Human immunodeficiency virus type 1 capsid protein is a substrate of the retroviral proteinase while integrase is resistant toward proteolysis.

The capsid protein of human immunodeficiency virus type 1 was observed to undergo proteolytic cleavage in vitro when viral lysate was incubated in the presence of dithiothreitol at acidic pH. Purified HIV-1 capsid protein was also found to be a substrate of the viral proteinase in a pH-dependent manner; acidic pH (<7) was necessary for cleavage, and decreasing the pH toward 4 increased the degree of processing. Based on N-terminal sequencing of the cleavage products, the capsid protein was found to be cleaved at two sites, between residues 77 and 78 as well as between residues 189 and 190.

Covalently crosslinked complexes of human immunodeficiency virus type 1 (HIV-1) gp120 and CD4 receptor elicit a neutralizing immune response that includes antibodies selective for primary virus isolates.

Specific conformational changes in the envelope glycoprotein gp120 of the human immunodeficiency virus type-1 (HIV-1) may be critical for eliciting a broadly neutralizing immune response against primary virus isolates. Since the interaction of gp120 with its receptor, CD4, induces conformational perturbations in both molecules, gp120-CD4 complexes should present unique immunogenic features that may include novel epitopes for broadly neutralizing antibodies. To test this hypothesis, we raised polyclonal antiserum against covalently crosslinked gp120-CD4 complexes in a goat and examined the ability of the anti-complex antibodies to neutralize primary and laboratory-adapted HIV-1 isolates.

Monoclonal antibodies raised against covalently crosslinked complexes of human immunodeficiency virus type 1 gp120 and CD4 receptor identify a novel complex-dependent epitope on gp 120.

The binding of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein, gp120, to its cell surface receptor, CD4, represents a molecular interaction involving distinct alterations in protein structure. Consequently, the pattern of epitopes presented on the gp120-CD4 complex should differ from those on free gp120. To investigate this concept, mice were immunized with covalently crosslinked complexes of viral HIV-1IIIBgp120 and soluble CD4.

Detection of HIV-1 infection in vitro using NASBA: an isothermal RNA amplification technique.

Establishment of a sensitive infection assay for HIV-1 is essential for successful screening of antiviral agents and neutralizing antibodies. In this report, an infection assay is described which measures the expression of viral genomic RNA and spliced mRNA intermediates in infected cells by an amplification-based technique called NASBA. The extreme sensitivity of this method permits the detection of viral RNA in peripheral blood mononuclear cells (PBMC) within 48 h of infection by a low dose of virus.

Mechanism of enzyme inhibition mediated by anti-reverse transcriptase antibodies from HIV type 1-infected individuals.

We have examined the mechanisms of reverse transcriptase (RT) inhibition mediated by anti-RT antibodies, isolated by affinity chromatography, from four HIV-1-positive individuals. In kinetics assays, anti-RT immunoglobulin (Ig) obtained from three of the sera mediated a noncompetitive type of inhibition against template primer; two of these three also mediated noncompetitive inhibition with respect to deoxyribonucleoside triphosphate. Such inhibition did not require that the Ig be preincubated with RT prior to the addition of reaction components.

Enzyme immunoassay using native envelope glycoprotein (gp160) for detection of human immunodeficiency virus type 1 antibodies.

An enzyme immunoassay using the purified native gp160 for the detection of human immunodeficiency virus type 1 (HIV-1) antibody was developed. This assay was determined to be highly specific, since (i) 157 serum samples that were confirmed negative by Western blot (immunoblot) (WB) were negative, (ii) 41 serum samples from populations with medical conditions that might cause nonspecific assay reactivity were all negative, and (iii) all 15 serum samples that showed false-positive reactions in one or more commercial HIV-1 screening tests were negative. The assay gave 100% specificity with a randomly selected and unlinked panel of 1,000 serum samples from healthy blood donors.

Glycoprotein of human immunodeficiency virus type 1 synthesized in chronically infected Molt3 cells acquires heterogeneous oligosaccharide structures.

Diversity of oligosaccharide structures on the glycoprotein of HIV-1 was studied in individual clones of Molt3 cells chronically infected with HIV-1IIIB. A glycoprotein of molecular weight 140 kD (gp140) was found to be shed into the medium from one of these clones, which unlike normally processed gp120, contained significant proportions of endo H resistant oligosaccharides. Treatment of infected cells with the inhibitors of oligosaccharide trimming enzymes affected the glycosylation pattern as well as the secretion of the glycoprotein into the medium.

Conformational perturbation of the envelope glycoprotein gp120 of human immunodeficiency virus type 1 by soluble CD4 and the lectin succinyl Con A.

We have studied perturbation of the gp120/gp41 envelope complex of HIV-1 in the presence of the mannose-specific lectin succinyl Con A (SC) and compared the effect with that observed in the presence of soluble CD4 (sCD4). SC did not inhibit the binding of gp120 to CD4. Both sCD4 and SC inhibited syncytium formation induced by HIV-1-infected Molt3/HIV-1IIIB cells.

High prevalence of serum antibodies to equine infectious anemia virus reverse transcriptase.

The immunogenicity of the equine infectious anemia virus (EIAV) reverse transcriptase (RT) was examined by immunoblot assay with recombinant EIAV RT. All of the 19 sera from EIAV-infected horses tested contained antibodies that recognized EIAV RT and directly inhibited the polymerase activity of the enzyme. An examination of sera obtained sequentially from two experimentally infected animals revealed that anti-RT antibodies arise early in infection and increase in level.

Delineation of immunoreactive, conserved regions in the external glycoprotein of the human immunodeficiency virus type 1.

Immunization of mice and rats with purified external glycoprotein gp120 from two divergent human immunodeficiency virus type 1 (HIV-1) isolates resulted in the development of seven hybridomas secreting monoclonal antibodies able to recognize regions of gp120 which are common among divergent strains of HIV-1. These monoclonal antibodies cross-reacted with env glycoproteins from one African (Rutz), one Haitian (RF), and three North American viral isolates, namely IIIB, MN, and 451 by either immunoblot or radioimmunoprecipitation assays. All recognized denatured gp120 in immunoblots with the exception of one which required a conformationally intact glycoprotein for reactivity.

Identification of a major growth factor for AIDS-Kaposi's sarcoma cells as oncostatin M.

Conditioned medium from human T cell leukemia virus type 2 (HTLV-II)-infected T cells supports the growth and long-term culture of cells derived from acquired immunodeficiency syndrome (AIDS)-associated Kaposi's sarcoma lesions (AIDS-KS cells). A protein of 30 kilodaltons was purified from conditioned medium that supports the growth of AIDS-KS cells. The amino-terminal sequence of this protein was identical to the amino-terminal sequence of Oncostatin M, a glycoprotein that inhibits the growth of a variety of cancer cells.

Characterization of a neutralizing monoclonal antibody to the external glycoprotein of HIV-1.

The major neutralizing epitope on the external glycoprotein of HIV-1 was studied with an envelope-specific monoclonal antibody and with a human serum positive for antibodies to HIV-1 proteins, both of which were able to neutralize virus infectivity. The monoclonal antibody reacted specifically with gp120 from HIV-1IIIB, and was shown to neutralize infection of CEM cells by cell-free virions, and inhibited the formation of syncytia normally observed when uninfected cells are cocultured with HIV-1-infected cells. Similar neutralization of viral infection and inhibition of syncytia formation was also demonstrated by the HIV-1-antibody-positive human serum.

Purification and partial characterization of equine infectious anemia virus reverse transcriptase.

Previously we raised a rabbit monospecific antibody (C2003) against a synthetic peptide derived from a sequence within the C-terminal portion of the reverse transcriptase (RT) of the human immunodeficiency virus type 1 (HIV-1). This sequence is found to be conserved in the predicted amino acid sequence of a related lentivirus, the equine infectious anemia virus (EIAV). It was previously determined that the C2003 antibody could cross-react with native EIAV RT and directly inhibit the DNA polymerase activity of the enzyme.

Brefeldin A inhibits the processing and secretion of envelope glycoproteins of human immunodeficiency virus type 1.

The processing and secretion of the envelope glycoproteins of human immunodeficiency virus type 1 (HIV-1) were studied in chronically infected T cells and in primary macrophages treated with an antiviral antibiotic brefeldin A (BFA). BFA blocks the egress of proteins from the endoplasmic reticulum and has a profound effect on the structure of cis/medial Golgi. In MOLT-3 cells infected with the IIIB strain of HIV-1 (MOLT-3/IIIB), BFA inhibited the intracellular processing of gp160.

Effect of Evans blue and trypan blue on syncytia formation and infectivity of human immunodeficiency virus type I and type II in vitro.

Polyanionic compounds were used to inhibit infectivity of human immunodeficiency virus in vitro. Suramin, Evans blue, and Trypan blue were shown to inhibit syncytia formation normally observed when HIV-1-infected cells are cocultured with CD4+ cells. The inhibition was more pronounced with Evans blue than with any of the other polyanions studied.

Lateral diffusion of CD4 on the surface of a human neoplastic T-cell line probed with a fluorescent derivative of the envelope glycoprotein (gp120) of human immunodeficiency virus type 1 (HIV-1).

The envelope glycoprotein (gp120) of HIV-1 was labeled with fluorescein by using 6-[4,6-dichlorotriazinyl]aminofluorescein. The labeled glycoprotein was found to bind to CD4-positive CEM cells. Monoclonal antibody OKT4a but not OKT4 blocked this binding.

Interaction of C-terminal sequences of human immunodeficiency virus reverse transcriptase with template primer.

We have raised a rabbit monospecific antibody (designated C2003) against a synthetic peptide (CTP66) derived from a conserved sequence in the C-terminal portion of the p66 component of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) (DeVico, A.L., Copeland, T.

Mode of action of 5'-linked cholesteryl phosphorothioate oligodeoxynucleotides in inhibiting syncytia formation and infection by HIV-1 and HIV-2 in vitro.

A phosphorothioate homocytidine 10-mer containing a cholesteryl moiety covalently linked to the 5'-end (Chol-SdC10) inhibited syncytium formation in susceptible T cells induced by HIV-1 and HIV-2. The syncytium inhibition effect was minimal with unmodified cytidine homopolymer of the same net charge. Chol-SdC10 was shown to protect CEM cells against infection by cell-free HIV-1 particles without any apparent toxicity to the growth of CD4+ T cells.

Differential viral gene expression and its effect on the biological properties of the cell clones of an HIV-1-infected cell line.

We have shown that 6D5 cells infected with the HIV-1 strain HTLV-III451 (6D5(451)) secreted viral envelope proteins gp160 and gp120 into the culture medium. Single cell cloning of 6D5(451) cells separated three distinct phenotypes. All clones secreted unprocessed env protein gp160 along with gp120.

Myristoylation of gag proteins of HIV-1 plays an important role in virus assembly.

The gag proteins of HIV-1 are modified by the addition of myristic acid to the amino terminal glycine residue. Site-directed mutagenesis was used to construct a mutant of HIV-1 in which this glycine residue was changed to an alanine. Upon transfection into cos-1 cells, the mutant genome directed the synthesis of the full complement of HIV-1 proteins, but p17 and p17-containing polyproteins were not myristoylated.

Characterization of the secreted, native gp120 and gp160 of the human immunodeficiency virus type 1.

We have previously shown that the cell line 6D5(451) chronically infected with the HIV-1 isolate HTLV-III(451), secretes the HIV-1 envelope glycoproteins gp120 and gp160 in the extracellular medium. The HTLV-III(451) gp120 and gp160 were purified by sequential affinity chromatographic steps using a monoclonal antibody to HIV-1 gp41 and an anti-HIV-1-positive human serum. Amino acid sequence analysis of gp120 and gp160 showed the loss of the signal peptide.

Monoclonal antibodies to HTLV-III451 gp41: delineation of an immunoreactive conserved epitope in the transmembrane region of divergent isolates of HIV-1.

We report on the development of monoclonal antibodies directed against the transmembrane portion of the envelope of HTLV-III451 gp41. One of these monoclonal antibodies, designated M71/2B4, was found to cross-react with transmembrane proteins from other independent isolates of HIV-1, namely IIIB, MN, and RF. Thus, this monoclonal antibody identifies an epitope located in a region of gp41 that is conserved among all these isolates.