Characterization of a naphthalene derivative inhibitor of retroviral integrases.

The retroviral integrase protein (IN) is essential for virus replication and, therefore, an attractive target for the development of inhibitors to treat human immunodeficiency virus (HIV) infection. Diverse classes of compounds that are active against this protein have been discovered using in vitro assays. Here we describe the synthesis of a novel compound, 3,8-dibromo-7-amino-4-hydroxy-2-naphthalenesulfonic acid (2BrNSA), which inhibits the in vitro activities of the full-length HIV-1 and avian sarcoma virus (ASV) integrases, and the isolated catalytic core fragment of the ASV protein (residues 52-207). The compound also inhibits retroviral reverse transcriptase in vitro, but the IC(50) for the HIV-1 enzyme is almost two orders of magnitude higher than for HIV-1 integrase. The inhibitor was found to be active in cell culture, preventing reporter gene transduction of HeLa cells by both ASV and HIV-1 vectors. Neither viral attachment nor uptake into cells appeared to be affected in these transfections, whereas accumulation of vector DNA and its joining to host DNA were both drastically reduced in the presence of the inhibitor. Propagation of two different strains of replication-competent HIV-1 in human peripheral blood mononuclear cells (PBMCs) was also reduced by the inhibitor, allowing survival of a substantial number of cells in the treated cultures. Based on these and other results we speculate that binding of 2BrNSA to integrase in infected cells interferes not only with its catalytic activity but also with critical interactions that are required for the formation or function of the reverse transcriptase complex. Its activity in cell culture suggests that this inhibitor may provide a valuable new lead for further development of drugs that target early steps in the HIV life cycle.

Prostratin: activation of latent HIV-1 expression suggests a potential inductive adjuvant therapy for HAART.

Prostratin is a unique phorbol ester that stimulates protein kinase C activity but is nontumor promoting. Remarkably, prostratin is also able to inhibit de novo human immunodeficiency virus type 1 (HIV-1) infection yet up-regulate viral expression from latent proviruses. Prostratin's lack of tumor promotion, coupled with its ability to block viral spread yet induce latent proviral expression, prompted studies to determine whether this compound could serve as an inductive adjuvant therapy for patients treated with highly active antiretroviral therapy (HAART). The current experiments indicate that prostratin is a potent mitogen for mononuclear phagocytes possessing many of the activities of phorbol myristate acetate (PMA) with notable functional differences. Prostratin, like PMA, accelerates differentiation of the myeloid cell-lines, HL-60 and THP-1, as well as mononuclear phagocytes from bone marrow and peripheral blood. Enzyme-linked immunosorbent assay and gene array analyses indicate significant changes in the expression of proteins and messenger RNA after treatment of cells with prostratin, consistent with phagocyte activation and differentiation. Prostratin blocks HIV-1 infection relating to down-regulation of CD4 receptor expression. The array analysis indicates a similar down-regulation of the HIV-1 coreceptors, CXCR4 and CCR5, and this may also reduce viral infectivity of treated host cells. Finally, prostratin is capable of up-regulating HIV-1 expression from CD8+ T lymphocyte-depleted peripheral blood mononuclear cells of patients undergoing HAART. This novel observation suggests the agent may be an excellent candidate to augment HAART by inducing expression of latent HIV-1 with the ultimate goal of eliminating persistent viral reservoirs in certain individuals infected with HIV-1.

Pathogenesis of HIV-1 infection within bone marrow cells.

Mononuclear phagocytic cells and CD4+ T lymphocytes represent the major targets for infection by HIV-1 in vivo. The most severe pathogenic features associated with HIV-1 infection can be attributed to malfunction or premature death of these cells that are of hematopoietic origin. Patients with acquired immunodeficiency syndrome (AIDS), suffer from many hematologic disorders, particularly those persons with long-term infection of HIV-1. These disorders include anemia, lymphocytopenia, thrombocytopenia and neutropenia. The mechanisms that lead to the induction of these disorders are multi-factorial. However, sufficient evidence has accumulated which suggests that HIV-1 infection of cells within the microenvironment of the bone marrow can lead to the induction of hematopoietic deficits. Most studies indicate that marrow-derived hematopoietic stem cells cannot be infected by HIV-1 until they undergo modest differentiation in order to express the appropriate receptors to enable virus entry and subsequent replication. Some cells within the mixed environment of the marrow stroma appear to support HIV-1 replication however. These cells include marrow microvascular endothelial cells, sometimes referred to as blanket cells, stromal fibroblasts, as well as mononuclear phagocytes. Our recent experiments suggest that the HIV-1 accessory protein, Vpr, plays some role in the activation of marrow-derived mononuclear phagocytes which appears to result in premature phagocytosis of non-adherent marrow cells present in the in vitro cultures. This phenomenon could account, in part, for the induction of cytopenias that are typical of individuals infected by HIV-1.

Inhibition of HIV-1 replication and infectivity by expression of a fusion protein, VPR-anti-integrase single-chain variable fragment (SFv): intravirion molecular therapies.

OBJECTIVES: To deliver antiretroviral agents or other foreign proteins into progeny virions and evaluate their inhibitory effect on human immunodeficiency virus type 1 (HIV-1) replication. STUDY DESIGN/METHODS: HIV-1 encodes proteins in addition to gag, pol, and env, some of which are packaged into virus particles. One essential retroviral enzyme is integrase (IN), which has been used as a target for developing agents that inhibit virus replication. In previous studies, we demonstrated that intracellular expression of single-chain variable antibody fragments (SFvs), which bind to IN, results in resistance to productive HIV-1 infection in T-lymphocytic cells. Because the highly conserved accessory HIV-1 Vpr protein can be packaged within virions in quantities similar to those of the major structural proteins, this primate lentiviral protein may be used as a fusion partner to deliver antiviral agents or other foreign proteins into progeny virions. In these studies, the fusion proteins Vpr-chloramphenicol acetyl transferase (CAT) and Vpr-SFv-IN have been developed. Stable transfectants expressing these fusion proteins were generated from PA317 cells and SupT1 T-lymphocytic cells and analyzed using immunofluorescence microscopy. After challenge of SupT1 cells with HIV-1, p24 antigen expression was evaluated. The incorporation of these fusion proteins were evaluated by immunoprecipitation of virions using a Vpr antibody. RESULTS: Expression of the fusion proteins was confirmed by immunofluorescent staining in PA317 cells transfected with the plasmids expressing Vpr-CAT and Vpr-SFv-IN proteins. Stable transfectants expressing these fusion proteins were generated from SupT1 T-lymphocytic cells. When challenged, HIV-1 replication, as measured by HIV-1 p24 antigen expression, was inhibited in cells expressing Vpr-SFv-IN. It was demonstrated that Vpr-chloramphenicol acetyl transferase (Vpr-CAT and Vpr-SFv-IN proteins can be efficiently packaged into the virions and that Vpr-SFv-IN also decreases the infectivity of virions into which it is encapsidated. CONCLUSIONS: An anti-integrase single-chain variable fragment moiety can be delivered into HIV-1 virions by fusing it to Vpr. Vpr-SFv-IN decreases HIV-1 production in human T-lymphocytic cells. The benefits of "intravirion" gene therapy include immunization of target cells as well as decreasing infectivity of HIV-1 virions harboring the fusion construct. Thus, this approach to anti-HIV-1 molecular therapies has the potential to increase inhibitory effects against HIV-1 replication and virion spread.

Human immunodeficiency virus type 1 Vpr alters bone marrow cell function.

Vpr, a 96 amino acid protein, encoded by the human immunodeficiency virus type I (HIV-1), is important for efficient infection of mononuclear phagocytic cells. These cells are abundant in whole bone marrow, which can easily be cultured in vitro to support hematopoiesis. Our experiments indicate that Vpr plays a role in the potent activation of murine and human mononuclear phagocytic cells within a hematopoietic microenvironment. In murine cultures, avid erythrophagocytosis is triggered by transduction of marrow cells with supernatant derived from PA317 cells transfected with a murine retroviral delivery vector bearing a Vpr expression cassette. Supernatants derived from cells transfected with the same vector carrying sequences for the expression of other relevant viral and nonviral proteins do not induce erythrophagocytosis to any marked degree. The effect on human marrow cells is similar, where treatment promotes adhesion of mononuclear phagocytic cells to culture plates in association with other nucleated and nonnucleated cells that undergo subsequent engulfment. The differential effects of Vpr point and deletion mutants in both marrow culture systems fortify the view that the effect is specific to HIV-1 Vpr. Addition of low molar quantities of purified Vpr to marrow cultures is also capable of promoting cell adhesion and phagocytosis, suggesting that extracellular Vpr is the effector of the phenomenon. Accelerated phagocytosis is a hallmark of promonocyte, monocyte, and macrophage activation and its occurrence within a hematopoietic microenvironment may account for critical in vivo pathogenic features of HIV-1 infection. First, activation of mononuclear phagocytes may promote productive viral infection; and second, premature phagocytosis could provide, at least in part, a molecular explanation for the induction of the idiopathic cytopenias that are typical of individuals infected with HIV-1.

A novel Vpr peptide interactor fused to integrase (IN) restores integration activity to IN-defective HIV-1 virions.

A novel approach to complement human immunodeficiency virus type I (HIV-1) integrase (IN)-defective virions has been identified. The approach involves fusion of a 23-amino-acid stretch to the N-terminus of wild-type IN and coexpression of this chimera with the IN-defective proviral template in virus producing cells. The 23-amino-acid peptide represents a Vpr "interactor," referred to as the the WxxF or WF domain, which apparently leads to docking of the domain along with the fusion partner onto HIV-1 Vpr, thus permitting virion incorporation of the chimeric protein when expressed, in trans, with other viral products. Transfection of the WF-IN expression plasmid along with HIV-1 viral clones that produce Vpr, but bear an IN mutation, results in the release of a proportion of viral particles that are competent for integration. The extent of complementation was assessed using the MAGI cell assay, where integration of viral DNA results in the eventual appearance of easily visible multinucleated blue syncytia. The efficiency of dWF-IN (double copy of WF domain) complementation is not improved markedly by incorporation of a HIV-1 protease cleavage site (PR) between the dWF domain and IN (dWF-PR-IN), unlike that observed with Vpr fusions to IN. Furthermore, the ability of Vpr-PR-IN and dWF-PR-IN to complement IN-defective proviral clones, both of which bear an intervening protease cleavage site, appear comparable. Western blotting analyses using virions isolated through sucrose cushions demonstrate clearly the incorporation of the dWF-IN fusion protein into Vpr containing HIV-1 particles but not in Vpr-deficient virions. Additional Western blotting analyses indicate that all Vpr-IN and dWF-IN chimeras, with or without a PR site, are packaged into virions. The efficiency of virion incorporation of Vpr-IN and dWF-IN chimeras appears approximately comparable by Western blotting analysis. The ability of dWF-IN to complement IN-defective proviruses with efficiency similar to that of Vpr-PR-IN and dWF-PR-IN indicates that dWF-IN retains the full complement of functions necessary for integration of proviral DNA and is likely due to the benign nature of this small domain at the amino-terminus of IN.

Intracellular expression of single-chain variable fragments to inhibit early stages of the viral life cycle by targeting human immunodeficiency virus type 1 integrase.

Integration of viral DNA into a chromosome of the infected host cell is required for efficient replication of a retroviral genome, and this reaction is mediated by the virus-encoded enzyme integrase (IN). As IN plays a pivotal role in establishing infection during the early stages of the retroviral life cycle, it is an attractive target for therapeutic intervention. However, the lack of effective antiviral drug therapy against this enzyme has led to the testing of other novel approaches towards its inhibition. In these studies, a panel of anti-human immunodeficiency virus type 1 (anti-HIV-1) IN hybridomas has been used in the construction of single-chain variable antibody fragments (SFvs). The monoclonal antibodies produced by these hybridomas, and derived SFvs, bind to different domains within IN. We now demonstrate that intracellular expression of SFvs which bind to IN catalytic and carboxy-terminal domains results in resistance to productive HIV-1 infection. This inhibition of HIV-1 replication is observed with SFvs localized in either the cytoplasmic or nuclear compartment of the cell. The expression of anti-IN SFvs in human T-lymphocytic cells and peripheral blood mononuclear cells appears to specifically neutralize IN activity prior to integration and, thus, has an effect on the integration process itself. These data support our previous studies with an anti-HIV-1 reverse transcriptase SFv and demonstrate further that intracellularly expressed SFvs can gain access to viral proteins of the HIV-1 preintegration complex. This panel of anti-HIV-1 IN SFvs also provides the tools with which to dissect the molecular mechanism(s) directly involved in integration within HIV-1-infected cells.

Activities and substrate specificity of the evolutionarily conserved central domain of retroviral integrase.

The retroviral integrase (IN) is a virus-encoded enzyme that is essential for insertion of viral DNA into the host chromosome. In order to map and define the properties of a minimal functional domain for this unique viral enzyme, a series of N- and C-terminal deletions of both Rous sarcoma virus (RSV) and human immunodeficiency virus (HIV) INs were constructed. The RSV IN deletion mutants were first tested for their ability to remove two nucleotides from the end of a substrate representing the terminus of viral DNA in order to assess the contribution of N and C regions towards this reaction, referred to as processing. The results suggest that C-terminal amino acids of the intact RSV protein are required to maintain specificity of the processing reaction. Though deficient for processing, the RSV deletion mutants exhibited a secondary endonucleolytic activity that was indistinguishable from that of wild-type IN, demonstrating that all retained some enzymatic activity. RSV, and a larger set of HIV-1, IN deletion mutants were then tested for their ability to perform an intramolecular, concerted cleavage-ligation reaction using an oligodeoxynucleotide substrate that mimics the intermediate viral-host DNA junction found prior to the final step of covalent closure. The composite results from such analyses define a minimal functional central region of approximately 140 amino acids for each enzyme that includes the highly conserved D,D(35)E domain. Results with HIV-1 and HIV-2 IN also indicate that the efficiency of concerted cleavage-ligation depends upon the presence of CA/GT base pairs within the viral component of the DNA substrate at the reaction site. Even the isolated central region of HIV-1 IN exhibited this sequence requirement for optimal activity. We conclude that this evolutionarily conserved central region of IN not only encodes residues that are required for the catalytic activity of the enzyme but also harbors some or all of the determinants responsible for recognition of the CA/GT dinucleotides that are present at the ends of all retroviral DNAs.

Monoclonal antibodies against HIV type 1 integrase: clues to molecular structure.

Eleven murine hybridoma clones were selected for their ability to produce anti-HIV-1 integrase (IN) antibodies. Competition and epitope mapping studies allowed segregation of the monoclonal antibodies (MAbs) into four distinct classes. The five MAbs that comprise the first class showed high affinity for epitopes within an N-terminal domain of 58 amino acids that includes a conserved zinc finger motif. The second class, with two MAbs, showed high affinity for epitopes within 29 amino acids at the C terminus. Another two MAbs, which constitute the third class, displayed moderate affinities for epitopes that mapped to regions within the highly conserved catalytic core referred to as the D,D(35)E domain. One of these MAbs showed significant cross-reactivity with HIV-2 IN and weak, but detectable, cross-reactivity with RSV IN. The remaining two MAbs, which comprise the fourth class, exhibited fairly low binding affinities and appeared to recognize epitopes in the zinc finger motif domain as well as the C-terminal half of the IN protein. The MAbs can be used for immunoprecipitation and immunoblotting procedures as well as for purification of HIV-1 IN protein by affinity chromatography. We show that several can also be used to immunostain viral IN sequences in HIV-1-infected T cells, presumably as a component of Gag-Pol precursors. Finally, analysis of our mapping and competition data suggests a structure for mature IN in which the C terminus approaches the central core domain, and the N and C termini touch or are proximal to each other. These MAbs should prove useful for further analyses of the structure and function of IN both in vitro and in vivo.

Molecular mechanism of retroviral DNA integration.

The integration of retroviral DNA appears to be obligatory for the efficient replication of retroviruses in their respective host cells. During a natural infection, integration takes place in a process that includes biochemically and temporally discrete steps. These are: (1) the removal of two nucleotides from the 3' ends of newly synthesized linear viral DNA in the host cell cytoplasm; (2) transport of the trimmed viral DNA to the nucleus within a viral protein/DNA complex; and (3) insertion of the viral DNA into host cell DNA via a concerted cleavage and ligation reaction. The cleavage of viral DNA and its subsequent joining to host DNA are catalyzed by the retroviral enzyme, integrase (IN). Elucidation of the mechanistic details of these catalytic activities of IN has relied heavily upon the use of relatively simple in vitro assays which recapitulate the in vivo reactions. These assays and the information derived from them should also facilitate the search for potential inhibitors of IN with the ultimate goal of providing a means to halt retroviral infections, such as that which causes the acquired immunodeficiency syndrome (AIDS), effectively.

Requirement for a conserved serine in both processing and joining activities of retroviral integrase.

Retroviruses encode a protein, the integrase (IN), that is required for insertion of the viral DNA into the host cell chromosome. IN alone can carry out the integration reaction in vitro. The reaction involves endonucleolytic cleavage near the 3' ends of both viral DNA strands (the processing step), followed by joining of these new viral DNA ends to host DNA (the joining step). Based on their evolutionary conservation, we have previously identified at least 11 amino acid residues of IN that may be essential for the reaction. Here we report that even conservative replacements of one of these residues, an invariant serine, produce severe reductions in both the processing and joining activities of Rous sarcoma virus IN in vitro. Replacement of the analogous serine of the type 1 human immunodeficiency virus IN had similar effects on processing activity. These results suggest that this single conserved serine is a component of the active site and that one active site is used for both processing and joining. Replacement of this serine with certain amino acids resulted in a loss or reduction in DNA binding activities, while other replacements at this position appeared to affect later steps in catalysis. All of the defective Rous sarcoma virus INs were able to compete with the wild-type protein, which supports a model in which IN functions in a multimeric complex.

Residues critical for retroviral integrative recombination in a region that is highly conserved among retroviral/retrotransposon integrases and bacterial insertion sequence transposases.

Our comparison of deduced amino acid sequences for retroviral/retrotransposon integrase (IN) proteins of several organisms, including Drosophila melanogaster and Schizosaccharomyces pombe, reveals strong conservation of a constellation of amino acids characterized by two invariant aspartate (D) residues and a glutamate (E) residue, which we refer to as the D,D(35)E region. The same constellation is found in the transposases of a number of bacterial insertion sequences. The conservation of this region suggests that the component residues are involved in DNA recognition, cutting, and joining, since these properties are shared among these proteins of divergent origin. We introduced amino acid substitutions in invariant residues and selected conserved and nonconserved residues throughout the D,D(35)E region of Rous sarcoma virus IN and in human immunodeficiency virus IN and assessed their effect upon the activities of the purified, mutant proteins in vitro. Changes of the invariant and conserved residues typically produce similar impairment of both viral long terminal repeat (LTR) oligonucleotide cleavage referred to as the processing reaction and the subsequent joining of the processed LTR-based oligonucleotides to DNA targets. The severity of the defects depended upon the site and the nature of the amino acid substitution(s). All substitutions of the invariant acidic D and E residues in both Rous sarcoma virus and human immunodeficiency virus IN dramatically reduced LTR oligonucleotide processing and joining to a few percent or less of wild type, suggesting that they are essential components of the active site for both reactions.(ABSTRACT TRUNCATED AT 250 WORDS)

Retroviral integrase domains: DNA binding and the recognition of LTR sequences.

Integration of retroviral DNA into the host chromosome requires a virus-encoded integrase (IN). IN recognizes, cuts and then joins specific viral DNA sequences (LTR ends) to essentially random sites in host DNA. We have used computer-assisted protein alignments and mutagenesis in an attempt to localize these functions within the avian retroviral IN protein. A comparison of the deduced amino acid sequences for 80 retroviral/retrotransposon IN proteins reveals strong conservation of an HHCC N-terminal 'Zn finger'-like domain, and a central D(35)E region which exhibits striking similarities with sequences deduced for bacterial IS elements. We demonstrate that the HHCC region is not required for DNA binding, but contributes to specific recognition of viral LTRs in the cutting and joining reactions. Deletions which extend into the D(35)E region destroy the ability of IN to bind DNA. Thus, we propose that the D(35)E region may specify a DNA-binding/cutting domain that is conserved throughout evolution in enzymes with similar functions.

Induction of chronic human immunodeficiency virus infection is blocked in vitro by a methylphosphonate oligodeoxynucleoside targeted to a U3 enhancer element.

Oligodeoxynucleosides with internucleoside methylphosphonate linkages complementary to regions within U3 of human immunodeficiency virus type 1 were evaluated for their ability to block phorbol myristate acetate upregulation of virus in chronically infected promonocytic and T-lymphoblastoid cell lines. One such oligomer, targeted to an NF-kappa B enhancer element, inhibited phorbol myristate acetate induction of viral replication and tat-mediated trans activation of the human immunodeficiency virus long terminal repeat. The effect of this construct is contrasted with classical antisense methylphosphonate-derivatized oligomers complementary to initiation codon and splice acceptor sites of human immunodeficiency virus structural and regulatory genes. Its activity suggests a novel application of the modified oligonucleotide strategy in the blockade of viral induction from latently infected cells.

The avian retroviral IN protein is both necessary and sufficient for integrative recombination in vitro.

The integration of viral DNA into the host cell chromosome is an essential feature of the retroviral life cycle. The integration reaction requires cis-acting sequences at the ends of linear viral DNA and a trans-acting product of the pol gene, the integration protein (IN). Previously, we demonstrated that avian sarcoma-leukosis virus (ASLV) IN is able to carry out the first step in the integration process in vitro: nicking of the ends of linear viral DNA. In this paper, using two independent assays, we demonstrate that IN, alone, is sufficient to carry out the second step: cleavage and joining to the target DNA. These results demonstrate that the retroviral IN protein is an integrase.

A soluble inhibitor of T lymphocyte function induced by HIV-1 infection of CD4+ T cells: characterization of a cellular protein and its relationship to p15E.

Soluble suppressor factor (SSF), first described in association with HIV-1 infection in vivo, is a molecule(s) capable of inhibiting T cell-dependent immune reactivity. Its relationship to human immunodeficiency virus (HIV) was further defined as supernatants of mononuclear cell cultures from HIV-1-seropositive carriers, CD4+ T lymphocytes infected with HIV-1 in vitro, and a T cell hybridoma incorporating CD4+ lymphocytes from an HIV-1-seropositive individual were shown to elaborate factors with similar activity profiles. These factors were recognized antigenically by certain antibodies directed against epitopes of p15E, a transmembrane protein of murine leukemia virus which shares regions of identity with proteins deduced from human endogenous retroviral envelope transcripts as well as HIV. These reagents precipitated a single-chain, nonglycosylated, nonviral protein of molecular weight 57,000 Da from SSF-producing cells. There was no cross-reactivity with antisera recognizing the IL-2R alpha-chain (CD25) or tumor necrosis factor. This molecule was present in very low levels in PHA-activated T lymphocytes and was upregulated following their infection with HIV-1. Isolation of HIV-linked SSF should permit comparisons with other virion, cellular, and serum inhibitory substances described in AIDS, and perhaps suggest therapeutic strategies.

Terminal nucleotides of the preintegrative linear form of HIV-1 DNA deduced from the sequence of circular DNA junctions.

The termini of integrated retroviral DNAs are characterized by highly conserved dinucleotide sequences: 5'TG...//...CA3'. For the avian and murine C-type retroviruses, the dinucleotide sequences reside two deoxynucleotides (usually AA and TT) from the LTR ends of unintegrated viral DNA (5' AATG...//...CATT3'). The number and identity of terminal deoxynucleotides in unintegrated HIV-1 linear DNA that extend beyond the conserved dinucleotides have not been determined. However, they had been presumed to consist of a single nucleotide (5'CTG...//...CAG3') on each end, based on inspection of the nucleotide sequence between the end of the supposed primer sites for retroviral DNA synthesis and the conserved proviral termini. We have utilized the polymerase chain reaction (PCR) to amplify segments representing the joined ends of linear DNA present in covalently closed circular HIV-1 DNA molecules isolated from infected T cells. These fragments were cloned and the nucleotide sequence of the LTR-LTR circle junction of several independent clones determined. Based upon the results, we predict that, like the avian and murine viruses, HIV-1 linear DNA contains two nucleotides beyond the conserved dinucleotides: 5'ACTG...//...CAGT3'. Models for the origin of these termini and other observed junction sequences are proposed.

HIV DNA integration: observations and interferences.

DNA integration appears to be an obligatory step for the efficient replication of retroviruses. Studies of the integration reaction for the avian and murine C-type retroviruses have defined the importance of: i) LTR terminal sequences, which are joined to host DNA, and ii) the activity of the retroviral integration protein (IN). The specific requirements for the integration of HIV DNA have not yet been defined. In this review, we survey studies pertinent to this reaction for HIV and extrapolate a mechanism for HIV DNA integration based upon knowledge of the integrative recombination reaction of the C-type retroviruses.

PolyI.polyC12U-mediated inhibition of loss of alloantigen responsiveness viral replication in human CD4+ T cell clones exposed to human immunodeficiency virus in vitro.

Two alloreactive human CD4+ T cell clones, recognizing HLA-DR2 and HLA-DR1 determinants, lost their specific proliferative capacity after infection with HIV. This system was used to explore the effect of polyI.polyC12U on HIV replication and immune suppression. The mismatched double-stranded RNA blocked HIV-associated particulate reverse transcriptase activity and viral-mediated cytopathic effects. Also, polyI.polyC12U preserved the alloreactivity of T cell clones after exposure to HIV.PolyI.polyC12U appeared to act at a level subsequent to host cell infection and reverse transcription. It had no effect on the enhancement of gene expression by the HIV transcription unit tatIII. These findings indicate that early in the course of infection of CD4+ T lymphocytes, HIV can directly abrogate proliferation to specific allodeterminants, and that this function is preserved in the presence of polyI.polyC12U. They also provide insight into the mechanism of antiviral action of a class of agent with potential clinical utility in AIDS.

Characterization and clinical association of antibody inhibitory to HIV reverse transcriptase activity.

Reverse transcriptase activity of the human immunodeficiency virus (HIV) was blocked in vitro by immunoglobulin G (IgG) derived from certain individuals infected with this retrovirus. A heterogeneous immune response for inhibition of enzyme function was noted. Catalytic activity was depressed by 50% or more with the use of 10 micrograms of IgG from 11 of 16 HIV-seropositive asymptomatic carriers, but from 0 of 8 seronegative controls and 2 of 12 patients with acquired immune deficiency syndrome (AIDS) or the AIDS-related complex (ARC). The inhibitor was confined to the F(ab')2 fragment. It was not directed against the poly(rA) X oligo(dT) template, nor against major envelope or structural viral antigens, and did not cross-react with bacterial, avian, or other mammalian DNA polymerases. It did not correlate with recognition of polymerase antigens by radioimmunoprecipitation. Loss of this inhibitor may be associated with development of clinical disease. Ten asymptomatic HIV-seropositive carriers with high titers of IgG antibodies to reverse transcriptase were followed for a mean of 3 years. All of four lost inhibitory capability prior to development of AIDS or ARC, while titers persist in the six who remain clinically healthy.