Effect of serum components on syncytium formation and virus production by cells infected with human immunodeficiency virus in vitro.

Previously it has been reported that cocultivation of human immunodeficiency virus type 1 (HIV-1)-infected cells with uninfected cells results in formation of multinuclear giant cells, generated via an interaction of gp120 on the surface of infected cells with CD4 on the uninfected cells. Formation of multinuclear giant cells as occurring in the presence of normal fetal calf serum was not observed when HIV-infected MOLT-4 or MOLT-3 cells (chronically infected with HTLV-IIIB) and uninfected cells were cocultured in both serum-free medium and fibrinogen-depleted serum. Addition of sera (human and rabbit) as well as of fibrinogen (human and bovine), fibronectin (human), and alpha-globulin (human), but not of albumin, transferrin or gamma-globulin to serum-free medium caused formation of multinuclear giant cells. In contrast, HIV production from MOLT-3 cells proceeds also in the absence of serum. In control experiments it was established that the cells maintained at reduced serum concentration, or in serum-free medium without or with fibrinogen are viable even though displaying a lower metabolic rate (ATP formation and DNA synthesis). From these findings we conclude that serum components (e.g., fibrinogen, fibronectin, and alpha-globulin) are absolutely required for syncytium formation but are not essential for virus release.

Formation of a small ribonucleoprotein particle between Tat protein and trans-acting response element in human immunodeficiency virus-infected cells.

The trans-acting response element (TAR) within the long terminal repeat of human immunodeficiency virus (HIV) is present in all 5' termini of HIV mRNAs and is recognized by the viral Tat protein. Now we describe that the 59-nucleotide-long TAR-RNA exists as a ribonucleoprotein particle in polysomal and heterogeneous nuclear RNP fractions of HIV-1-infected HeLa-T4+ cells. Applying an immunoprecipitation technique this Tat.TAR complex could be isolated from total cell extracts as well as from polysomal or heterogeneous nuclear RNP fractions. The chain length and the identity of the TAR-RNA were established by RNase protection assays while the Tat protein was confirmed by Western blotting technique. The TAR-RNA in this complex was sequenced and found to comprise nucleotides +2 to +61 and hence includes the 3-nucleotide bulge (nucleotides +23 to +25) and the loop sequence of the TAR stem-and-loop structure. The Tat.TAR complex is present in cells at low abundance (12.5 x 10(3) copies/cell). In contrast to the TAR-containing mRNAs, which decay very rapidly after incubation of cells with actinomycin D (half-life of approximately 120 min) the half-life of TAR in the Tat.TAR complex is greater than 180 min. Alignment studies revealed that TAR-RNA (positive strand) has a potential binding ability to the U5 region within the long terminal repeat (DNA negative strand; nucleotides +107 to +147); a complementary binding with a continuous homology of 16 nucleotides was identified. It is proposed that the Tat.TAR complex functions as a small ribonucleoprotein particle during transcription initiation of HIV mRNA.

Evidence for a direct interaction of Rev protein with nuclear envelop mRNA-translocation system.

The interaction of the Rev protein from human immunodeficiency virus type 1 (HIV-1) with the nucleocytoplasmic mRNA-transport system was investigated. In gel-shift assay, the recombinant Rev protein used in this study selectively bound to the Rev-responsive element (RRE) region of HIV-1 env-specific RNA. Nitrocellulose-filter-binding studies and Northern/Western-blotting experiments revealed an association constant of approximately 1 x 10(10) M-1. The Rev protein also strongly bound to isolated nuclear envelopes from H9 cells, containing the poly(A)-binding site (= mRNA carrier) and the nucleoside triphosphatase (= NTPase), which are thought to be involved in nuclear export of poly(A)-rich mRNA. Binding of 125I-Rev to a 110-kDa nuclear-envelope protein, the putative mRNA carrier, could be demonstrated in in vitro experiments. Both efflux of cellular poly(A)-rich RNA, such as actin RNA [but not efflux of poly(A)-free RNA] from isolated nuclei and the nuclear-envelope NTPase activity were strongly inhibited by Rev protein. On the other hand, transport of viral env RNA, containing the Rev-responsive element, was increased in the presence of Rev. Studying the release of RNA from closed nuclear-envelope vesicles containing entrapped RNA, the action of Rev was found to occur at the level of translocation of RNA through the nuclear pore. Evidence is presented that Rev down-regulates the NTPase-driven transport of mRNA lacking the RRE, most likely via binding to the mRNA carrier within the envelope. In contrast to the efflux of RRE-free RNA, ATP-dependent efflux of RRE-containing RNA from resealed nuclear-envelope vesicles was found to be increased, if the RNA was entrapped in the vesicles together with Rev protein. In addition, it was found that phosphorylated Rev, which is transported together with RRE-containing RNA out of the vesicles, becomes dephosphorylated during transport. In the vesicle experiments it is demonstrated for the first time that a protein selectively channels a specific mRNA across the nuclear-envelope pore complex.

Human immunodeficiency virus: novel enzyme-linked immunoassays for quantitation of envelope glycoprotein 120.

Two novel enzyme-linked immunoassays (ELISA) for the quantitation of human immunodeficiency virus type 1 (HIV-1) coded glycoprotein with an Mr 120 (gp120) are described. These are based on the highly specific interaction between gp120 and the mannose-specific lectins from Narcissus pseudonarcissus (NPL) and Galanthus nivalis (GNL). Two systems were developed: (1) an HIV-protein ELISA using HIV-protein (also containing HIV-gp120) for the solid phase and NPL as a detector and (2) a lectin-ELISA using the NPL bound to the solid phase and GNL as detector. The HIV-protein ELISA was validated for quantitation of gp120 within the range 3 to 600 ng/ml; the lectin-ELISA for concentrations between 0.6 and 20000 ng gp120/ml. Serum components did not interfere with the binding of gp120 to the lectins. The ELISAs were used for the quantitation of gp120 in HIV-infected CEM cells in vitro. It was found that gp120 appeared in the medium earlier after infection than HIV-p24 and reverse transcriptase, suggesting that gp120 is released as free glycoprotein. Moreover, the ELISAs were also applied successfully for the detection of compounds that bind to gp120 and for the identification of antibodies directed against the highly pathogenic mannan portion of gp120. These ELISAs are considered to be suitable also for the detection of gp120 in the serum of HIV-infected individuals.

Cordycepin analogues of 2',5'-oligoadenylate inhibit human immunodeficiency virus infection via inhibition of reverse transcriptase.

Analogues of 2',5'-oligoadenylates (2-5A), the cordycepin (3'-deoxyadenosine) core trimer (Co3) and its 5'-monophosphate derivative (pCo3), were shown to display pronounced anti-human immunodeficiency virus type 1 (HIV-1) activity in vitro. Treatment of HIV-1 infected H9 cells with 1 microM Co3 or pCo3 resulted in an almost 100% inhibition of virus production. The compounds were encapsulated in liposomes targeted by antibodies specific for the T-cell receptor molecule CD3. Substitution of one or two cordycepin units in Co3 or pCo3 decreased the antiviral activity of the compounds. pCo3 did not stimulate 2-5A-dependent ribonuclease L activity and displayed no effect on the amount of cellular RNA and protein. At a concentration of 10 microM the cellular DNA polymerases alpha, beta, and gamma were almost insensitive toward Co3 or pCo3. In contrast, these compounds reduced the activity of HIV-1 reverse transcriptase (RT) by 90% at a concentration of 10 microM if the viral RNA genome and the cellular tRNALys.3 was used as template/primer system; if the synthetic poly(A).(dT)10 was used as template/primer, no marked inhibition was observed. Dot-blot, gel-retardation, and cross-linking assays showed that Co3 or pCo3 interfere with the binding site of tRNALys.3 to RT. These results indicate that inhibition of RT at the level of initiation of the enzymic reaction is a novel approach to inhibit HIV-1 replication.

Antibodies against defined carbohydrate structures of Candida albicans protect H9 cells against infection with human immunodeficiency virus-1 in vitro.

In this study we raised antibodies against Candida albicans mannans of serotype A and B which comprise mannose alpha(1----2)-and mannose alpha(1----3)-linked residues. These antibodies inhibited human immunodeficiency virus type IIIB (HIV-IIIB) infection of H9 cells in vitro; 5 micrograms/ml of antibodies against mannan from serotype A and 10 micrograms/ml of antibodies against serotype B mannan were sufficient to inhibit infection by almost 100 and 85%, respectively, after an incubation period of 4 days. During a prolonged incubation period (8-12 days), the amount of HIV particles (as measured by reverse transcriptase activity in the culture medium) increased again in assays with antibodies raised against serotype B, but only little in assays containing antibodies against serotype A. Applying the Western blotting technique and a novel enzyme-linked immunosorbent assay system it was established that the antibodies reacted with the gp120 of HIV-1 exclusively. Immunofluorescence inspection using a confocal laser scanning microscope revealed that the gp120 protein is exposed on the outer surface of H9 cells where it is recognized by the anti-mannan antibodies. These results indicate that mannan residues of C. albicans can serve as antigens to raise neutralizing antibodies against HIV infection.

Susceptibility of primary human glial fibrillary acidic protein-positive brain cells to human immunodeficiency virus infection in vitro: anti-HIV activity of memantine.

Primary human glial fibrillary acidic protein-positive (GFAP+) brain cells (enriched population) have successfully been infected with human immunodeficiency virus type 1 (HIV-1) in vitro, when cocultivated with HIV-1-producing H9 cells. Direct incubation of brain cells with HIV-1 resulted only in a limited infection. The percentage of HIV+ cells increased from 5% in passage 1 to 40% in passage 8. Simultaneously with the increase of infected cells, the reverse transcriptase activity in the culture medium increased and reached maximal values in passage 8. The infected cells also produced intact viral particles. In the early phase of cultivation the HIV-infected cells displayed a significantly higher proliferation rate than the uninfected controls. At passage number 8 the HIV-infected GFAP+ cells had almost totally lost the ability to grow, while the controls proliferated at a rate almost unimpaired from the beginning of the cultivation. Up to 10 to 15% of the HIV-infected GFAP+ cells contained at passage number 5 more than 3 nuclei. Memantine (1-amino-3,5-dimethyladamantane), a blocker of the N-methyl-D-aspartate receptor channels, was found to display a significant anti-HIV effect (at a concentration of 1 microgram/ml) on enriched cultures of GFAP+ cells in vitro.

Potent inhibition of hepatitis B virus production in vitro by modified pyrimidine nucleosides.

2',3'-Dideoxy-3'-fluorothymidine (FddThd), 2',3'-didehydro-2',3'- dideoxythymidine (ddeThd), and 3'-fluoro-5-methyl-deoxycytidine (FddMeCyt) are, in their triphosphate forms, selective inhibitors of human immunodeficiency virus type 1 reverse transcriptase. We report that 0.3 microM FddThd, FddMeCyt, or ddeThd as well as 3'-chloro-5-methyl-deoxycytidine (ClddMeCyt) or 3'-amino-5-methyl-deoxycytidine (AddMeCyt) almost completely blocked production of hepatitis B virus (HBV) particles by HBV DNA-transfected cell line 2.2.15 in vitro. Only at an at least 10-fold-higher concentration was a cytotoxic effect observed. These results indicate that FddThd, FddMeCyt, ClddMeCyt, AddMeCyt, and ddeThd are potent anti-HBV agents in vitro.

Sulphoevernan, a polyanionic polysaccharide, and the narcissus lectin potently inhibit human immunodeficiency virus infection by binding to viral envelope protein.

Sulphoevernan is a sulphated alpha-1----3, 1----4 polyglucan (Mr 20,000) with a helical structure. This compound effectively inhibits both human immunodeficiency virus type 1 (HIV-1) and type 2 infection of cells in vitro at concentrations around 0.5 micrograms/ml. Moreover, the compound completely inhibits HIV-1-induced syncytium formation at a concentration of 1 microgram/ml. Competition experiments with 35S-labelled sulphoevernan revealed that the mannose-specific lectin from Narcissus pseudonarcissus prevented binding of sulphoevernan to HIV-1, whereas the antibody OKT4A did not reduce the amount of sulphoevernan bound to MT-2 cells. These data indicate that the non-cytotoxic polymer sulphoevernan binds to the virus rather than to the host cell. In vivo studies, using Rauscher leukaemia virus in NMRI mice, revealed that, at a daily dose of 20 mg/kg, the animals were protected against virus-induced increases in spleen weight. From these in vitro and in vivo data we conclude that sulphoevernan has potential in the treatment of acquired immunodeficiency syndrome.

Alteration of DNA topoisomerase II activity during infection of H9 cells by human immunodeficiency virus type 1 in vitro: a target for potential therapeutic agents.

Infection of H9 cells with human immunodeficiency virus type 1 (HIV-1) was found to decrease the phosphorylation of DNA topoisomerase II during the initial phase of infection. Simultaneously, with a later overshoot of phosphorylation and the subsequent activation of DNA topoisomerase II, the production of HIV-1 started. Applying three new protein kinase C inhibitors from the class of O-alkylglycerophospholipids we demonstrated that inhibition of protein kinase C-mediated phosphorylation of DNA topoisomerase II resulted in an inhibition of HIV-1 production. Based on the differential effect of the two protein kinase C activators, phorbol ester and bryostatin, we conclude that phosphorylation of DNA topoisomerase II is mediated by the form alpha and gamma of protein kinase C. These data suggest that agents which inhibit these two forms of protein kinase C are also potential candidates for an anti-HIV therapy.

Intracellular signal transduction pathways in sponges.

Sponges are the lowest multicellular eukaryotic organisms. Due to the relatively low specialization, and concomitantly the high differentiation and dedifferentiation potency of their cells, the sponge cell system has proven to be a useful model to study the mechanism of cell-cell adhesion on molecular levels. Results of detailed biochemical and cell biological studies with the main cell adhesion molecules, the aggregation factor (AF) and the aggregation receptor, led to the formation of the modulation theory of cell adhesion. The events of cell adhesion are contigent on a multiplicity of precisely coordinated intracellular signal transduction pathways. Using the marine sponge Geodia cydonium we showed that during the initial phase of cell-cell contact the AF causes a rapid stimulation of the phosphatidylinositol pathway, resulting in an activation of protein kinase C and a subsequent phosphorylation of DNA topoisomerase II. As one consequence of these processes, the cells undergo a phase of high DNA synthesis. However, at later stages, the AF loses its mitogenic activity; this function is then taken over by the matrix lectin. During this switch, the lectin receptor associates in the plasma membrane with the ras oncogene product. The description of these processes is subject of this review article.