Cell-specific differences in activation of NF-kappa B regulatory elements of human immunodeficiency virus and beta interferon promoters by tumor necrosis factor.

Three aspects of the involvement of tumor necrosis factor in human immunodeficiency virus (HIV) pathogenesis were examined. Tumor necrosis factor alpha (TNF-alpha) mRNA production was analyzed by polymerase chain reaction amplification in monocytic U937 cells and in a chronically HIV infected U937 cell line (U9-IIIB). TNF-alpha RNA was undetectable in U937 cells, whereas a low constitutive level was detected in U9-IIIB cells. Paramyxovirus infection induced a 5- to 10-fold increase in the steady-state level of TNF-alpha RNA in U9-IIIB cells compared with U937 cells, suggesting that HIV-infected monocytic cells produced higher levels of TNF-alpha than did normal cells after a secondary virus infection. The effects of TNF-alpha on gene expression were examined by transient expression assays using reporter chloramphenicol acetyltransferase plasmids linked to regulatory elements from the HIV long terminal repeat (LTR) and the beta interferon promoter. In U937 and Jurkat T lymphoid cells, the inducibility of the different hybrid promoters by TNF-alpha or phorbol ester varied in a cell type- and promoter context-specific manner; the levels of gene activity of NF-kappa B-containing plasmids correlated directly with induction of NF-kappa B DNA-binding activity. Although the intact beta interferon promoter was only weakly stimulated by phorbol ester or TNF-alpha, multimers of the PRDII NF-kappa B-binding domain were inducible by both agents. TNF-alpha was able to increase expression of the HIV LTR in T cells, but in monocytic cells, TNF-alpha did not induce the HIV LTR above a constitutive level of activity. This level of NF-kappa B-independent activity appears to be sufficient for virus multiplication, since TNF-alpha treatment had no effect on the kinetics of de novo HIV type 1 (HIV-1) infection and viral RNA production in U937 cells. However, in Jurkat cells, TNF-alpha dramatically enhanced the spread of HIV-1 through the cell population and increased viral RNA synthesis, indicating that in T cells HIV-1 multiplication was stimulated by TNF-alpha treatment.     

Coexistence of fusion receptors for human T-cell leukemia virus type-I (HTLV-I) and human immunodeficiency virus type-1 (HIV-1) on MOLT-4 cells.

Human immunodeficiency virus type-1 (HIV-1) and human T-cell leukemia virus type-I (HTLV-I) have a similar tropism for target cell types, especially for CD4+ T cells. In this study, we provide evidence that receptors of these two viruses exist independently on the target cell. We established an HTLV-I-producing CD8+ T cell line (ILT-8M2) with a remarkable cell fusion capacity. When cocultured with MOLT-4 cells, ILT-8M2 cells induced giant syncytia more efficiently than any other tested HTLV-I-producer cell lines. In contrast to other HTLV-I-producers, ILT-8M2 cells were minimally susceptible to cytopathic effects of HIV-1 due to very low expression of CD4, although they were able to be persistently infected by HIV-1. The indicator MOLT-4 cells are known to respond well to HIV-1-induced cell fusion, but they lose this ability if they become persistently infected with HIV-1 because of the reduction of CD4 receptor expression. ILT-8M2 was, however, still capable of inducing syncytia with the MOLT-4 cells persistently infected by HIV-1 (MOLT-4/IIIB). This syncytium formation was dependent on the HTLV-I-envelope, as it was inhibited by HTLV-I-positive human sera or a monoclonal antibody to HTLV-I gp46 but not by monoclonal antibodies to HIV-1 gp120 or CD4. Moreover, ILT-8M2 cells persistently infected by HIV-1 (ILT-8M2/IIIB) induced both HTLV-I- and HIV-1-mediated syncytia with uninfected MOLT-4 cells. These results suggest that HTLV-I induces cell fusion utilizing receptors on the target cells independent of HIV-1-receptors.     

Changes in both gp120 and gp41 can account for increased growth potential and expanded host range of human immunodeficiency virus type 1.

Virus derived from an infectious molecular clone of the ELI strain of human immunodeficiency virus type 1 (HIV-1) replicates well in peripheral blood mononuclear cells and in some CD4-positive cell lines but exhibits a delayed time course of infection in CEM and H9 cells and fails to infect SupT1 and U937 cells. If the virus that emerges from infected H9 cells is used to infect CEM and H9 cells, the time course of infection is accelerated and the virus is able to infect U937 and SupT1 cells. In this study, we used the technique of polymerase chain reaction-single-strand conformation polymorphism to localize changes in both the extracellular gp120 and the transmembrane gp41 components of the envelope gene associated with adaptation to growth in tissue culture cell lines. Specifically, mutations were identified both in a region of gp120 implicated in CD4 binding and in the amino-terminal portion of gp41 adjacent to the region involved in fusion. No changes were found in the V3 loop of gp120, a region previously shown to be involved in viral tropism. When these mutations were introduced into the original molecular clone, they conferred an enhanced replicative capacity on ELI. These results demonstrate that two additional determinants in the HIV-1 envelope protein influence viral tropism and growth in vitro. They also may have important implications for the generation of viruses with increased growth potential and expanded host range seen in the late stages of HIV disease.     

Early activation events render T cells susceptible to HIV-1-induced syncytia formation. Role of protein kinase C.

In human immunodeficiency virus-1 (HIV-1)-infected cell cultures, cell-to-cell fusion and the formation of multinucleated giant cells (syncytia) are induced as a consequence of interactions between the viral envelope glycoprotein on infected cells and cell surface CD4 molecules on uninfected cells. Although activated CD4+ T cells rapidly form syncytia when cultured with HIV-1 envelope glycoprotein expressing (env+) cells, freshly isolated, unstimulated CD4+ T cells do so more slowly. In these studies, we sought to explore the role of T cell activation in rendering CD4+ T cells susceptible to HIV-1-mediated syncytia formation. Our results indicate that within 2 h of exposure to immunologic stimuli, CD4+ T cells acquire the ability to form syncytia with HIV-1 env+ cells. Both cholera toxin, an inhibitor of protein kinase C (PKC) through its effects on inositol triphosphate and diacylglycerol production, and 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride, a noncompetitive inhibitor (with respect to ATP) of PKC, prevented unstimulated but not previously stimulated CD4+ T cells from forming syncytia with HIV-1 env+ cells. 1-Oleoyl-2-acetyl glycerol, an analog of the PKC activator, diacylglycerol, enhanced syncytia formation whereas ionomycin, a calcium ionophore, had no effect. These results suggest that activation of PKC is essential for previously unstimulated CD4+ T cells to become fusogenic.     

Tetraspanins CD9 and CD81 modulate HIV-1-induced membrane fusion

Protein organization on the membrane of target cells may modulate HIV-1 transmission. Since the tetraspanin CD81 is associated to CD4, the receptor of HIV-1 envelope protein (Env; gp120/gp41), we have explored the possibility that this molecule may modulate the initial steps of HIV-1 infection. On the other hand, CD81 belongs to the tetraspanin family, which has been described as organizers of protein microdomains on the plasma membrane. Therefore, the role of CD81 and other related tetraspanin, CD9, on the cell-to-cell fusion process mediated by HIV-1 was studied. We found that anti-tetraspanin Abs enhanced the syncytia formation induced by HIV-1 envelope proteins and viral entry in human T lymphoblasts. In addition, anti-CD81 Abs triggered its clustering in patches, where CD4 and CXCR4 were included. Moreover, the knocking down of CD81 and CD9 expression resulted in an increase in syncytia formation and viral entry. Accordingly, overexpression of CD81 and CD9 rendered cells less susceptible to Env-mediated syncytia formation. These data indicate that CD9 and CD81 have an important role in membrane fusion induced by HIV-1 envelope.     

The leukocyte adhesion glycoprotein CD18 participates in HIV-1-induced syncytia formation in monocytoid and T cells

mAb 60.3 and IB4 to CD18, the common beta-subunit of the human leukocytic cell adhesion molecule family, efficiently inhibit syncytium formation induced by the interaction of HIV type 1 (HIV-1)-infected monocytoid cells and CD4+ T cells. The antibodies also interfere with cellfree HIV-1 infection of U-937 clone 16 cells. Virus-induced aggregation of these cells and the subsequent syncytia formation leading to massive cell death are efficiently blocked, and the number of infected cells remains at a very low level, 2 to 5%, for the entire culture period. However, anti-CD18 mAb do not inhibit binding of the viral envelope glycoprotein gp120 to the cell surface receptor CD4. The results indicate participation of CD18, or of the protein complex CD11a-c/CD18, in addition to CD4, in the infection and cytopathic effect of HIV-1. They also suggest that intercellular adhesion contributes to virus transmission from cell to cell and may be an important mechanism for virus spreading.     

Diagnostic surface expression of SWAP-70 on HIV-1 infected T cells

Following immunization with HIV-1 infected cells, a hybridoma cell line termed 9F11 was established from the P3U-1 myeloma line fused with lymphocytes from a trans-chromosome (TC) mouse, that harbors human chromosomes containing immunoglobulin genes. The 9F11 human IgM monoclonal antibody (9F11 Ab) reacts with HIV-1 infected MOLT4 cells but not with uninfected MOLT4 cells, and causes immune cytolysis with homologous human complement at a concentration as low as 0.4 microg/ml. This Ab was used to perform immunoscreening of a cDNA expression library derived from HIV-1 infected cells. All positive cDNA clones contained SWAP-70 cDNA. SWAP-70 RNA and protein expression are much stronger in HIV-1 infected cells. SWAP-70 was also detected on the surface of HIV-1 infected cells by flow cytometric analysis. The monocyte cell line U937 cells expresses SWAP-70 on its cell surface regardless of whether it was infected with HIV-1. Furthermore, among PBMCs surface expression of SWAP-70 was detected on CD21+, CD56+ and CD14+ cells. Although CD3+ cells scarcely express SWAP-70 on their surface, once activated, they become positive. SWAP-70 may therefore serve as a marker for T cell differentiation as well as for HIV-1 infection.     

Requirement for delivery of signals by physical interaction and soluble factors from accessory cells in the induction of receptor-mediated T cell proliferation. Effectiveness of IFN-gamma modulation of accessory cells for physical interaction with T cells

We analyzed the mechanism by which accessory cells support the induction of the proliferation of human peripheral blood T cells by a monoclonal anti-CD3 antibody, OKT3. Cross-linking of T cell receptor/CD3 complex by anti-CD3 coupled to latex beads and the addition of IL-1 are not enough to induce the IL-2 production and proliferation of T cells extensively depleted of accessory cells, while the addition of both the culture supernatant of macrophages or a monoblastic cell line, U937 cells, and the paraformaldehyde-fixed macrophages or U937 cells which had been precultured with interferon-gamma before fixation into the culture of the T cells with anti-CD3-latex did induce the T cell proliferation. Lack of the addition of either one of these did not induce the response. These results indicate that the signal(s) delivered by soluble factors released from the accessory cells and that delivered by the physical interaction between accessory cells and T cells are both required for the induction of IL 2 production and proliferation of T cells by anti-CD3-latex. Importantly, the macrophages or U937 cells had to be cultured with Con A-stimulated lymphocyte culture supernatant or IFN-gamma prior to fixation with paraformaldehyde, suggesting that a molecule(s) inducible on accessory cells surface by IFN-gamma or other lymphokine is necessary for the effective accessory cell-T cell interaction to induce the T cell response. It was further revealed that the activity of the culture supernatant of accessory cells may be mediated synergistically by IL 1 and a certain other factor(s) and was actually shown to be replaced by the combined addition of rIL-1 and rIL-6 but not by rIL-1 alone. The experimental system described here will be very useful for dissecting the accessory functions for T cell activation.     

Lack of the trans-receptor mechanism of HIV-1 infection: CD4- and coreceptor-independent incorporation of HIV-1-resistant cells into syncytia induced by HIV-1

Human immunodeficiency virus type 1 (HIV-1) infects cells through an interaction of HIV-1 envelope protein with CD4 and an appropriate coreceptor on target cells. This interaction often leads to cell fusion, and formation of syncytia. HIV-1-resistant cells expressing either CD4 or a coreceptor are often surrounding HIV-1-susceptible cells, expressing both CD4 and a compatible coreceptor, in vivo. It is therefore worthwhile to investigate whether these HIV-1-resistant cells could cooperate in HIV-1 infection or cell fusion leading to their incorporation into syncytia. When CD4-positive, coreceptor-negative cells were co-cultured with CD4-negative, coreceptor-positive cells and exposed to HIV-1, HIV-1 infection was not established, indicating that CD4 and the coreceptor expressed on different cell surfaces could not cooperate in HIV-1 entry. However, when HIV-1-resistant cells expressing CD4 or a coreceptor or lacking both were mixed with HIV-1-susceptible cells and inoculated with HIV-1, all these HIV-1-resistant cells were similarly incorporated into syncytia induced by HIV-1, indicating a CD4- and coreceptor-independent incorporation of HIV-1-resistant cells into syncytia. This incorporation was impaired by the transfection of these cells with siRNAs for adhesion molecules. Our study demonstrates that HIV-1-resistant cells can be incorporated into syncytia induced by HIV-1 and this incorporation may partially be mediated through adhesion molecules.     

Decrease in CD93 (C1qRp) expression in a human monocyte-like cell line (U937) treated with various apoptosis-inducing chemical substances

Human CD93, a receptor for complement component 1, subcomponent q phagocytosis (C1qRp), has been shown to be selectively expressed by cells of a myeloid lineage and was originally reported to be involved in the C1q-mediated enhancement of phagocytosis in innate and adaptive immune responses. The modulation of CD93 expression has been investigated in various cells, particularly in granulocytes and monocytes . We previously reported that a protein kinase C activator (PKC), phorbol myristate acetate (PMA), effectively up-regulated CD93 expression on several cultured cell lines and that its regulation was mainly controlled by a PKC delta-isoenzyme. However, the expression pattern of CD93 in myeloid cells with apoptotic properties remains poorly understood. In this study, we examined the modulation of CD93 expression on a human monocyte-like cell line (U937) treated with various apoptosis-inducing chemical substances : an RNA-synthesis inhibitor, actinomycin D (ActD); a DNA topoisomerase I inhibitor, camptothecin (CPT); a protein-synthesis inhibitor, cycloheximide (CHX); a DNA topoisomerase II inhibitor, etoposide (EPS); and a DNA-synthesis inhibitor, mitomycin C (MMC). Apoptosis was monitored using two-color flow cytometry with Annexin V and 7-amino actinomycin D (7AAD). The above-mentioned substances sufficiently induced the early and late stages of apoptosis, identified as Annexin V positive (+)/7AAD negative (-) cells and Annexin V positive (+)/7AAD positive (+) cells, respectively, in U937 cells after 6 hr of treatment. The modulation of CD93 expression on U937 cells during the early stage of apoptosis, gated as Annexin V positive (+)/7AAD negative (-) cells, was then investigated using a CD93 mAb (mNI-11), originally established in our laboratories, and flow cytometry using a fluorescence-activated cell sorter (FACS). The mean fluorescence intensity (MFI) of the cells that stained positive for CD93 mAb (mNI-11) among the treated U937 cells showed a dramatic decrease in expression. In addition, the expressions of HLA-class I (HLA-A, B, C), HLA-class II (HLA-DR), CD18 (lymphocyte function-associated antigen-1 beta; LFA-1beta) and CD54 (intercellular adhesion molecule-1; ICAM-1) were also markedly decreased on the treated U937 cells identified as Annexin V positive (+)/7AAD negative (-) cells (early stage of apoptosis). Interestingly, the expression patterns of CD93 on the U937 cells treated with the above-mentioned chemical substances closely resembled those of HLA-class I (HLA-A, B, C). An immunoblotting analysis showed that the expression of a surface antigen (molecular size, about 97 kDa) targeted by the CD93 mAb (mNI-11) on the U937 cells treated with various apoptosis-inducing chemical substances had clearly decreased. On the other hand, an enzyme-linked immunoassay (EIA) showed that although PMA-treated U937 cells had strongly secreted soluble CD93 (sCD93) into the culture supernatant, the secretion of sCD93 in the culture supernatant of the U937 cells treated with the above-mentioned chemical substances was not enhanced, compared with that of untreated U937 cells. Importantly, however , the U937 cells with apoptotic properties induced by various apoptosis-inducing chemical substances also rapidly (in 30 min) and strongly secreted sCD93 into the culture supernatant in the presence of PMA. Taken together, these findings indicate that the expression of the CD93 molecule identified by CD93 mAb (mNI-11) is dramatically decreased on U937 cells with apoptotic properties, and that the decrease in CD93 expression on U937 cells treated with apoptosis-inducing chemical substances may be a good model for analyzing the regulation of CD93 expression on apoptotic myeloid cells.     

Differentiation of Promonocytic U937 Subclones into Macrophagelike Phenotypes Regulates a Cellular Factor(s) Which Modulates Fusion/Entry of Macrophagetropic Human Immunodeficiency Virus Type 1

Monocytes/macrophages (M/M) and CD4⁺ T cells are two important targets of human immunodeficiency virus (HIV) infection. Different strains of HIV-1 vary markedly in their abilities to infect cells belonging to the M/M lineage. Macrophagetropic (M-tropic) HIV-1 strains replicate well in primary lymphocytes as well as in primary macrophages; however, they generally infect T-cell lines poorly, if at all. Although promonocytic cell lines such as U937 have been used as in vitro models of HIV-1 infection of M/M, these cell lines are susceptible to certain T-cell-tropic (T-tropic) HIV-1 strains but are resistant to M-tropic HIV-1. In this study, we demonstrate that (i) certain U937 clones (“plus” clones), which are susceptible only to T-tropic HIV-1, become highly susceptible to M-tropic HIV-1 upon differentiation with retinoic acid (RA); (ii) other U937 clones (“minus” clones), which are resistant to both T- and M-tropic HIV-1, remain resistant to both viruses; and (iii) RA treatment induces expression of CCR5, a fusion/entry cofactor for M-tropic HIV-1 in both types of U937 clones, and yet enhances the fusogenicity of the plus clones, but not the minus clones, with M-tropic Env’s. These results indicate that the major restriction of M-tropic HIV-1 infection in promonocytic cells occurs at the fusion/entry level, that differentiation into macrophage-like phenotypes renders some of these cells highly susceptible to infection with M-tropic HIV-1, and that CD4 and CCR5 may not be the only determinants of fusion/entry of M-tropic HIV-1 in these cells.     

Amino acid changes in the fourth conserved region of human immunodeficiency virus type 2 strain HIV-2ROD envelope glycoprotein modulate fusion.

The fourth conserved region (C4) of human immunodeficiency virus type 1 (HIV-1) surface glycoprotein has been shown to participate in CD4 binding and to influence viral tropism (A. Cordonnier, L. Montagnier, and M. Emerman, Nature [London] 340:571-574, 1989). To define the role of the corresponding region of HIV-2, we introduce single amino acid changes into the C4 sequence of HIV-2ROD. The effects of these mutations on glycoprotein function and on virus infectivity have been examined. We have shown that the tryptophan residue at position 428 is necessary primarily for CD4 binding. The isoleucine residue at position 421 is necessary for the establishment of productive infection in the promonocytic cell line U937, while it is dispensable to some extent for infection of primary T lymphocytes or the lymphocytic cell line SUP-T1. This replication defect correlated with the failure of the Ile-421-to-Thr (Ile-421-->Thr) mutant glycoprotein to form syncytia in U937 cells. DNA analysis of revertant viruses revealed that a strong selective pressure was exerted on residue 421 of the surface glycoprotein to allow HIV-2 infection of U937 cells. These results demonstrate that this region of HIV-2 plays an important role in determining fusion efficiency in a cell-dependent manner and consequently can influence viral tropism.     

A monoclonal antibody (12G5) directed against CXCR-4 inhibits infection with the dual-tropic human immunodeficiency virus type 1 isolate HIV-1(89.6) but not the T-tropic isolate HIV-1(HxB).

We used a monoclonal antibody (12G5) directed against an extracellular domain of CXCR-4 to investigate the role of this receptor in infection of immortalized lymphoid cell lines, peripheral blood mononuclear cells (PBMCs), and primary brain microglia with a dual-tropic strain of human immunodeficiency virus (HIV-1(89.6)) and a T-tropic strain (HIV-1(IIIB)). Addition of antibody 12G5 to cells prior to and during infection with HIV-1(89.6) inhibited p24 production 100- to 10,000-fold in CEMx174 and 174-CD4 cells and about 10-fold in PBMC cultures but had no activity against infection of either monocyte-derived macrophages or brain microglia. In contrast, 12G5 had little or no effect on infection of CEMx174 cells with HIV-1(IIIB) or HIV-1(HxB). To identify the region of the HIV-1(89.6) envelope that confers sensitivity to 12G5, we used chimeric molecular clones. Chimeras containing the V3 loop region of HIV-1(89.6) were inhibited by 12G5 to the same degree as wild-type HIV-1(89.6) whereas replication of those viruses containing the V3 loop of HIV-1(HxB) was not inhibited by the antibody. A similar pattern was seen in infections of a U87 glioblastoma line that coexpresses CD4 and CXCR-4. Antibody 12G5 was also able to block fusion between HeLa-CD4 cells and CEMx174 cells chronically infected with HIV-1(89.6) but had no effect on fusion mediated by cells chronically infected with HIV-1(IIIB). Taken together, these results suggest that different strains of HIV-1 may interact with different sites on CXCR-4 or may have different binding affinities for the coreceptor.     

Productive and lytic infection of human CD4+ type 1 helper T cells with macrophage-tropic human immunodeficiency virus type 1.

It is generally recognized that macrophage-tropic human immunodeficiency virus type 1 (HIV-1) is the predominant population during the acute and asymptomatic phases of HIV-1 infection. Here, we compared the proliferation and syncytium-inducing activities of different HIV-1 strains in primary CD4+ T cells expressing various helper T (Th)-type cytokine profiles. The macrophage-tropic HIV-1 strains HIV-1JR-CSF, HIV-1NFN-SX, and HIV-1SF162 could proliferate vigorously and generate syncytia in primary CD4+ T cells irrespective of their Th subtype, in contrast to the T-cell-line-tropic HIV-1 strains HIV-1NL4-3 and HIV-1IIIB, which favored non-type 1 Th conditions. These results indicate that macrophage-tropic HIV-1 may be more invasive and virulent, since it kills more CD4+ Th1 cells than T-cell-line-tropic HIV-1 during the early stages of HIV-1 infection, when the Th1 immune response is dominant.