Herpes simplex virus type 1 can either suppress or enhance human immunodeficiency virus type 1 replication in CD4-positive T lymphocytes

It was proposed recently that CEM CD4-positive T cells infected chronically by herpes simplex virus type 1 (HSV-1) and human immunodeficiency virus type 1 (HIV-1) (CEM(HSV/HIV)) may be used as a model for studying HIV/HSV interactions. To ascertain whether HSV-HIV coinfection of T lymphocytes has a role in promoting progression of lentiviral infection, T cells infected chronically by either HSV-1 (CEM(HSV)) or HIV-1 (CEM(HIV)) were challenged with a superinfecting dose of HIV-1 or HSV-1. The results show a positive influence on HIV growth when CEM(HIV) cells were superinfected with HSV-1 to an extent that was dependent on the multiplicity of superinfection used. In contrast, HIV superinfection of CEM(HSV) cells resulted in a delay of HIV-1 production and in a lack of HSV-mediated LTR transactivation. These effects were due to cell growth inhibition and apoptosis, resulting from persistent HSV-1 infection. Treatment of CEM(HSV) with acyclovir inhibited completely the HSV-1 cytopathic effects and allowed efficient HIV-1 replication. These data may be relevant in clarifying the role of HIV/HSV interaction in the pathogenesis of AIDS.     

Human immunodeficiency virus infection of monocytic and T-lymphocytic cells: Receptor modulation and differentiation induced by phorbol ester

The monocytic leukemic cell line U937 can be infected with human immunodeficiency virus type 1 (HIV-1) to become permanently infected virus producers. Uninfected U937 cells express T4 (CD4) antigen and form syncytia when mixed with HIV-1 producing cells. Anti-T4 monoclonal antibodies block syncytium formation indicating that the HIV-1 receptors on U937 cells include T4 antigen. The promyelocytic leukemic cell line HL60, while expressing only low amounts of surface T4 and not forming syncytia on exposure to HIV-1, can be infected by HIV-1 at lower efficiency than U937 and T-cell lines. 12-O-Tetradecanoylphorbol-13-acetate (TPA) treatment induces the differentiation of U937 cells into macrophages. HIV-infected U937 cells retain the ability to differentiate, though less efficiently, as shown by the appearance of monocyte/macrophage surface markers. T4 antigen on both U937 and T-cell lines is down regulated by TPA treatment. Functional receptors for HIV-1, assayed by syncytium induction and pseudotype plating, are lost concomitantly with T4 antigen following TPA treatment of U937 cells and T cells.     

Productive infection of CD4+ cells by selected HIV strains is not inhibited by anti-CD4 monoclonal antibodies.

Differential susceptibility of four diverse HIV strains to inhibition of infection of CD4+ CEM cells by anti-CD4 monoclonal antibodies was studied. The highly cytopathic HIV-1 246 and NDK strains were able to infect CEM cells and undergo several cycles of replication at saturating doses of LEU3-A, OKT4-A, and 13B8-2 monoclonal antibodies, whereas propagation of reference HIV-1 BRU and weakly cytopathic strain HIV-1 PAS was inhibited. Postadsorption treatment by anti-CD4 antibodies had stronger inhibitory effect than did treatment during the virus adsorption period. In parallel experiments, the same monoclonal antibodies successfully blocked syncytium formation between uninfected MT4 cells and CEM cells infected by all four HIV-1 virus strains tested. To explain these seemingly contradictory data we have postulated that anti-CD4 antibodies efficiently inhibit cell-to-cell but not virus-to-cell infection.     

Restriction of HIV-1 replication in promonocytic cells: a role for IFN-alpha

A comparative study of the replication kinetics of human immunodeficiency virus type 1 (HIV-1) was performed in the promonocytic U937 cells and in the T lymphoblastoid H9 cells. If a productive HIV-1 infection of both cell types could be established, the time which elapses before most of the cells could express viral proteins is always proportionally longer for U937 cells than for H9 cells. Indeed, when U937 cells are infected with HIV-1, this nonproductive phase is followed by a lag phase during which the percentage of virus-producing cells is slowly increasing when compared to H9 cells. The restriction of HIV-1 replication in U937 cells might be consecutive to the lower adsorption of viral particles to these cells, even though the same percentage of U937 and H9 cells was expressing the CD4 molecule. Furthermore, we demonstrate that HIV-1 replication in U937 cells is mainly restricted by endogenous IFN-alpha. Indeed, addition of anti-IFN-alpha antibodies at the time of infection, during the nonproductive phase of the viral replication cycle, or during the lag phase leads to an earlier expression of viral proteins and/or to a rapid increase in the percentage of virus-producing cells. Likewise, the treatment of cultures of HIV-1 chronically infected U937 cells with the same antibodies induces an increased production of viral particles. Thus, IFN-alpha appears to be involved in the persistence of HIV-1 in the monocytes/macrophages of infected individuals.     

Cytokine-induced interleukin-8 production is depressed in chronic as opposed to acute human immunodeficiency virus 1 infection of promonocytic cells

Spontaneous secretion of interleukin 8 (IL-8) was higher in latently infected U1 cells than in acutely infected or uninfected parental U937 cells. However, the induction of IL-8 by various cytokines (IL-1 alpha, TNF-alpha, IL-6, TNF-beta, GM-CSF, IFN-gamma) was significantly reduced in U1 cells. Cytokine modulation of IL-8 production in U937 cells acutely infected with a T cell-tropic strain (IIIB) or monocytotropic strain (ADA) of human immunodeficiency virus 1 (HIV-1) (HIV-1IIIB and HIV-1ADA) was variable and showed strain-specific differences. The obtained results showed that the in vitro induction of IL-8 is impaired in promonocytic cells latently infected with HIV-1 and is differently modulated under acute conditions of infection depending on the IL-8 inducing cytokine and on the infecting virus strain.     

Interference patterns of human immunodeficiency viruses HIV-1 and HIV-2

The ability of cells infected with a retrovirus to interfere with superinfection by another retrovirus usually involves blockage, by the primary virus, of the receptors for the superinfecting virus. Retroviruses using different receptors do not interfere with each other, and this property has been used to classify various types of retroviruses. Different isolates of human immunodeficiency virus (HIV) were subjected to this type of analysis, and it was found that all HIV-1s cross-interfere with each other in T cells as well as in U937 promonocytic cells, substantiating further that all isolates use the same receptor on these cells. An HIV-2 isolate was found to interfere with HIV-1s, but HIV-1s only partially interfered with HIV-2 superinfection, indicating that inherent differences in receptor interactions exist between HIV-1s and HIV-2. For comparison, interference patterns of D-type primate retroviruses (SRVs) and murine amphotropic and xenotropic retroviruses revealed that each virus fell within distinct interference groups demonstrating that human T cells possess at least four distinct receptors for retroviruses. The mechanism of HIV interference was found to be due to receptor blockage in productively infected cells and to receptor elimination in latently infected T cells. Our findings that all HIV-1s completely interfere with each other and that interference occurs rapidly following acute infection suggests that a cell infected with HIV-1 will not permit reinfection by progeny or by other exogenous HIVs. This, in turn, suggests that progeny reinfection may not be the source of the large amount of unintegrated viral DNA observed following HIV cytopathic infection.     

Inhibition of HIV-1 transmission by interferon and 3'-azido-3'-deoxythymidine during de novo infection of promonocytic cells.

HIV-1 infection of promonocytic U937 cells was used to examined induction of IFN-alpha/beta gene expression and to determine the inhibitory effects of IFN-alpha 2 and/or AZT on de novo HIV-1 infection, initiated either by coculture of virus-shedding U9-IIIB cells with uninfected cells or by incubation of U937 cells with virus-containing supernatants. Usually 21-28 days were required to transmit virus to greater than 90% of the cell culture. HIV-1 infection did not stimulate constitutive production of endogenous IFN-alpha 1/alpha 2 or IFN-beta genes, although induction of IFN RNA was observed following coinfection with the paramyxovirus Sendai. Exogenous rIFN-alpha 2 treatment decreased the intracellular accumulation of viral RNA 5- to 20-fold as determined by Northern blot analysis and the extracellular levels of HIV-1 as measured by p24 ELISA antigen capture. The effect was most dramatic at a time coincident with the rapid increase in virus spread through the cell culture (Days 10-18). During the fourth week of infection, HIV-1 multiplication was able to overcome the IFN-induced block in virus spread. AZT was not effective in limiting virus spread in the cocultivation experiments. When U937 cells were infected with virus-containing supernatants from U9-IIIB cells, IFN and AZT acted in combination to limit the number of infected cells and to inhibit intracellular accumulation of viral RNA. These experiments suggest that subtle differences in the mode of virus transmission in monocytic cells may alter the efficacy of combination antiretroviral therapy.     

Antibody-mediated infection of macrophages and macrophage-like cell lines with 17D-yellow fever virus

The 17D vaccine strain of yellow fever virus (17D-YF) produces a safe human arboviral infection that can provide antisera of well-defined specificity under chronologically defined conditions. We studied 17D-YF growth in human peripheral blood macrophages and in two continuous Fc receptor-bearing, macrophage-like cell lines, P388D of mouse origin and U937 of human origin. Cells were infected with virus in the presence or absence of antibody to 17D-YF and to two related flaviviruses, St. Louis encephalitis (SLE) and dengue 2 (D2V). The virus 17D-YF grew in the three cell types when infection was established without antibody; viral yields were increased by addition of antibody to 17D-YF, SLE, and D2V. Increased titers of virus were accompanied by an increased number of infected cells by immunofluorescent assay. Enhancing activity was present in the IgG but not the IgM fractions of immune sera. Infection without cytopathic effect was observed in U937.     

Glycans and proteoglycans are involved in the interactions of human immunodeficiency virus type 1 envelope glycoprotein and of SDF-1alpha with membrane ligands of CD4(+) CXCR4(+) cells

We demonstrate that human immunodeficiency virus HIV-1(LAI) envelope glycoprotein 120 (gp120(LAi)) specifically interacts with several membrane ligands on lymphoid CEM or monocytic U937 cells in addition to its previously identified receptor, CD4, and CXCR4, its coreceptor. In its native state, gp120(LAI) is able to elicit specific multimolecular complexes with these membrane ligands at the surface of the cells; most of the interactions are abolished by mannan or heparin but not by dextran. Similarly, stromal cell-derived factor (SDF)-1alpha interacts not only with CXCR4 expressed by CXCR4(+) CD4(+) U937, CEM, and HOS-CD4(+) CXCR4(+) cells but also with CD4 expressed by intact U937, CEM, and HOS-CD4(+) CXCR4(+/-) cells or electroblotted onto Immobilon. SDF-1alpha binding to CD4(+) CXCR4(+/-) cells, or soluble CD4 electroblotted onto Immobilon, is significantly inhibited by sCD4, whereas truncated sCD4 lacking D3 and D4 domains had no significant effect, which indicates that SDF-1 binds to CD4 but at regions different from the HIV-gp120-binding site. Heparin and mannan also inhibit SDF-1alpha binding to intact CD4(+) CXCR4(+/-) cells, and electroblotted soluble CD4. Heparitinase treatment of such cells reduced SDF-1alpha binding. These data demonstrate that glycans and glycosaminoglycans are directly or indirectly involved in the interactions of HIV-1 gp120(LAI) and of SDF-1alpha with membrane ligands of CD4(+) CXCR4(+) cells and thus could play a role both in HIV-1 infection and in the physiology of SDF-1alpha.     

Cell surface antigens and function of monocytes and a monocyte-like cell line before and after infection with HIV

The human immunodeficiency virus (HIV-1) preferentially infects cells that express the CD4 molecule, including monocytes and cells of the monocyte lineage. The monocyte-like cell line U937 and monocytes isolated from peripheral blood lymphocytes (PBL) were infected with HIV-1. Cell surface antigen expression was determined in infected and noninfected cells as was the ability to stimulate in mixed lymphocyte reaction. The CD4 antigen decreased in infected cells U937 and PBL monocytes. MHC class II antigens HLA-DR, HLA-DQ, and HLA-DP increased in HIV-1 infected U937 cells. In infected PBL-derived monocytes, HLA-DR increased, HLA-DQ decreased, and HLA-DP was unchanged. Infected U937 and PBL monocytes were capable of stimulating allogeneic lymphocytes, thus demonstrating retention of the alloantigen presentation function of HIV-1-infected monocytes.     

A comparison of 2 T-lymphoid cell lines persistently infected with the human immunodeficiency virus (HIV-1) with different productive capacities]

T-lymphoid cell lines (H9/CBL-4 and CEM/CBL-4) persistently infected with HIV-1 were observed simultaneously for 6.5 months. The virus activity was characterized by such parameters as the number of infected cells determined by fluorescent antibody technique, the total level of virus--specific protein synthesis determined by immune blotting method, and the capacity to infect H9 and CEM cells. A comparative analysis of the two cell lines helped define the evaluation criteria for high and low productivity cultures. It was shown that a short-term virus persistence could exist in high-productivity cultures and long-term persistence in low-productivity cultures. The cytopathic activity of virus in cultures could be judged by accumulation of virus protein p24 in cell-free supernatants, this being one of the factors defining the efficacy of infection of H9 and CEM T-lymphoid cells.     

Characterization of herpes simplex virus persistence in a human T lymphoblastoid cell line.

Persistent, dynamic-state infection with herpes simplex virus (HSV) type 1 has been maintained in human T lymphoblastoid (CEM) cells for many months after initial infection with the wild-type virus (HSV0) (input virus/cell multiplicity of 1.0). Persistently infected cells grew as well as uninfected cells, except during occasional periods of crisis (increased viral replication and cytopathic effect). Cells could survive the crisis when they were maintained for twice the usual time interval (8 to 10 rather than 4 to 5 days) before subculture. Interferon was not detectable in the cultures. HSV0 was compared with HSVp1, a small plaque-forming isolate from persistently infected CEM cells. Primary infection of CEM cells with HSV0 at a low input multiplicity (0.01) led to abortive replication, whereas infection with HSVp1 at the same multiplicity resulted in either rapidly lytic or persistent infection depending upon the time interval of subculture. Approximately 55% of plaque-purified clones of HSVp1, as compared with only 5% of HSV0 clones, displayed temperature-sensitive growth in Vero cells. Defective interfering virus was not detectable in uncloned HSVp1 by interference assay. Persistently infected cultures "cured" by treatment with HSV antiserum or incubation at 39 degrees C were resistant to reinfection with HSV but permissive for vesicular stomatitis virus replication, suggesting that these treatments modulated a shift from the dynamic-state of the static-state, latent infection. These studies provide a model for characterization of HSV persistence and latency in a highly differentiated human cell line.     

Viral and cellular gene expression in CD4+ human lymphoid cell lines infected by the simian immunodeficiency virus, SIV/Mne.

Our laboratory has undertaken an analysis of cellular and viral gene expression in CD4+ human lymphoid cell lines infected by the human and simian immunodeficiency viruses, HIV-1 and SIV/Mne, respectively. The purpose of the current study was to: (i) examine the effects of SIV/Mne infection on host macromolecular synthesis and compare the results to those in the HIV-1 system; and (ii) investigate the mechanisms responsible for the restriction of SIV/Mne infection in CD4 positive lymphoid cells which are readily infected by HIV-1. First we determined that SIV does not impose selective blocks on host macromolecular synthesis, unlike HIV-1, which induces both the selective inhibition of cellular protein synthesis and the degradation of cellular mRNAs (Agy, M., Wambach, M., Foy, K., and Katze, M. G., 1990, Virology 177, 251-258). No such selective reduction in cellular mRNA stability or protein synthesis was observed in cells infected by SIV/Mne. Additional differences between SIV and HIV-1 were observed using a panel of CD4+ human cell lines. While HIV-1-infected all cell lines. SIV/Mne efficiently infected only the MT-4, C8166, and 174 x CEM cell lines. Repeated efforts to infect CEM or Jurkat cells were unsuccessful as determined by PCR analysis of viral DNA. HUT 78 cells supported a limited infection detectable only by PCR analysis. These data suggest the block in viral replication in the nonsusceptible cell lines is at an early step. Interestingly, all the SIV susceptible cells were virally transformed, C8166 and MT-4 by HTLV-1, and 174 x CEM by Epstein-Barr virus. Furthermore FACS analysis revealed that all susceptible cells expressed two B cell associated markers, B7/BB1 and CD40. These observations taken together highlight differences between the HIV and SIV viruses, and suggest that for efficient replication, SIV/Mne may require an additional cell surface molecule, cofactors provided by transforming viruses, or a complex interplay between the two.     

Human complement component factor B rescues HIV-1-infected leukemic T cells from cytopathic death

The addition of 5-10% of fresh normal human serum (NHS) from normal individuals into a culture of primarily HIV-1-infected CD4+ leukemic T-cell lines CEM and MT4 was found to rescue the infected cells from cytopathic death, enabling the cells to achieve growth within 10 days. The HIV-1-infected cells cultured in ordinary medium with fetal calf serum (FCS) all died within 10 days. The effect of NHS was ascribed to human complement component factor B and one or more factor B-dependent heat-labile co-factors. The cells which survived in the presence of NHS rapidly lost surface expression of CD4 and became completely resistant to rechallenge by HIV-1. Viral genomes were dramatically reduced in surviving cells within 30 days, and one cell-line CEM completely expelled them during this period. The results suggest that factor B has protective and potential therapeutic significance in HIV-1 infection.     

Type I interferon is a powerful inhibitor of in vivo HIV-1 infection and preserves human CD4(+) T cells from virus-induced depletion in SCID mice transplanted with human cells.

Although several studies are available on the in vitro inhibitory activities of type I interferon (IFN) on HIV-1 replication, the role of these cytokines in the pathogenesis of AIDS is still matter of conjecture. Both beneficial and adverse effects have been envisaged and considered as a possible rationale for the development of either IFN or anti-IFN therapies in HIV-1-infected patients. In the present study, we have evaluated the efficacy of human type I IFN on HIV-1 infection and virus-induced depletion of human CD4 T cells in two models established in SCID mice. In SCID mice transplanted with human U937 cells (U937-SCID mouse model), continuous treatment with type I consensus IFN (CIFN) resulted in a total suppression of HIV-1 infection. This inhibitory effect was superior to that obtained after AZT treatments. Results from an ensemble of experiments in SCID mice transplanted with either control or genetically modified human U937 cells transduced with a Tat-inducible IFN-alpha gene (LTR-IFN-A2 U937) indicated that low levels of IFN-alpha, produced locally as a result of virus infection, were extremely effective in inhibiting acute HIV infection and virus replication. Of interest, LTR-IFN-A2 U937 cells conferred a strong anti-HIV-1 protection to coinjected bystander U937 cells. Notably, experiments with SCID mice reconstituted with human PBL (hu-PBL-SCID mouse model) showed that treatment with CIFN inhibited HIV-1 replication more effectively than AZT treatment. Remarkably, treatment with CIFN resulted in a clear-cut protection from the virus-induced depletion of human CD4 T cells, which was also associated with the generation of an antibody response toward HIV-1 antigens in 50% of the virus-injected xenografts. These results suggest that type I IFN efficiently preserves human CD4(+) cells from virus-induced damage in hu-PBL-SCID mice, not only by inducing an antiviral state in target cells but also by stimulating anti-HIV-1 human immune responses in vivo.     

Titration of human immunodeficiency virus type 1 (HIV-1) and quantitative analysis of virus expression in vitro using liquid RNA-RNA hybridization

An assay is described for titration of human immunodeficiency virus type 1 (HIV-1) and for quantitative analysis of virus expression in vitro. The assay utilizes a liquid RNA-RNA hybridization method coupled with reversible target capture (RTC) on oligo(dT) derivatized magnetic particles. The assay provides a rapid, specific, and sensitive method for quantitation of HIV-1 RNA molecules present either in cells or in viral particles from cell-free culture media. Chronically infected monocytoid U1.1 cells were found to carry 52 pg HIV-1 RNA per 200,000 cells (160 HIV-1 RNA molecules per cell). In contrast, acutely infected CEM and H9 cells carried 3010 and 4370 pg HIV-1 RNA per 200,000 cells (9040 and 13,110 HIV-1 RNA molecules per cell, respectively). No hybridization was observed with uninfected cells or cells infected with HIV-2, HTLV-I, HTLV-II, or EBV. Use of liquid HIV-1 RNA hybridization in association with HIV-1 protein detection methods permits more complete characterization of HIV-1 expression in host cells than either method alone, and also provides a method for standardizing preparations of virus.