Different fates of intracellular glutathione determine different modalities of apoptotic nuclear vesiculation.

U937 monocytic cells show two main apoptotic nuclear morphologies, budding and cleavage, that are the result of two independent morphological routes, since they never interconvert one into the other, and are differently modulated by stressing or physiological apoptogenic agents [Exp Cell Res 1996; 223:340-347]. With the aim of understanding which biochemical alterations are at the basis of these alternative apoptotic morphologies, we performed an in situ analysis that showed that in U937 cells intracellular glutathione (GSH) is lost in cells undergoing apoptosis by cleavage, whereas it is maintained in apoptotic budding cells. Lymphoma cells BL41 lose GSH in apoptosis, and show the cleavage nuclear morphology; the same cells latently infected with Epstein Barr Virus (E2r line) undergo apoptosis without GSH depletion and show the budding nuclear morphology. GSH depletion is not only concomitant to, but is the determinant of the cleavage route, since the inhibition of apoptotic GSH efflux with cystathionine or methionine shifts the apoptotic morphology from cleavage to budding. Accordingly, cystathionine or methionine antagonizes apoptosis in the all-cleavage BL41, without affecting the all-budding E2r.

Fluorescent poliovirus for flow cytometric cell surface binding studies.

Specific cell-surface binding is the essential first step for cellular invasion by viruses. To understand this process, various methods to evaluate binding properties of viruses to cells have been developed. However, many rely on radioactive labeling or indirect immunofluorescence. The development of a novel fluorescence binding assay for poliovirus is described. Poliovirus (type 1 Mahoney or Sabin) was labeled directly with fluorescein using a commercially available fluoresceination kit. Fluorescently labeled poliovirus was bound to its specific receptor on Hela or U937 cells and detected by flow cytometric analysis. Specific binding and infectivity was retained, although reduced, depending on the extent of fluoresceination. Therefore, depending on the users' requirements, the extent of fluoresceination must be titrated carefully to achieve maximal fluorescence and minimal functional destruction. It is likely that this method may be useful with other viruses.

Parvovirus H-1-induced cell death: influence of intracellular NAD consumption on the regulation of necrosis and apoptosis.

The autonomous parvovirus H-1 exerts tumor-suppressive effects in living organisms and has been shown to specifically interfere with the survival of transformed cells in culture. The mechanism(s) by which H-1 virus induces death of transformed cells is not yet well understood. It has recently been reported that H-1 virus induces apoptotic cell death in the human monocytic U937 cell line, as assessed by biochemical and morphological changes of infected cells (Rayet, B., Lopez-Guerrero, J.-A., Rommelaere, J., Dinsart, C., 1998. Induction of programmed cell death by parvovirus H-1 in U937 cells: connection with the TNFalpha signalling pathway. J. Virol. 72, 8893-8903). Here we show that parvovirus H-1 infection induced early biochemical changes pointing to apoptotic events also in the transformed human keratinocyte cell line, HeLa, and the transformed rat fibroblast cell line, P1. Morphologic changes, however, and in particular the early breakdown of plasma membrane integrity, suggested that apoptosis did not go to completion, leading to necrotic cell death as the major result of parvovirus infection of HeLa and P1 cells. Parvovirus infection of these, and to a significantly lesser extent of U937 cells, was accompanied by rapid depletion of intracellular NAD stores. Inhibition of NAD-consuming enzymes interfered with parvovirus-induced NAD depletion and increased the proportion of H-1 virus-infected cells displaying apoptotic features of cell death. In contrast, a similar prevention of NAD depletion through stimulation of NAD production had little influence on the cell death pathway, suggesting that NAD-consuming enzymes may promote necrosis in a direct way rather than through inducing the overall drop of intracellular NAD.

9-nitrocamptothecin selectively inhibits human immunodeficiency virus type 1 replication in freshly infected parental but not 9-nitrocamptothecin-resistant U937 monocytoid cells.

We have previously reported that 9-nitrocamptothecin (9NC) inhibited human immunodeficiency type 1 (HIV-1) replication in latently HIV-1-infected T lymphocytic ACH-2 cells stimulated with the cytokine tumor necrosis factor alpha (TNF-alpha) (Moulton et al., AIDS Res Hum Retroviruses 1998;14:39). 9NC induced an accelerated apoptosis in HIV-1-infected, but not uninfected, lymphocytic cells. The present study demonstrates that 9NC selectively inhibits release of HIV-1 from freshly infected monocytoid U937 cells in a dose-response manner. Significant inhibition was achieved with concentrations of 9NC that were not toxic. In contrast, HIV-1 replication in 9NC-resistant monocytoid cells, derived from U937, was not inhibited by similar doses of 9NC. Importantly, sensitivity of HIV-1 replication to 9NC correlated with the effect of 9NC on topoisomerase I (topo I) activity. In a 9NC-sensitive subline, 9NC induced posttranslational activation of the nuclear transcription factor kappaB (NF-kappaB) after the drug treatment. This activation was neither related to selective 9NC suppression of HIV-1 replication, nor was it sufficient for the 9NC-induced toxicity in the drug-sensitive monocytoid cells. Taken together, the selective inhibition of HIV-1 replication in both lymphoid and monocytoid cells lends further credence to the potential development of 9NC as an alternative drug for treating HIV-1 infection.

Long-term culture of HIV-1-infected cells with the transcription inhibitor K-37.

We have previously reported that the fluoroquinoline derivative K-37 is a potent and selective inhibitor of HIV-1 replication in both acutely and chronically infected cells. K-37 blocks the HIV-1 transcription process through the inhibition of still unknown cellular factor(s). To gain further insight into the target of K-37 for HIV-1 replication, we have conducted long-term culture of acutely infected cells in the presence of K-37. When MOLT-4 and U937 cells were infected with HIV-1 and cultured in the absence of K-37, the p24 antigen levels in the culture supernatants reached a plateau within 12 days. In the presence of K-37 (0.25 and 0.5 microM), the elevation of p24 antigen levels was delayed but reached a similar plateau level on day 16 or later. At a concentration of 1 microM, K-37 markedly suppressed HIV-1 replication. However, viral breakthrough was observed after 1 month of the culture period in both MOLT-4 and U937 cells. We established MOLT-4 cell lines chronically infected with the breakthrough viruses (M(1) and U(1)) or the corresponding wild-type strains (M(0) and U(0), respectively), and K-37 was examined for its inhibitory effects on HIV-1 replication in these cell lines. No substantial difference in anti-HIV-1 activity was observed between the two cell lines. However, acute infection experiments revealed that the infectivity of M(1) and U(1) was much lower than that of M(0) and U(0), respectively. Furthermore, both M(1) and U(1) had a G to T nucleotide mutation at position -215 in the second nuclear factor of activated T-cells-binding domain (-215 to -203) of the HIV-1 long terminal repeat.

Outbreak of fatal childhood viral infection in Sarawak, Malaysia in 1997: inocula of patients' clinical specimens induce apoptosis in vitro.

Identification of the aetiologic agent(s) associated with an outbreak of fatal childhood viral infection in Sarawak, Malaysia, in mid 1997 remains elusive. It is reported here that African green monkey kidney (Vero) and human monocytic (U937) cells treated with inocula derived from clinical specimens of some of these fatal cases showed the presence of cellular genomic DNA degradation when the extracted DNA was separated by pulsed field gel electrophoresis (PFGE), oligonucleosomal DNA ladders characteristic of apoptotic cells when the infected cells' DNA was separated by agarose gel electrophoresis, and apoptotic cellular DNA fragmentation when cells were stained using terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL). These results suggest that inocula derived from the patients' clinical specimens contain factors which stimulate apoptotic cellular responses in vitro.

Transcription-dependent gene looping of the HIV-1 provirus is dictated by recognition of pre-mRNA processing signals.

HIV-1 provirus, either as a chromosomal integrant or as an episomal plasmid in HeLa cells, forms a transcription-dependent gene loop structure between the 5'LTR promoter and 3'LTR poly(A) signal. Flavopiridol-mediated inhibition of RNA polymerase II elongation blocks 5' to 3'LTR juxtaposition, indicating that this structure is maintained during transcription. Analysis of mutant or hybrid HIV-1 plasmids demonstrates that replacement of the 5'LTR promoter with CMV or the 3'LTR poly(A) signal with a synthetic element (SPA) permits gene loop formation, suggesting that these interactions are not retroviral specific. In addition, activation of the 5'LTR poly(A) signal or inactivation of the 3'LTR poly(A) signal abolishes gene loop formation. Overall, we demonstrate that both ongoing transcription and pre-mRNA processing are essential for gene loop formation, and predict that these structures represent a defining feature of active gene transcription.

In vitro and in vivo characterization of recombinant Ebola viruses expressing enhanced green fluorescent protein.

To facilitate an understanding of the molecular aspects of the pathogenesis of Zaire ebolavirus (ZEBOV) infection, we generated 2 different recombinant viruses expressing enhanced green fluorescent protein (eGFP) from additional transcription units inserted at different positions in the virus genome. These viruses showed in vitro phenotypes similar to that of wild-type ZEBOV (wt-ZEBOV) and were stable over multiple passages. Infection with one of the viruses expressing eGFP produced only mild disease in rhesus macaques, demonstrating a marked attenuation in this animal model. However, in mice lacking signal transducer and activator of transcription 1, both viruses expressing eGFP caused lethal cases of disease that were moderately attenuated, compared with that caused by wt-ZEBOV. In mice, viral replication could be easily tracked by the detection of eGFP-positive cells in tissues, by use of flow cytometry. These findings demonstrate that the incorporation of a foreign gene will attenuate ZEBOV in vivo but that these viruses still have potential for in vitro and in vivo research applications.

Immunoglobulin g antibody-mediated enhancement of measles virus infection can bypass the protective antiviral immune response.

Antibodies to viral surface glycoproteins play a crucial role in immunity to measles by blocking both virus attachment and subsequent fusion with the host cell membrane. Here, we demonstrate that certain immunoglobulin G (IgG) antibodies can also enhance the entry of measles virus (MV) into monocytes and macrophages. Antibody-dependent enhancement of infectivity was observed in mouse and human macrophages using virions opsonized by a murine monoclonal antibody against the MV hemagglutinin (H) glycoprotein, polyclonal mouse anti-MV IgG, or diluted measles-immune human sera. Neither H-specific Fab fragments nor H-specific IgM could enhance MV entry in monocytes or macrophages, indicating involvement of a Fc gamma receptor (FcgammaR)-mediated mechanism. Preincubation with an anti-fusion protein (anti-F) monoclonal antibody or a fusion-inhibitory peptide blocked infection, indicating that a functional F protein was required for viral internalization. Classical complement pathway activation did not promote infection through complement receptors and inhibited anti-H IgG-mediated enhancement. In vivo, antibody-enhanced infection allowed MV to overcome a highly protective systemic immune response in preimmunized IfnarKo-Ge46 transgenic mice. These data demonstrate a previously unidentified mechanism that may contribute to morbillivirus pathogenesis where H-specific IgG antibodies promote the spread of MV infection among FcgammaR-expressing host cells. The findings point to a new model for the pathogenesis of atypical MV infection observed after immunization with formalin-inactivated MV vaccine and underscore the importance of the anti-F response after vaccination.

Poliovirus modulates Bcl-xl expression in the human U937 promonocytic cell line.

Poliovirus induces bcl-2-independent apoptosis in the human U937 promonocytic cell line [28]. Here we describe that this cell death, induced after viral infection, correlates with the modulation of the protooncoprotein Bcl-xl. Furthermore, poliovirus infection decreases the detected Bcl-xl in a U937 clone that overexpresses this protein (U937bcl-xl). Although in U937bcl-xl cells, Bcl-xl was not as highly regulated as in parental U937 cells, correlation between Bcl-xl modulation and the cleavage of poly(ADP-ribose)polymerase could be observed. Nevertheless, the induction of shutoff after infection of transfected control U937neo or U937bcl-xl clones was not significantly altered. Finally, production of new viral particles was slightly restricted in Bcl-xl-overexpressing U937 cells. Taken together, these results suggest that Bcl-xl is modulated during the induction of apoptosis in the promonocytic cell line U937 after poliovirus infection, although modulation of this protooncogene was not sufficient to modify the course of infection.

Human immunodeficiency virus replication induces monocyte chemotactic protein-1 in human macrophages and U937 promonocytic cells.

We have recently described a significant correlation between human immunodeficiency virus-1 (HIV-1) RNA replication and monocyte chemotactic protein-1 (MCP-1) levels in the cerebrospinal fluid (CSF) of individuals with the acquired immunodeficiency syndrome (AIDS) with HIV encephalitis (E). Because local macrophages (microglia) are the cells predominantly infected in the brain, we investigated whether in vitro HIV infection affects MCP-1 production in mononuclear phagocytes (MP). MCP-1 secretion and expression were consinstently upregulated over constitutive levels in human monocyte-derived macrophages (MDM) infected with the M-tropic R5 BaL strain of HIV-1. HIV replication was required for this effect, as demonstrated by the absence of chemokine upregulation after infection in the presence of 3'-azido-3'-deoxythimidine (AZT) or cell-exposure to heat-inactivated (triangle up degrees ) virus. MCP-1 induction was not restricted to HIV-1 BaL, but was also observed during productive infection of MDM with two primary isolates differing for entry coreceptor usage and of U937 cells with the X4 HIV-1 MN strain. Based on the observation that exogenous HIV-1 Tat induced MCP-1 expression in astrocytes, we also investigated its role in MDM and U937 cells. Exogenous Tat induced MCP-1 production from MDM in a concentration-dependent manner, however, it was not effective on uninfected U937 cells or on the chronically infected U937-derived cell line U1. Transfection of Tat-expressing plasmids moderately activated HIV expression in U1 cells, but failed to induce MCP-1 expression in this cell line or in uninfected U937 cells. HIV replication-dependent expression of MCP-1 in MP may be of particular relevance for the pathogenesis of HIV infection in nonlymphoid organs such as the brain.

HIV-1 tat molecular diversity and induction of TNF-alpha: implications for HIV-induced neurological disease.

Activation and infection by HIV-1 of glial cells and infiltrating macrophages are cardinal features of AIDS-related neurological disease. Tumor necrosis factor-alpha (TNF-alpha) is released by these cell types, and increased TNF-alpha mRNA and protein levels are associated with the development and severity of HIV-induced neurological disease. HIV-1 proteins have been implicated in HIV neuropathogenesis including Tat which has been shown to be a potent inducer of TNF-alpha. We review our data showing the induction of TNF-alpha by Tat in primary human fetal astrocytes, human peripheral blood mononuclear cells, macrophages, and astrocytic and macrophage cell lines. TNF-alpha induction was NF-kappaB dependent and was eliminated by inhibiting protein kinase A, phospholipase C and protein tyrosine kinase activity. In addition, we examined the molecular diversity of the tat genome in the brains of HIV-infected patients from different HIV-1 clades. Comparison of matched brain- and spleen-derived tat sequences indicated that homology among brain-derived clones was greater than that between the brain- and spleen-derived clones. The brain-derived tat sequences were markedly heterogeneous in regions which influence viral replication and intracellular transport. Future studies using Tat, encoded by different sequences, will be necessary to determine the functional significance of tat molecular diversity. Nonetheless, these studies suggest that Tat is an important inducer of TNF-alpha production and thus may play a key role in the pathogenesis of HIV-related neurological disease.

Intracellular accumulation of human immunodeficiency virus protease inhibitors.

Intracellular accumulation of the protease inhibitors (PIs) saquinavir (SQV), ritonavir (RTV), and indinavir (IDV) was determined in 50 human immunodeficiency virus-positive patients. Following extraction, PIs were quantified by mass spectrometry. Paired plasma and intracellular samples were collected over a full dosing interval from patients (13 on SQV, 6 on RTV, 8 on IDV, 16 on SQV plus RTV, 7 on IDV plus RTV) with a plasma viral load of <400 copies/ml. Data were expressed as intracellular/plasma drug concentration ratios. A hierarchy of intracellular accumulation was demonstrated by the following medians: 9.45 for SQV > 1.00 for RTV > 0.51 for IDV. Coadministration of RTV did not boost ratios of SQV or IDV within the cell or in plasma, although absolute plasma and intracellular SQV concentrations were increased by RTV. Seven individuals receiving SQV in hard-gel capsule form (median, 32 months) had higher intracellular/plasma drug ratios than all other patients receiving SQV (median, 17.62 versus 4.83; P = 0.04), despite consistently low plasma SQV concentrations. How this occurs may provide insight into the mechanisms that limit adequate drug penetration into sanctuary sites.

HIV-1 preferentially binds receptors copatched with cell-surface elastase.

Human leukocyte elastase (HLE) interacts with HIV-1 glycoprotein (gp)41, suggesting a nonenzymatic receptor function for HLE in the context of HIV-1. HLE is found localized to the cell surface, but not granules in HIV permissive clones, and to granules, but not the cell surface of HIV nonpermissive clones. Inducing cell-surface HLE expression on HLE null, HIV nonpermissive clones permits HIV infectivity. HIV binding and infectivity diminish in proportion to HLE RNA subtraction. HIV binding and infectivity show dose dependence for the natural HLE ligand alpha1 proteinase inhibitor (alpha1antitrypsin, alpha1PI). Chemokines prevent, whereas alpha1PI promotes, copatching of HLE with the canonical HIV receptors. Recent demonstration that decreased viral RNA is significantly correlated with decreased circulating alpha1PI in HIV seropositive individuals is consistent with a model in which HLE and alpha1PI can serve as HIV coreceptor and cofactor, respectively, and potentially participate in the pathophysiology of HIV disease progression.