B'-protein phosphatase 2A is a functional binding partner of delta-retroviral integrase.

To establish infection, a retrovirus must insert a DNA copy of its RNA genome into host chromatin. This reaction is catalysed by the virally encoded enzyme integrase (IN) and is facilitated by viral genus-specific host factors. Herein, cellular serine/threonine protein phosphatase 2A (PP2A) is identified as a functional IN binding partner exclusive to δ-retroviruses, including human T cell lymphotropic virus type 1 and 2 (HTLV-1 and HTLV-2) and bovine leukaemia virus (BLV). PP2A is a heterotrimer composed of a scaffold, catalytic and one of any of four families of regulatory subunits, and the interaction is specific to the B' family of the regulatory subunits. B'-PP2A and HTLV-1 IN display nuclear co-localization, and the B' subunit stimulates concerted strand transfer activity of δ-retroviral INs in vitro. The protein-protein interaction interface maps to a patch of highly conserved residues on B', which when mutated render B' incapable of binding to and stimulating HTLV-1 and -2 IN strand transfer activity.

Processing of gag precursor polyprotein of human T-cell leukemia virus type I by virus-encoded protease.

The biological activity encoded in the putative protease gene (pro) of human T-cell leukemia virus type I was investigated by using a vaccinia virus expression vector. The 53-kilodalton gag precursor polyprotein was processed into the mature p19, p24, and p15 gag proteins when the gag and protease-coding sequence was expressed under the control of a vaccinia virus promoter, suggesting that the protease may be synthesized through the mechanism of ribosomal frame shifting. The processing defect of a protease mutant could be complemented by cointroduction of a wild-type construct into the cell, demonstrating that the pro gene encodes the biologically active protease molecules which are capable of processing the gag precursor polyprotein in vivo in trans. A study involving the use of a variety of mutants constructed in vitro revealed that the protease consists of a nonessential carboxy-terminal region and a part essential for its activity, including the putative catalytic residue, aspartic acid. Furthermore, a cluster of adenine residues positioned at the overlapping region between the gag and pro genes was shown to be involved in the ribosomal frameshifting event for the synthesis of protease. To mimic the formation of the 76-kilodalton gag-pro precursor polyprotein formed by ribosomal slipping, the coding frames of the gag and pro gene were adjusted. The processing of the gag-pro precursor polyprotein depended on an intact protease gene, implying that a cis-acting function of human T-cell leukemia virus type I protease may be necessary to trigger the initial cleavage event that leads to the release of protease from the precursor protein.

Inhibition of retroviral protease activity by an aspartyl proteinase inhibitor.

Retrovirus protease is an enzyme that cleaves gag and gag-pol precursor polyproteins into the functional proteins of mature virus particles. The correct processing of precursor polyproteins is necessary for the infectivity of virus particles: in vitro mutagenesis which introduces deletions into the murine leukaemia virus genome produces a protease-defective virus of immature core form and lacking infectivity. A therapeutic drug effective against disease caused by retrovirus proliferation could likewise interfere with virus maturation. The primary structure has so far been determined for the protease of avian myeloblastosis virus, and of murine, feline and bovine leukaemia viruses. Amino acid sequencing of the retrovirus proteases, either after their purification or from prediction from the nucleotide sequence, shows that they possess the Asp-Thr-Gly sequence characteristic of the aspartyl proteinases. In this report we show that retrovirus proteases belong to the aspartyl proteinase group and demonstrate an inhibition by the aspartyl proteinase-specific inhibitor, pepstatin A, on the activity of bovine leukaemia, Moloney murine leukaemia and human T-cell leukaemia virus proteases.

Inhibition of human immunodeficiency virus type I reverse transcriptase by suramin-related compounds.

Ninety analogues of suramin have been examined for their ability to inhibit the exogenous reverse transcriptase (RT) of human immunodeficiency virus type I (HIV-I). Of these compounds, 57 inhibited the poly(rC).oligo(dG)-dependent RT activity. Three classes of dose-response curves could be discriminated. Allocation of a compound to one class did not correspond with obvious structural features. Twenty-four substances were superior to suramin in our RT inhibition assay. The RT-inhibitory activity of these compounds did not correlate with their effect against filariae or trypanosomes. Preliminary antiviral evaluation in susceptible human T cells inoculated with HIV-I demonstrated in vitro therapeutic efficacy for some compounds with lower drug-related cellular toxicity than suramin. Certain structural features relevant for the RT-inhibitory effect of these compounds were recognized. Predictions are made for the design of more effective RT inhibitors. Such compounds will help to understand the molecular mechanism of reverse transcription and might be useful in the therapy of retroviral infections.

Polymerase activity in lymphocyte culture supernatants from patients with Kawasaki disease.

Kawasaki disease (KD) is an acute vasculitis of infancy and early childhood characterized by high fever, rash, mucositis, lymphadenopathy and coronary artery damage. Large epidemics have been described in Japan and the United States and the number of cases reported annually is steadily increasing. The aetiology of KD is unknown. During the acute phase of the disease marked immunologic alterations occur including generalized T-cell lymphocytopenia, activation of circulating T4+ helper T cells, decreased numbers of T8+ suppressor T cells and marked B-cell activation. We postulated that a lymphotropic virus with affinity for endothelial and lymphoid cells might explain the vasculitis and immunological abnormalities in KD. We report here our study of the particulate fraction from culture supernatants of peripheral blood mononuclear cells (PBMC) for evidence of retrovirus-associated reverse transcriptase (RT) activity. Activity was found in the supernatants from KD patients but not control cultures. This RT activity was transmitted to an established T-cell line (HUT-78) and thus may be due to an exogenous agent infecting KD lymphocytes.

Expression of reverse transcriptase activity of human T-lymphotropic virus type III (HTLV-III/LAV) in Escherichia coli.

The pol gene from a biologically active clone of the human T-cell lymphotropic virus type III provirus was inserted into a bacterial expression vector. The resulting gene fusion induced the formation of active reverse transcriptase that could be readily detected in extracts of bacterial cells. The activity exhibited the template and divalent cation requirements of the authentic enzyme. These constructs will be useful for safe and rapid analysis of potential inhibitors of this important enzyme.

Suramin antiviral therapy in the acquired immunodeficiency syndrome. Clinical, immunological, and virologic results.

The human T-lymphotropic virus type III/lymphadenopathy-associated virus (HTLV-III/LAV) requires reverse transcriptase for viral replication. We treated 12 patients who had acquired immunodeficiency syndrome and active HTLV-III/LAV viremia with suramin, a potent competitive inhibitor of reverse transcriptase, in six weekly induction doses of 1 g, followed by weekly maintenance doses of 500 mg. Three of eleven evaluable patients had complete inhibition of viral reverse transcriptase levels, lasting at least 18 weeks in each. Two additional patients had marked reduction in reverse transcriptase activity. Nadir serum suramin levels at the end of the induction phase correlated with the level of reverse transcriptase reduction. Toxicity included hepatic transaminase elevation, fever, malaise, rash, proteinuria, paresthesias, reversible neutropenia, and adrenal insufficiency. Objective clinical improvement was documented in 1 patient, but no patient had improvement in immune function and 7 patients had recurrent opportunistic infections. Although suramin may suppress HTLV-III/LAV viremia, its significant toxicity and lack of effect on immune variables indicate that alternative therapy will be required.

Detection of human T-cell lymphotropic virus type III-related antigens and anti-human T-cell lymphotropic virus type III antibodies by anticomplementary immunofluorescence.

Techniques presently available for detection of human T-cell lymphotropic virus type III (HTLV-III) antigens and antibodies are laborious or relatively nonsensitive. We adapted anticomplementary immunofluorescence (ACIF) for these purposes. In HTLV-III-infected cells, specific ACIF was demonstrated by a diffuse speckling pattern that often resulted in a peripheral cellular rim of fluorescence. A 97% concordance was demonstrated between the ACIF assay and other sensitive tests for HTLV-III antibody detection (Western blot and membrane immunofluorescence and fixed-cell immunofluorescence tests). The ACIF assay was both more sensitive and more specific when compared with the enzyme-linked immunosorbent assay. For detection of HTLV-III antigens, the ACIF assay appeared to be as sensitive as the reverse transcriptase assay and more sensitive, with less background reactivity, than the conventional immunofluorescence assay. The ACIF assay often detected low levels of HTLV-III antigens within 3 days of infection in vitro, compared with 5 to 7 days with the indirect immunofluorescence assay, and generally paralleled the reverse transcriptase assay. The ACIF assay is a simple, sensitive, and specific assay for detection of HTLV-III-related antigens and antibodies. It should prove useful in the diagnosis of HTLV-III infection, as well as in studies of pathogenesis.

Serological relationship between reverse transcriptases from human T-cell lymphotropic viruses defined by monoclonal antibodies. Evidence for two forms of reverse transcriptases in the AIDS-associated virus, HTLV-III/LAV.

The immunological relationship between reverse transcriptases purified from human T-cell lymphotropic viruses (HTLV-I, HTLV-II, HTLV-III) was defined using monoclonal antibodies specific for HTLV-III reverse transcriptase, secreted by a mouse/mouse hybridoma clone (4F8) developed in our laboratory. The viral proteins from HTLV-I and HTLV-II do not bear any cross-reactive epitope to antibodies secreted by this clone. These antibodies specifically cross-react with HTLV-III reverse transcriptase. The antibodies failed to neutralize the catalytic activity of reverse transcriptase; however, after immunoprecipitation with a magnetic conjugate of goat anti-mouse IgG, the residual activity was completely inhibited. This shows that the antibodies are not directed towards the catalytic active center of the enzyme. Using an immunoblotting technique (Western blotting), we have found two cross-reactive proteins with HTLV-III lysate with molecular masses of 53 and 66 kDa. This suggests that HTLV-III possesses two reverse transcriptase activities with a common determinant recognized by the same epitope.

PPi analogs as inhibitors of human T-lymphotropic virus type III reverse transcriptase.

Twenty-six PPi analogs were tested for inhibitory effects on human T-lymphotropic virus type III reverse transcriptase. The structural requirements for inhibition and mechanism of action of the most active inhibitors have been investigated. Foscarnet (phosphonoformic acid) was the most potent inhibitor of human T-lymphotropic virus type III reverse transcriptase with 50% inhibition at 0.5 microM. The mechanism was a noncompetitive type of inhibition of a (riboadenylic acid)n . (deoxythymidylic acid)12-18 [(rA)n(dT)12-18]-directed transcription at varied dTTP concentration. At constant substrate (dTTP) concentration and varied amounts of template, (rA)n(dT)12-18, the inhibitory action of foscarnet was of an uncompetitive type. The same pattern of inhibition was seen when the less active inhibitor carbonyldiphosphonate was studied under identical conditions. The structural requirements for inhibition of human T-lymphotropic virus type III reverse transcriptase by PPi analogs were similar to those shown by other reverse transcriptases.

Protease gene structure and env gene variability of the AIDS virus.

The protease gene structure and the env gene variability have been precisely compared between the AIDS virus and members of the HTLV/BLV family. The conserved amino acid sequence (LVDT) which is repeated in the proteases of the HTLV/BLV family is not repeated in AIDS virus. Comparative analysis of the env gene sequences reveals the striking fact that the env gene of AIDS virus is 8-12-times more variable than those of the HTLV/BLV family. Within the AIDS virus env gene, the surface glycoprotein region is more liable to vary than is the transmembrane region; unexpectedly, however, this liability is not a characteristic feature of the AIDS virus because it is more prominent in other retroviruses including members of the HTLV/BLV family.

Stability and inactivation of HTLV-III/LAV under clinical and laboratory environments.

The stability of human T-cell lymphotropic virus type III/lymphadenopathy-associated virus (HTLV-III/LAV) under environmental conditions encountered in a clinical or laboratory setting and its inactivation by commonly used chemical disinfectants were investigated. Under our experimental conditions utilizing a highly concentrated viral preparation, virus with an initial infectious titer of approximately 7 log10 tissue culture infectious dose (TCID50) per milliliter can be recovered for more than a week from an aqueous environment held at room temperature (23 to 27 degrees C) or at 36 to 37 degrees C. Virus recovery is reduced at a rate of approximately 1 log10TCID50 per 20 minutes when held at 54 to 56 degrees C. Dried and held at room temperature, HTLV-III/LAV retains infectivity for more than three days with a reduction of approximately 1 log10TCID50 per nine hours. Viral infectivity is undetectable and reduced more than 7 log10TCID50 within one minute with 0.5% sodium hypochlorite, 70% alcohol, or 0.5% nonidet-P40, and within ten minutes with 0.08% quaternary ammonium chloride or with a 1:1 mixture of acetone-alcohol. These results help provide a rational basis to prevent the accidental spread of HTLV-III/LAV in the laboratory or clinical setting.

Characterization of highly immunogenic p66/p51 as the reverse transcriptase of HTLV-III/LAV.

Approximately 80 percent of all human sera that react with antigens of HTLV-III, the etiologic agent of the acquired immune deficiency syndrome (AIDS), recognize protein bands at 66 and 51 kilodaltons. A mouse hybridoma was produced that was specific to these proteins. Repeated cloning of the hybridoma did not separate the two reactivities. The p66/p51 was purified from HTLV-III lysates by immunoaffinity chromatography and subjected to NH2-terminal Edman degradation. Single amino acid residues were obtained in 17 successive degradation cycles. The sequence determined was a perfect translation of the nucleotide sequence of a portion of the HTLV-III pol gene. The purified p66/51 had reverse transcriptase activity and the monoclonal immunoglobulin G specifically removed the enzyme activity from crude viral extract as well as purified enzyme.

Biochemical heterogeneity of reverse transcriptase purified from the AIDS virus, HTLV-III.

The reverse transcriptase from AIDS virus, HTLV-III, was purified and characterized. The purified enzyme has a very high affinity for template primers (rC)n X (dG)12 and (rCm)n X (dG)12 compared to that for (rA)n X (dT)12. In addition, the HTLV-III reverse transcriptase was able to transcribe (rAm)n X (dT)12 very efficiently. The ionic requirements are unique in the sense that HTLV-III reverse transcriptase prefers Mg2+ as divalent ions to transcribe (rC)n X (dG)12 and (rA)n X (dT)12. The Mr of the enzyme is 95 000-98 000. Unlike the HTLV-I reverse transcriptase, the HTLV-III enzyme is highly stable and has a much higher activity in the presence of (rC)n X (dG)12; the Vmax for HTLV-III reverse transcriptase is several-fold higher than that for HTLV-I enzyme. The enzyme activity of the purified reverse transcriptase from HTLV-III was resolved into two peaks on a preparative isoelectric column, one at pH 5.75 and the other at pH 6.25. This leads us to conclude that the reverse transcriptase of HTLV-III is biochemically heterogeneous.

HTLV-III gag protein is processed in yeast cells by the virus pol-protease.

The gag-pol gene of HTLV-III (human T-lymphotropic virus), the virus linked to AIDS (acquired immune deficiency syndrome), was expressed in yeast, and processing of the gag precursor into proteins of the same size as those in the virion was observed. Processing of the gag gene in yeast cells mimics the process that naturally occurs in mammalian cells during maturation of virions. Therefore it was possible to perform mutational analysis of the virus genome to localize the gene that codes for the protease function to the amino terminal coding region of the pol gene. Since this region overlaps the gag gene, it is likely that ribosomal frameshifting occurs from gag to pol. Antibodies in all of the AIDS patients' sera tested recognized the yeast synthesized gag proteins, although the sera showed differences in relative reactivity to the individual gag proteins and the precursor. This yeast system should be valuable not only for production of viral proteins for diagnostic or vaccine purposes but also for analysis of the genetics and biochemistry of viral gene functions--parameters that are difficult to study otherwise with this virus.

Characterization of the AIDS-associated retrovirus reverse transcriptase and optimal conditions for its detection in virions.

The RNA-dependent DNA polymerase of the AIDS-associated retrovirus (ARV) gives highest activity with the synthetic template, poly(rA)oligo(dT) and prefers Mg2+ over Mn2+ as a divalent cation. It can use other template-primer combinations but with poly(rCm)oligo(dG), it prefers Mn2+ over Mg2+. Detection of ARV reverse transcriptase in culture fluids is substantially increased with an optimal KCl concentration and a special combination of EGTA and reducing agents. Our results indicate some distinguishing characteristics of the ARV reverse transcriptase and optimal conditions for its detection.

AIDS-associated retroviruses (ARV) can productively infect other cells besides human T helper cells.

We have examined the host range of AIDS-associated retroviruses (ARV) that are known to infect human T cells of the helper subset. We have observed that the virus cannot infect fibroblast and epithelial cell lines of many different animal species. It is infectious and replicates efficiently in peripheral mononuclear cells (PMC) of chimpanzee and at low levels in baboon and rhesus monkey PMC. Most importantly, it has been found to replicate in established lines of human B cells, monocytes, and promyelocytes. This ability to infect these other cell types appears to be associated, in most cases, with the presence of the Leu 3 T helper cell antigen on the cell surface. Other mechanisms for virus infection, however, may be involved. The results suggest that ARV will be found in other cells of AIDS patients, besides T cells, and that these cells could be the reservoir for continual virus spread in the host. Variations in the replicative ability of ARV isolates in human cells have also been noted; they could reflect potentially important pathogenic differences among these human retroviruses.

Infection of human T-lymphotropic virus type-I (HTLV-I)-bearing MT-4 cells with HTLV-III (AIDS virus): chronological studies of early events.

Early events in the infection of the human T-lymphotropic virus type-I (HTLV-I)-positive MT-4 cell line by the acquired immune deficiency syndrome (AIDS) retrovirus HTLV-III were investigated. The virus was adsorbed completely to the cells within 60 min incubation after inoculation of the virus. Then, infected MT-4 cells started to produce the HTLV-III-specific antigens between 12 and 24 hr postinfection. Synthesis of the viral antigens consisting of 120K, 46K, 24K, and 17K polypeptides was suppressed by the treatment of the virus-infected MT-4 cells with cytosine arabinoside (Ara-C) or by the treatment of the virus with anti-HTLV-III-positive sera. The progeny of the virus released from the infected MT-4 cells was titrated by a newly developed plaque-forming assay method and reverse transcriptase activity. The maximum activity of HTLV-III (3 X 10(5) PFU/ml) was observed on Days 4 and 5 p.i. Most of the viral activities in this preparation were ascribed to HTLV-III, and not to HTLV-I. No phenotypic mixing between HTLV-III and HTLV-I was discerned, although MT-4 cells were HTLV-I-producer cell line. Thus, HTLV-III-infected MT-4 cells are thought to be useful in further study of the interaction between host cells and the virus, and appear to be a good viral source for the analysis of the virus.