Lethal mutagenesis of HIV with mutagenic nucleoside analogs.

The human immunodeficiency virus (HIV) replicates its genome and mutates at exceptionally high rates. As a result, the virus is able to evade immunological and chemical antiviral agents. We tested the hypothesis that a further increase in the mutation rate by promutagenic nucleoside analogs would abolish viral replication. We evaluated deoxynucleoside analogs for lack of toxicity to human cells, incorporation by HIV reverse transcriptase, resistance to repair when incorporated into the DNA strand of an RNA.DNA hybrid, and mispairing at high frequency. Among the candidates tested, 5-hydroxydeoxycytidine (5-OH-dC) fulfilled these criteria. In seven of nine experiments, the presence of this analog resulted in the loss of viral replicative potential after 9-24 sequential passages of HIV in human CEM cells. In contrast, loss of viral replication was not observed in 28 control cultures passaged in the absence of the nucleoside analog, nor with other analogs tested. Sequence analysis of a portion of the HIV reverse transcriptase gene demonstrated a disproportionate increase in G --> A substitutions, mutations predicted to result from misincorporation of 5-OH-dC into the cDNA during reverse transcription. Thus, "lethal mutagenesis" driven by the class of deoxynucleoside analogs represented by 5-OH-dC could provide a new approach to treating HIV infections and, potentially, other viral infections.

Activation of the HIV long terminal repeat and viral production by H2O2-vanadate.

The long terminal repeat (LTR) of human immunodeficiency virus type 1 (HIV-1) contains sequences required for the initiation of gene transcription. Among the substances known to activate the HIV-1 LTR is hydrogen peroxide (H2O2). We report here that H2O2-induced activation of the LTR in the macrophage cell line THP-1 and the lymphocyte cell line, Jurkat, is greatly increased by vanadate. Activation of the LTR by phorbol myristate acetate, tumor necrosis factor alpha, lipopolysaccharide, or Staphylococcus epidermidis extract was not increased by vanadate, indicating some selectivity for H2O2. H2O2 and vanadate also acted synergistically to increase the production of HIV-1 virions by the latently infected macrophage cell line U-1 as determined by p24 antigen release and the detection of intact virions by electron microscopy. Effects were observed at H2O2 and vanadate concentrations down to 3 x 10(-6) M, with high concentrations leading to cell toxicity. Catalase was strongly inhibitory when added prior to the interaction of H2O2 and vanadate, but was considerably less inhibitory when the H2O2 and vanadate were allowed to preincubate prior to the catalase addition. H2O2 reacts with vanadate to form peroxides of vanadate that have potent biological effects. Our findings suggest that among these is the activation of the HIV-1 LTR.

Inactivation of human immunodeficiency virus type 1 by the amine oxidase-peroxidase system.

Human immunodeficiency virus type 1 (HIV-1) is rapidly inactivated by exposure to a naturally occurring antimicrobial system consisting of peroxidase, H2O2, and a halide. Among the potential sources of H2O2 is the amine oxidase system in which mono-, di-, and polyamines are oxidatively deaminated with the formation of H2O2. The polyamine spermine is present at exceptionally high concentrations in semen. We report here that spermine, spermidine, and, to a lesser degree, the synthetic polyamine 15-deoxyspergualin are viricidal to HIV-1 when combined with amine oxidase and myeloperoxidase. Antiviral activity required each component of the spermine-amine oxidase-peroxidase system and was inhibited by azide (a peroxidase inhibitor) and by catalase but not by superoxide dismutase. Heat treatment of catalase largely abolished its inhibitory effect. These findings implicate H2O2 formed by the amine oxidase system in the antiviral effect and raise the possibility that the polyamine-amine oxidase-peroxidase system influences the survival of HIV-1 in semen and in the vaginal canal.

Activation of the human immunodeficiency virus long terminal repeat in THP-1 cells by a staphylococcal extracellular product.

Staphylococcal strains can release a factor that strongly activates the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) in THP-1 cells transfected with the HIV-1 LTR-driven luciferase reporter gene (THP-1 LTRluc). The factor is present in the overnight culture fluid and is readily released from the organisms into aqueous medium by vigorous mixing. Staphylococcal extracellular material is a complex mixture of polysaccharide and protein containing peptidoglycan and teichoic acid, released in part by cell wall turnover. The importance of the carbohydrate component is emphasized by concanavalin A (Con A) inhibition of staphylococcal product-induced LTR activation but not of activation by phorbol 12-myristate 13-acetate or tumor necrosis factor. The effect of Con A was decreased or abolished by sugars in the order methyl alpha-D-mannopyranoside > methyl alpha-D-glucopyranoside > mannose > glucose = fructose > N-acetylglucosamine. Wheat germ agglutinin was less inhibitory than Con A; in this instance N-acetylglucosamine decreased inhibition, whereas methyl alpha-D-mannopyranoside or methyl alpha-D-glucopyranoside did not. The induction of luciferase activity in THP-1 LTRluc by the staphylococcal extracellular product also was inhibited by fetal bovine and normal human serum. A comparison of 31 staphylococcal isolates (9 Staphylococcus aureus, 11 Staphylococcus epidermidis, 2 Staphylococcus haemolyticus, 4 Staphylococcus hominis, 2 Staphylococcus capitis, 2 Staphylococcus warneri, 1 Staphylococcus saprophyticus) revealed wide variation in LTR activating activity that did not correlate closely with slime production. Our findings, using induction of luciferase in THP-1 LTRluc as a model for upregulation of HIV infection, raise the possibility that staphylococci, as well as certain other microorganisms, release carbohydrate-containing exopolymers, which can activate the HIV-1 LTR, thus influencing progression of HIV infection.

Interleukin 4 and human immunodeficiency virus stimulate LFA-1-ICAM-1-mediated aggregation of monocytes and subsequent giant cell formation.

The effects of recombinant interleukin 4 (IL-4) on cell cluster and multinucleated giant cell (MGC) formation from human immunodeficiency virus (HIV)-infected and uninfected monocytes were examined. Human blood monocytes were isolated by centrifugal elutriation and monoclonal antibody-complement-dependent lysis of residual T cells, and infected with low passage HIV strains. Monocytes were exposed to recombinant IL-4 (1 to 20 ng/ml), continuously after inoculation with HIV. Monocyte expression of ICAM-1 but not LFA-1 was significantly enhanced by IL-4 although substrate adherence was a more potent stimulus. Monocyte cluster and MGC formation was quantified after fixation and staining with Giemsa. Clusters of HIV-infected and uninfected monocytes were consistently and significantly increased at 4 to 7 days after IL-4 stimulation. The combination of HIV and IL-4 was more stimulatory than either treatment alone. In two out of five uninfected and three out of seven HIV-infected monocyte cultures, MGC formation was also markedly increased at 10 to 14 days after stimulation. Incubation with anti-LFA-1 (anti-CD11a, anti-CD18) and anti-ICAM-1 (anti-CD54) monoclonal antibodies reduced IL-4-stimulated aggregation in HIV-infected and uninfected monocytes and subsequently reduced MGC formation. Anti-ICAM-1 was not as effective as anti-CD11a or anti-CD18 in inhibiting aggregation of HIV-infected monocytes and in these cultures anti-ICAM-2 was also inhibitory. Extracellular HIV antigen concentrations were not consistently reduced by anti-CD11a or anti-ICAM-1. Hence IL-4 markedly enhanced monocyte aggregation in both HIV-infected and uninfected monocytes, probably through enhanced LFA-1-ICAM-1 interactions in all cultures and LFA-1-ICAM-2 interactions in infected monocytes, leading subsequently to MGC formation in some cultures.

Human immunodeficiency virus type 1 infection of human placental macrophages in vitro.

Placental macrophages (Hofbauer cells) were isolated and cultured in vitro to investigate their susceptibility to human immunodeficiency virus type 1 (HIV-1) infection. Of adherent cells, 80% expressed CD14, and > 99% were nonspecific esterase-positive. CD4 antigen was expressed at very low levels. CD4 mRNA could be detected in the cells by reverse transcription followed by polymerase chain reaction. The macrophages were infected productively after inoculation with low-passage blood isolates of cell-free HIV-1. Peak virus titers were detected 3-7 days after infection by HIV-1 antigen ELISA and reverse transcriptase assay. Replication of HIV-1 in placental macrophages was less than in blood monocytes. HIV-1 RNA was detected in placental macrophages by in situ hybridization 16 days after infection. Multinucleated giant cells were identified in some cultures, indicative of an HIV-induced cytopathic effect. Thus, placental macrophages can be infected productively with clinical isolates of HIV-1, and such cells may act as a reservoir of virus for transmission to the fetus in utero.

Patients infected with human immunodeficiency virus type 1 have low levels of virus in saliva even in the presence of periodontal disease.

In two consecutive studies, 80 subjects human immunodeficiency virus (HIV)-1-seropositive (21 asymptomatic, 6 persistent generalized lymphadenopathy, 13 AIDS-related complex, and 40 AIDS) were examined for oral lesions. Paired serum and saliva specimens were tested for HIV isolation, DNA, and antigen. HIV antigen was detected in sera from 31 patients, but not in saliva. HIV was isolated from blood mononuclear cells of 83% and saliva supernatants of 21%. In the second study of 25 patients, HIV was detected in plasma of 56% (titers, 1/10 to > 1/1000) but not in diluted saliva supernatants, even in those with severe periodontal disease. HIV DNA was detected using polymerase chain reaction in 2 of 7 saliva cell pellets and 4 of 5 blood samples. Hence, infectious HIV and DNA was found at very low concentrations in 21% and 28% of HIV-seropositive patients, respectively, at all stages of HIV infection.

Identification of infection of an Australian resident with the human immunodeficiency virus type 2 (HIV-2).

OBJECTIVE: To present the first confirmed case of human immunodeficiency virus infection type 2 (HIV-2) in an Australian resident. CLINICAL FEATURES: HIV-2 infection in a west African man resident in Sydney was diagnosed in 1992 at Westmead Hospital, Sydney, by serological testing. He was asymptomatic and the blood CD4 T-lymphocyte concentration was not significantly reduced. Infection was probably acquired before migration to Australia. The patient was initially tested for HIV-1 antibody as part of an application for permanent residency. He was in no obvious risk group or transmission category. His serum was repeatedly positive by Genetic Systems enzyme immunoassay (EIA) and borderline by Abbott EIA, was reactive to the HIV-2 peptide on a synthetic envelope peptide assay, and was strongly reactive to all HIV-2 specific viral protein bands on an HIV-2 western blot test. HIV-2 was isolated by co-cultivation of the patient's peripheral blood mononuclear cells and identified by hybridisation using HIV-2 specific oligonucleotide probes, with further confirmation by polymerase chain reaction. INTERVENTION AND OUTCOME: The patient was counselled regarding the clinical course and prognosis of HIV-2 infection, the possible indications for zidovudine therapy, modes of transmission of the virus and safer sex precautions. CONCLUSIONS: This is the first documented case of HIV-2 infection diagnosed in Australia and raises the possibility of other undetected cases. The cost effectiveness of general testing for HIV-2 needs to be assessed and formal epidemiological sentinel programs should be established to monitor specific Australian populations.

Recombinant interleukin 4 stimulates human immunodeficiency virus production by infected monocytes and macrophages.

Recombinant interleukin 4 (IL-4) stimulated extracellular (EC) and intracellular (IC) production of human immunodeficiency virus (HIV) from infected human blood-derived monocytes and macrophages when incubated with the cells after but not before virus inoculation. Significant stimulation was observed in 20 of 27 experiments with monocytes (inoculated with HIV immediately after adherence) and 10 of 13 experiments with macrophages (inoculated after 5 days adherence) using a total of 30 normal donors of monocytes and macrophages, and 11 recent isolates of monocytotropic HIV strains (after one passage in mononuclear cells). Marked increases in EC and IC HIV antigen were observed in some experiments, which were comparable with the maximal stimulatory effects of other cytokines such as IL-2. IL-4 also had similar effects on infectious HIV concentration as measured by reverse transcriptase and TCID50 assays. Antibody to IL-4 prevented the stimulatory effect of the cytokine. The proportion of monocytes and macrophages infected by HIV, as determined by in situ hybridization, also increased after incubation with IL-4 for 7 days. The most marked effects were observed with HIV-infected macrophages, for which the proportion of unstimulated infected cells was lower (35 to 45% increasing to 66 to 70% with IL-4 treatment). There was also an increased proportion of cells with high granule concentrations, suggesting that IL-4 increases the intracellular concentration of viral nucleic acids. This was supported by semi-quantitative hybridization experiments showing that total HIV RNA increased in IL-4-stimulated monocytes 48 to 96 h after HIV inoculation. A marked increase in aggregates was observed on day 7 in HIV-infected monocytes treated with IL-4, compared to that in HIV-infected cells alone or IL-4-treated uninfected monocytes. These findings suggest that IL-4 stimulates HIV replication in the early phases of infection and may also facilitate virus transmission by aggregate formation.

HIV infection of monocytes inhibits the T-lymphocyte proliferative response to recall antigens, via production of eicosanoids.

Human monocytes infected in vitro with human immunodeficiency virus (HIV) soon after adherence to plastic substrate demonstrated a significantly decreased ability to restimulate autologous immune T-lymphocyte proliferation after exposure to soluble (tetanus toxoid) and particulate [herpes simplex virus (HSV)] antigen. Incubation with the cyclo-oxygenase inhibitor, indomethacin (2-5 microM), prevented inhibition of antigen-stimulated lymphocyte proliferation. The inhibitory activity was identified in ultrafiltrates containing the low molecular weight fraction (less than 3000 MW) of supernatants from HIV-infected monocyte cultures. This activity was significantly and markedly reduced in similar ultrafiltrates prepared from indomethacin-treated cultures. Increased concentrations of prostaglandin E2 (PGE2) were detected in ultrafiltrates from HIV-infected monocyte cultures compared with uninfected cultures and cultures preincubated with indomethacin. Ultrafiltrates were inhibitory when added during the presentation of antigen to T lymphocytes but not when removed from monocyte cultures prior to the addition of lymphocytes. In addition, ultrafiltrates inhibited antigen-stimulated lymphocyte proliferation and PHA-induced lymphocyte proliferation to the same extent. These data indicate that cyclo-oxygenase products of arachidonic acid, including PGE2, are produced in excess by HIV-infected monocytes and that PGE2 and perhaps other cyclo-oxygenase products are implicated in the inhibition of antigen-stimulated lymphocyte proliferation via a direct effect on T lymphocytes.

Variations in CD4 expression by human monocytes and macrophages and their relationships to infection with the human immunodeficiency virus.

The expression of CD4 antigen on the surface of LeuM3-positive human blood monocytes was found to be variable with 65 to 90% of cells from 46 normal human volunteers being positive by dual staining flow cytometry. When monocytes adhered to plastic (but not when cultured on Teflon), a marked decrease in CD4 expression was observed between 1 and 24 h post-adherence. CD4 expression could not be detected in macrophages adhered to plastic for 5 days by using four anti-CD4 monoclonal antibodies in flow cytometry or direct immunofluorescence. Conversely an increasing proportion of adherent cells expressed LeuM3 and OKM5 surface antigens over the 5 days. CD4 mRNA levels were measured by slot-blot and Northern hybridization, and total cellular CD4 protein levels by immunoprecipitation. Both cellular mRNA and CD4 levels remained constant throughout the 5 day period but membrane CD4 protein levels were greatly reduced indicating that the down-regulation of CD4 was post-translational. Infection with two of six fresh human immunodeficiency virus (HIV) isolates showed different kinetic patterns when tested on purified monocytes recently adhered to plastic and macrophages adherent for 5 days. HIV antigen and reverse transcriptase levels in infected monocyte cultures remained high for 3 to 4 weeks before detachment and necrosis of the cells occurred. Infection of macrophages generated much lower levels of antigen and reverse transcriptase which declined to very low or undetectable levels over 2 weeks, leaving persisting viable macrophages. One week after infection HIV nucleic acid was detected in 69 +/- 7% of monocytes and 6 +/- 3% of macrophages by in situ hybridization. Blocking experiments with anti-Leu3a monoclonal antibody suggested that HIV infection of 5 day adherent macrophages occurred mainly by a mechanism other than binding to CD4.

Effect of tuftsin on in vivo development of 3-methylcholanthrene-induced primary fibrosarcoma and Lewis lung carcinoma in mice.

The effect of tuftsin therapy on tumor development was examined in a murine primary fibrosarcoma and the Lewis lung carcinoma systems. Following im injection of 3-methylcholanthrene (CAS: 56-49-5) on day 0, C57BL/10ScSn mice were treated weekly with 3 ip tuftsin injections beginning on day 1 or day 60. Similar patterns of tumor development were observed regardless of whether tuftsin therapy was immediate or delayed. Only modest differences in experimental and control tumor incidences were found upon termination of studies; however, treated animals developed significantly fewer tumors than controls early during the observation periods. Thus mean tumor latent periods varied significantly when therapy began on day 1 (103.6 days in controls vs. 119.1 in treated mice; P = .02) or 2 months later (104.6 days in controls vs. 115.3 in treated mice; P = .01). One day subsequent to intra-footpad implantation of 10(5) Lewis lung carcinoma cells, C57BL/6 mice received at least 10 iv injections of tuftsin and were compared with controls for variations in survival or lung tumor development. The mean survival time in treated mice, 41.2 days, differed sharply from that (30.1 days) in controls (P = .00001). Similar groups of mice varied significantly in mean metastatic lung colony counts when examined on day 30; there were 15.1 colonies in controls and 8.0 in experimental animals (P = .03).