Differences in the antibody response to human immunodeficiency virus-1 envelope glycoprotein (gp160) in infected laboratory workers and vaccinees.

We have monitored fusion between cell pairs consisting of a single human immunodeficiency virus–1 (HIV-1) envelope glycoprotein–expressing cell and a CD4+ target cell, which had been labeled with both a fluorescent lipid in the membrane and a fluorescent solute in the cytosol. We developed a new three-color assay to keep track of the cell into which fluorescent lipids and/or solutes are redistributed. Lipid and solute redistribution occur as a result of opening a lipid-permissive fusion pore and a solute-permissive fusion pore (FPS), respectively. A synthetic peptide (DP178) corresponding to residues 643–678 of the HIV-1LAI gp120-gp41 sequence (Wild, C.T., D.C. Shugars, T.K. Greenwell, C.B. McDanal, and T.J. Matthews. 1994. Proc. Natl. Acad. Sci. USA. 91:12676–12680) completely inhibited FPS at 50 ng/ml, whereas at that concentration there was 20–30% fusion activity measured by the lipid redistribution. The differences detected in lipid mixing versus contents mixing are maintained up to 6 h of coculture of gp120-41–expressing cells with target cells, indicating that DP178 can “clamp” the fusion complex in the lipid mixing intermediate for very long time periods. A peptide from the NH2-terminal of gp41, DP107, inhibited HIV-1LAI gp120-gp41–mediated cell fusion at higher concentrations, but with no differences between lipid and aqueous dye redistribution at the different inhibitor concentrations. The inhibition of solute redistribution by DP178 was complete when the peptide was added to the fusion reaction mixture during the first 15 min of coculture. We have analyzed the inhibition data in terms of a fusion pore dilation model that incorporates the recently determined high resolution structure of the gp41 core.

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Separation and partial characterization of proteinases with substrate specificity for basic amino acids from human MOLT-4 T lymphocytes: identification of those inhibited by variable-loop-V3 peptides of HIV-1 (human immunodeficiency virus-1) envelope glycoprotein

Biochemical Journal ◽

10.1042/bj2920711 ◽

1993 ◽

Vol 292 (3) ◽

pp. 711-718 ◽

Cited By ~ 10

Author(s):

I T Harvima ◽

R J Harvima ◽

G Nilsson ◽

L Ivanoff ◽

L B Schwartz

Keyword(s):

Amino Acids ◽

Human Immunodeficiency Virus ◽

Ionic Strength ◽

Active Site ◽

Envelope Glycoprotein ◽

Gel Filtration ◽

V3 Loop ◽

Immunodeficiency Virus ◽

Hiv 1 ◽

Human Immunodeficiency Virus 1

The V3 loop of the HIV (human immunodeficiency virus)-1 envelope glycoprotein gp120 likely plays a role in HIV-1 infectivity. Although the amino acid sequence of the V3 loop is hypervariable, it contains a conserved region, Gly-Pro-Gly-Arg, that shows similarity to the active-site Gly-Pro-Cys-Arg sequence of inter-alpha-trypsin and trypstatin proteinase inhibitors. The purpose of the present work was to identify proteinases recognizing substrates with basic amino acids in the P1 substrate site that are present in MOLT-4 cells, a human CD4-positive T helper lymphocyte cell line, and to characterize these enzymes in terms of substrate, pH and ionic-strength preferences, size and susceptibility to various inhibitors, including 24- and 36-amino-acid-long V3 loop peptides. Extraction of MOLT-4 cells at low ionic strength solubilized nearly all of the trypsin-like activity, which was separable into five peaks of activity by chromatography on Mono-Q: Peaks 1, 2a, 2b, 3 and 4. All showed a neutral pH optimum, and all except Peak 4 showed optimal activity at high ionic strength. Peak 1 preferred Tos-Gly-Pro-Arg, p-nitroanilide (-pNA) substrate; Peaks 2-4 preferred benzyloxycarbonyl-Val-Leu-Gly-Arg-pNA. Peak 1, a zinc-dependent enzyme with serine and histidine in the active site, exhibited an M(r) of 75,000 on Superose 12 and was poorly inhibited by V3 loop peptides. Peak 2 contained two overlapping peaks, called 2a and 2b, that exhibited properties of zinc-dependent metalloproteinases. Gel filtration of Peak 2 activities revealed a major peak of activity at 81 kDa and a shoulder centred at 240 kDa. Each was modestly inhibited by V3 loop peptides. Peak 3, a zinc-dependent proteinase, exhibited a molecular mass of 100 kDa by gel filtration and was particularly sensitive to inhibition by V3 loop peptides. Peak 4 exhibited a molecular mass of 1100 kDa by gel filtration and was not inhibited by V3 loop peptides. None of these enzymes could be classified as mast-cell tryptase, and material in MOLT-4 cells cross-reactive with anti-(human tryptase) antibodies was not detected. Whether any of the MOLT-4 proteinases described in this study play a role in HIV-1 infectivity remains to be examined.

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Characterization of Simian-Human Immunodeficiency Virus Envelope Glycoprotein Epitopes Recognized by Neutralizing Antibodies from Infected Monkeys

Journal of Virology ◽

10.1128/jvi.72.10.8437-8445.1998 ◽

1998 ◽

Vol 72 (10) ◽

pp. 8437-8445 ◽

Cited By ~ 35

Author(s):

Bijan Etemad-Moghadam ◽

Gunilla B. Karlsson ◽

Matilda Halloran ◽

Ying Sun ◽

Dominik Schenten ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Antibody Response ◽

Envelope Glycoprotein ◽

Neutralizing Antibodies ◽

Neutralizing Antibody ◽

Virus Envelope ◽

Immunodeficiency Virus ◽

Variable Loops

ABSTRACT We characterized human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein epitopes recognized by neutralizing antibodies from monkeys recently infected by molecularly cloned simian-human immunodeficiency virus (SHIV) variants. The early neutralizing antibody response in each infected animal was directed mainly against a single epitope. This primary neutralizing epitope, however, differed among individual monkeys infected by identical viruses. Two such neutralization epitopes were determined by sequences in the V2 and V3 loops of the gp120 envelope glycoprotein, while a third neutralization epitope, apparently discontinuous, was determined by both V2 and V3 sequences. These results indicate that the early neutralizing antibody response in SHIV-infected monkeys is monospecific and directed against epitopes composed of the gp120 V2 and V3 variable loops.

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