration, differential centrifugation, and isopycnic sedimentation led to a progressively increasing stimulation of cell growth. This early stimulation was prevented by neutralizing the virus with soluble CD4 molecules. Study of cell growth in the presence of a purified membrane preparation indicated that membrane fragments contaminating the crude HIV supernatant were responsible for the observed growth inhibition. Interestingly, the stimulation of proliferation was also observed with heat-inactivated virus or after inhibition of viral replication with ZDV. In the presence of purified HIV virions, the rate of general protein synthesis was not inhibited, as is usually observed with crude viral supernatants. However, a marked reduction in protein content and increased protein degradation was found in cultures infected with either crude or purified HIV preparations.