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Electromagnetic signals of low frequency have been shown to be durably produced in aqueous dilutions of the Human Imunodeficiency Virus DNA. In vivo, HIV DNA signals are detected only in patients previously treated by antiretroviral therapy and having no detectable viral RNA copies in their blood. We suggest that the treatment of AIDS patients pushes the virus towards a new mode of replication implying only DNA, thus forming a reservoir insensitive to retroviral inhibitors. Implications for new approaches aimed at eradicating HIV infection are discussed.

The discovery of HIV in 1983 is a landmark of medical science in the 20th century. HIV is a fragile but stealthy virus that thrives within the cells of the immune system itself, subverting the body's defences against disease. Knowing the structure and life cycle of the virus is the key to understanding how it is transmitted, how it causes disease and how best to prevent or control infection.

A microtransfection method, using either the DEAE-dextran or the Ca.phosphate procedure has been developed. A plasmid expressing the luciferase-encoding gene under the control of the human immunodeficiency virus (HIV) LTR promoter was constructed. Transfections were performed in 96-well plates, allowing statistical evaluation of the results. This microtransfection method requires the use of 100- to 1000-fold less plasmid and cells than in a conventional chloramphenicol acetyl transferase (CAT) assay. A Luciferase index which takes into account cell viability after transfection has been defined using a semi-automated absorbance assay. A 20-h incubation period post-transfection is sufficient for optimal results. Basal long terminal repeat activity and autologous Tat transactivation were studied in various lymphoid, monocytic and adherent human cell lines. Infection of microtransfected cells by HIV activated luc expression. This assay can thus also be used for rapid detection and quantitation of HIV. Antiviral activities of drugs can be assessed in a two-day test.

The 177 N-terminal amino acids of CD4, the receptor of the human immunodeficiency virus (HIV), have been expressed in Escherichia coli as genetic fusions to the periplasmic maltose-binding protein (MalE) from this organism. A large fraction of the hybrid proteins can be released from the periplasm by osmotic shock and purified in one step on a cross-linked amylose column eluted with maltose under mild conditions. One hybrid protein binds HIV envelope protein gp160 and neutralizes the virus in vitro. This provides the first example of the production and one-step purification of an active form of an eukaryotic protein by fusion to MalE. The use of this system for mass screening of CD4 mutants, high-scale production of the hybrid protein for structural studies on CD4, testing antiviral compounds, and therapeutic assays is discussed.

Antibodies prepared against a peptide corresponding to the site of cyto-adherence of Mycoplasma genitalium adhesine inhibit or reduce the infectivity of the HIV-1BRU and HIV-2ROD strains of Human Immunodeficiency Virus in lymphoid cells. These results strengthen the hypothesis that some mycoplasmas may play an important part in HIV replication and pathogenicity.

A heterogenous population of envelope glycoprotein-specific cytotoxic effector cells are found in the peripheral blood of individuals infected with HIV-1, and in many cases env-specific lysis is not restricted by MHC molecules and is not blocked by antibody to CD3 (Rivière, Y. et al., J. Virol. 1989, 63:2270). In order to detect env-specific cytotoxic T lymphocytes (CTL) in fresh peripheral blood mononuclear cells of HIV-1-infected donors, a mutant env gene with deletion of the amino-terminal signal sequence was inserted into vaccinia virus. This deletion of the amino-terminal signal sequence was inserted into vaccinia virus. This deletion results in synthesis of an envelope protein that is not glycosylated and not expressed at the surface of infected cells. Target cells infected with this recombinant vaccinia virus are not lysed by antibody-mediated cellular cytotoxicity, but they are recognized by secondary CTL. Comparing lysis of target cells expressing gp160 of HIV-1 and the signal peptide deletion mutant, primary env-specific CTL were detected in some individuals infected with HIV-1.

The recently described human immunodeficiency virus type 2 (HIV2) is significantly divergent in sequence from the more frequently isolated human immunodeficiency virus type 1 (HIV1). Both HIV1 and HIV2 encode a transactivator that is capable of strongly stimulating expression directed by the viral long terminal repeat (LTR). Here, we define the region of the HIV2 genome encoding the transactivator and show that the specificity of the transactivator differs from that of HIV1. By deletion analysis of the HIV2-LTR, we show that both HIV1 and HIV2 transactivators require sequences within 35 to 53 bp downstream of the start of transcription. However, in order to stimulate expression at full efficiency, the HIV2 transactivator further requires sequences unique to the HIV2-LTR between nucleotides +53 and +99. Hence, HIV2 poorly transactivates the LTR of HIV1, while two divergent isolates of HIV1 will efficiently transactivate the LTR of either HIV1 or HIV2. Nonetheless, in vivo competition between the transactivators of HIV1 and HIV2 suggests that they use a common mechanism.

HIV-infected individuals progress toward AIDS despite the early elicitation of a specific immune response. Analysis of the isotypic distribution of HIV-specific antibodies appears of special interest for two reasons: first, isotypic diversity is partly under the control of antigen-specific T-helper cells, the very cells infected by HIV; second, isotype determines antibody functions, effector (neutralization, antibody-dependent complement, or cell-mediated cytotoxicity) as well as blocking functions. We have investigated by Western blot analysis the isotypic profile of the antibody response to HIV structural proteins (env, gag, pol) and to the nonstructural protein F (3' orf), which is absent from the virion and might primarily target infected cells. In 115 asymptomatic individuals, infected by sexual contact (homosexual men) or intravenously (hemophiliacs), the response to gag-products was polyisotypic, including IgM, IgG1, IgG3 and IgA; the response to F was more restricted (IgM, IgG1, IgA) and the response to env strikingly restricted to the IgG1 isotype, suggesting different regulatory mechanisms in the B-cell response to these proteins. The isotypic distribution was also influenced by the route of infection, IgG4 and IgE (gag-specific) being exclusively elicited in the hemophiliac group. Finally, observations of potential diagnostic interest were made in a limited number of at-risk individuals; these included the presence of gag- and pol-specific IgM or IgA in the absence of any HIV-specific IgG isotypes; and the presence of gag- and F-specific antibodies in the absence of env-specific antibodies, suggesting the early occurrence of both isotypic and antigenic selection mechanisms during the course of HIV infection.

Purified streptococcal mitogens (SMs) including erythrogenic exotoxin were compared with phytohemagglutinin (PHA) for their ability to sustain lymphadenopathy-associated virus (LAV) replication after the stimulation of normal human peripheral blood mononuclear cells and purified CD4+ and CD8+ T cells infected with LAV. Both SM and PHA supported LAV production in peripheral blood mononuclear and CD4+ cells but not in CD8+ cells. LAV production assessed by the assay of reverse transcriptase in cell supernatants appeared earlier after stimulation with SM and was 6- to 10-fold greater than after stimulation by PHA.

The Human Immunodeficiency Virus (HIV) displays a selective tropism for cells expressing the CD4 molecule which, by itself, represents at least part of the specific receptor for this virus. However, modification of the activation state of each individual cell seems critical not only for virus replication but also for its binding and subsequent penetration into its target. We demonstrate here that Cyclosporin-A (CSA), a drug which inhibits IL-2 dependent T-lymphocyte proliferation and differentiation and which is known for its immunosuppressive activity, can prevent subsequent virus binding to cells otherwise susceptible to HIV. Normal T-lymphocytes were preincubated in vitro with CSA at concentrations that were in the same range than those reached in the serum of treated patients. This resulted in the complete disappearance of HIV receptors (HIV-R), as assessed by the direct measure of specific binding of fluoresceinated HIV (HIV-FITC), and in the subsequent inhibition of HIV replication in cultured cells. Moreover CSA pretreatment of IL-2 independent transformed cells derived from the CEM line, before their infection, strongly inhibited HIV adsorption as well as further virus replication. These results provide a new experimental basis for the potential application of CSA in the treatment of HIV-related diseases.

The neutralization properties of three independent HIV-2 isolates were examined in comparison with four diverse HIV-1 strains. Human sera containing antibodies specific to HIV-2 can cross-neutralize HIV-1. By contrast, HIV-1 sera are group-specific and have no neutralizing effect on HIV-2. Therefore, HIV-2 antigens may be important components for the development of broadly cross-protective AIDS vaccines.

gp120 and CD4 are two glycoproteins that are considered to interact together to allow the binding of HIV to CD4+ cells. We have utilized enzymatic digestion by endoglycosidases in order to analyze N-linked carbohydrate chains of these proteins and their possible role in the interaction of gp120 or gp160 with CD4. SDS denaturation was not necessary to obtain optimal deglycosylation of either molecule, but deglycosylation of CD4, nonetheless, depended on the presence of 1% Triton X-100. Endo H and Endo F that cleave high mannose type and biantennary glycans diminish the molecular mass of the glycoproteins from 120 or 160 Kd to 90 or 130 Kd, respectively; but these enzymes had no action on CD4 glycans. Endo F N-glycanase mixture, which acts on all glycan species, including triantennary chains, led to complete deglycosylation of gp120/160 and of CD4. Therefore, probably half of the glycan moieties of gp120/160 are composed of high mannose and biantennary chains, the other half being triantennary species. The carbohydrate structures of CD4 seems to be triantennary chains. To analyze the binding of gp120/160 to CD4, we used a molecular assay in which an mAb (110-4) coupled to Sepharose CL4B allowed the attachment of soluble gp120/160 to the beads; 125I-sCD4 was then added to measure the binding of CD4 to different amounts of gp120/160. Binding to gp160 was not modified when using completely deglycosylated 125I-sCD4, while deglycosylation of gp120 or of gp160 resulted in the decrease of the binding to native CD4 by two- and fivefold, respectively. Native and deglycosylated gp120/160 bound to CD4+ cells with comparable affinities. In addition, deglycosylated gp120 displaced 125I-gp160 binding to CD4+ cells and inhibited fusion of fresh Molt-T4 cells with CEM HIV1- or HIV2-infected cells to the same extent. Taken together, these results indicate that carbohydrates of CD4 and of gp120/160 do not play a significant role in the in vitro interaction between these two molecules.

Previous reports have shown the capacity of diphenylhydantoin (DPH) to attach to the membranes of lymphatic cells as a hapten and thus exert an unspecific influence on their ability to express certain recognition molecules. This led us to the hypothesis, that DPH might as well serve to manipulate the t-helper-lymphocytes in a way that the mode of infection of these cells by the HIV might be blocked. In order to verify this hypothesis, we exposed normal control lymphocytes as well as lymphocytes from DPH-treated patients (3 X 100-150 mg DPH/day, Phenhydan, for a minimum of 10 days) to radioactively labeled HIV (125I). Remaining radioactivity was assessed using a gamma-counter and measured 64.000-92.000 counts/min (n = 24, mean 80.000) for the control lymphocytes, while remaining radioactivity for the DPH-treated lymphocytes ranged between 2000 and 7000 counts/min (n = 24, mean 4.000, p less than 0.001). These results and similar experiments obtained with FITC-labeled HIV led us to the conclusion that DPH inhibits HIV recognition of T-lymphocytes and therefore might be used in therapy and prophylaxis of AIDS.

Infection by the human immunodeficiency virus (HIV) is initiated by the binding of its extracellular envelope glycoprotein, gp120, to the CD4 antigen on target cells. To map the residues of the HIV-1 glycoprotein that are critical for binding and to analyse the effects of binding on viral infectivity, we created 15 mutations in a region of gp120 that is important for binding to CD4 (refs 4,5). We find that substitution of a single amino acid (tryptophan at position 432) can abrogate CD4 binding and that virus carrying this mutation is non-infectious. By contrast, other amino-acid changes in the same region do not affect CD4 binding but restrict viral tropism: virions containing isoleucine substitutions at position 425 lose their ability to infect a monocyte cell line (U937 cells) but can still infect T-lymphocyte cell lines (CEM, SUP-T1) and activated human peripheral blood lymphocytes. These results indicate that cellular tropism of HIV can be influenced by a single amino-acid change in gp120.

Apart from the retroviral gag, pol and env the HIV genome contains the F (3' orf) gene which encodes a polypeptide of 206 amino acids which is myristylated at the N-terminal and whose function is unknown. We have expressed the F gene in Escherichia coli and from a recombinant vaccinia virus, VVTGfHIV. The F-protein produced in VVTGfHIV-infected mammalian cells is myristilated, and is phosphorylated by protein kinase C at a residue close to the N-terminus like pp60-src (ref. 5). Purified bacterial F-protein also shows the GTPase, autophosphorylation and GTP-binding activities reported for the ras gene product. Furthermore, we show that expression of F in a CD4+ cell line down-regulates the CD4(T4) antigen. These results suggest that F is important in the pathophysiology of AIDS (acquired immune deficiency syndrome).

The cellular receptor for HIV-1 is the leucocyte differentiation antigen, CD4. Blocking of HIV-1 infectivity can be achieved with monoclonal antibodies (MAbs) to some, but not all epitopes of this antigen. We demonstrate here, by inhibition of virus infection, blocking of syncytium formation and inhibition of pseudotype infection with a panel of CD4 MAbs, that HIV-1, HIV-2 and simian immunodeficiency virus (SIV) isolates share the same cellular receptor, the CD4 glycoprotein. It is also shown that very similar epitopes of this molecule are involved in virus binding. We infer from these data that the binding sites on these viruses are highly conserved regions, and may therefore make good targets for potential vaccines. In addition, we show that cell surface expression of CD4 is similarly modulated after infection of cell lines by all the viruses.

The HIV env glycoprotein mediates virus infection and cell fusion through an interaction with the CD4 molecule present at the surface of T4+ lymphocytes. Although env presents a major antigenic target, vaccinia recombinants expressing env elicit low titres of anti-env antibody (Kieny et al., Bio/Technology, 4, 790-795, 1986). To delimit the functional domains of env and to improve the immunogenicity of the vaccinia recombinants we constructed variants expressing env proteins in which the site permitting cleavage of the gp160 precursor to yield gp120 and gp41 was removed, the gp120 and gp41 moieties separated or in which the signal sequence and hydrophobic domains were replaced by equivalents from rabies virus G. Analysis of variants revealed that the gp120 moiety is alone capable of interacting with CD4 and of provoking aggregation of T4+ lymphocytes, whereas cell-associated gp41 liberated by gp160 cleavage was essential for cell fusion. The identity of the signal and transmembrane zones however appeared unimportant. Although removal of the consensus sequence permitting cleavage of gp160 prevented syncytium formation but not aggregation of T4+ lymphocytes, significant cleavage continued to take place. Removal of a second potential cleavage site blocked gp160 cleavage. The live viruses were examined for immunogenicity: recombinant 1139 which lacks both putative cleavage sites was found to elicit a 10-fold higher antibody response in experimental animals than the parental recombinant.

We report here the use of a simple, rapid, reproducible, and quantitative assay to detect the putative activity of anti-HIV agents. It can be employed with different T cell cultures, such as peripheral blood lymphocytes or CEM-C113 cells, a sensitive clone of CEM cells. It can be used alone as a rapid screening test in order to measure both cytotoxicity and the potential antiviral activity of large numbers of compounds. It can also be combined for thorough studies with other classic tests that detect virus production. Among these tests, a micro-method to measure reverse transcriptase activity is shown to be very useful. To illustrate the practicability of the assay, 3'-azido-2',3'-dideoxythymidine (AZT) and ribavirin are studied.

Sequence comparison of the human immunodeficiency virus type 1 and type 2 env genes revealed the presence of six linear regions in the extracellular glycoprotein that are highly conserved. To investigate the functional significance of these regions, we made short deletions in each and assayed the ability of the mutated proteins to bind CD4 antigen. Small deletions in four of the highly conserved regions drastically reduced receptor binding. Some deletions interfered with the maturation of the envelope glycoprotein, but maturation did not necessarily correlate with the ability to bind CD4 antigen.

HIV-1 infected Molt4-T4 cells provide an efficient system for the production of cellular precursor gp160 of HIV envelope glycoproteins, gp120 and gp41. The precursor gp160 was purified on an immuno-affinity column containing antibodies from sera of HIV-1-seropositive patients. The precursor gp160 was then isolated by preparative polyacrylamide gel electrophoresis. Two out of four Balb/c mice, immunized with these purified preparations of gp160, developed specific circulating antibodies. A hybridoma cell line was subsequently isolated producing monoclonal antibody KL49/19 (IgG1, K) specific for gp160. This monoclonal antibody can specifically immunoprecipitate gp160, existing in HIV-1-infected cells. In an immunoblotting assay, it identifies mainly gp160 and shows a slight affinity for the mature glycoprotein, gp120. The monoclonal antibody is probably directed against an epitope in the polypeptide residue of gp160 since it can recognize a deglycosylated polypeptide of molecular weight 90,000, a product of gp160 digestion by endoglycosidase H (Endo H). It does not cross-react with any protein of HIV-2 by immunoblot or immunoprecipitation assays. By virtue of its specificity, the monoclonal antibody KL49/19 might provide a powerful probe with which to detect gp160 in cells which might partially express the HIV-1 genes.

Using mAbs and genomic probe to the CD4 molecule, the HIV receptor, we demonstrated that HIV replication induces the disappearance of its functional receptor from the cell surface by two distinct mechanisms. First, after being expressed onto the cell surface, HIV envelope gp110 will complex CD4, efficiently masking the CD4 epitope used by the virus to bind its receptor. This phenomenon occurs on the surface of each infected cell and is not due to the release of soluble gp110; infection with recombinant HIV/vaccinia viruses expressing a mutated HIV env gene designed to prevent gp110 release from the cell surface induces a similar gp/CD4 complexes formation. Second, virus replication induces a dramatic and rapid loss of CD4 mRNA transcripts, preventing new CD4 molecules from being synthesized. These two mechanisms of receptor modulation could have been developed to avoid reinfection of cells replicating the virus as well as to produce more infectious particles. These results suggest that the classical virus interference documented for other retroviruses might not only be due to receptor/envelope interaction, but might also depend on receptor gene expression.

Human sera were tested for the presence of anti-nef antibodies by radioimmunoassay (RIA), with recombinant radiolabelled nef expressed in E. coli. Of the 300 HIV-positive sera tested by RIA, 70 +/- 5.3% were found to be anti-nef positive. Anti-nef antibodies bound to nef with a high affinity (K 0.5 = 2.2 x 10(-9) M). In 31 of the sera, the specificity of anti-nef antibodies was further analysed by enzyme-linked immunosorbent assay (ELISA) with large synthetic peptides ranging from 31 to 66 amino acid residues and spanning the total sequence of nef from HIV-1. The results obtained showed that the immunodominant antigenic sites of nef were located close to the N- and C-terminal regions of the molecule.

We have investigated the possible involvement in the interaction between HIV gp110 and its CD4 receptor of epitopes different from the currently known binding site(s) of the molecule. Four monoclonal antibodies (MAbs) to gp110 were used (Genetic Systems Corporation, Seattle, Washington, USA): one (110-1) recognized a peptide corresponding to the C-terminal part of gp110 (494-517); the other three (110-3, 110-4, 110-5) recognized the same peptide located at position 308-328. HIV or purified gp110 obtained from a vaccinia recombinant (Transgene S.A., Strasbourg, France) were pre-incubated with the MAb prior to addition to CD4+ cells. Specific binding of viral particles or of the soluble molecule was then determined by flow cytometer analysis, compared with that of control preparations where the MAb was added after HIV or gp 110 had been allowed to bind CD4+ cells. Significant inhibition of HIV binding was noted with the three MAbs to peptide (308-328), but not with 110-1. At the molecular level, these same MAbs decreased the affinity of interaction between CD4 and soluble gp110, although they could still label the latter molecule after it had bound to CD4+ cells. Therefore, steric hindrance may account for neutralization of HIV binding by antibodies that are actually directed to epitopes topographically distinct from the site of binding of gp110 to CD4.

Presents a study which compared immunoglobulin G antibodies to lymphadenopathy-associated virus in groups of differently treated French hemophiliacs and Belgian hemophiliacs. Methodology; Results; Discussion.