Structural and functional characterization of an epitope in the conserved C‐terminal region of HIV‐1 gp120

Abstract

Through an integrated study of the reactivity of a monoclonal antibody, 803-15.6, with synthetic peptides and native recombinant HIV-1 envelope glycoprotein gp120, we have obtained structure-functional information on a region of rgp120 not yet elucidated by X-ray crystallography. mAb 803-15.6 binds with high affinity and broad cross-clade specificity to the conserved C-terminal region (amino acids 502-516) of HIV-1 rgp120. Phage display selection from a random peptide library identified the core binding motif as AXXKXRH, homologous to residues 502-508. Using quantitative binding analyses, the affinity of mAb 803-15.6 for native, monomeric recombinant gp120HXB2 (rgp120) was found to be similar to that for the synthetic gp120 peptide (502-516). Circular dichroism studies indicate that the synthetic peptide largely has a random coil conformation in solution. The results therefore suggest that the 803-15.6 epitope is fully accessible on rgp120 and that this region of rgp120 is as flexible as the synthetic peptide. Residues 502-504 are on the edge of a putative gp41 binding site that has been postulated to change conformation on CD4 binding. However, the affinity of mAb 803-15.6 for rgp120 is not affected by binding of CD4 and vice-versa. These results suggest either that the 502-504 region does not change conformation upon CD4 binding, or that recombinant gp120 does not undergo the same changes as occur in the native viral gp120-gp41 oligomer. The detailed characterization of the 803-15.6 epitope may be useful for further study of the role of the C5 region of gp120 in the viral attachment and fusion process.

Screening Random Peptide Libraries with Subacute Sclerosing PanencephalitisBrain-Derived Recombinant Antibodies Identifies Multiple Epitopes in the C-Terminal Region of the Measles Virus Nucleocapsid Protein

“…In central nervous system (CNS) inflammatory diseases, where access to active diseased tissue is limited or where the levels of tissue antigen may be extremely low, phage peptide panning provides an alternative and sensitive avenue for antigen identification. Panning of phage-displayed random peptide libraries has successfully identified rheumatoid factor-specific mimotopes (22) and allergen mimotopes (19) and has mapped both linear and discontinuous viral epitopes recognized by antibodies specific for various infectious agents (5,10,11,16,20).Infectious and inflammatory diseases of the CNS are often characterized by increased intrathecal immunoglobulin G (IgG) synthesis that is seen as discrete bands of oligoclonal IgG when cerebrospinal fluid (CSF) or brain IgG is separated by isoelectric focusing. In CNS infectious diseases, such as subacute sclerosing panencephalitis (SSPE), neurosyphilis, mumps meningitis, progressive rubella panencephalitis, cryptococcal meningitis, and varicella zoster virus vasculitis, the oligoclonal IgG is directed largely against the infectious agent that causes the disease (reviewed in reference 12).…”

“…In central nervous system (CNS) inflammatory diseases, where access to active diseased tissue is limited or where the levels of tissue antigen may be extremely low, phage peptide panning provides an alternative and sensitive avenue for antigen identification. Panning of phage-displayed random peptide libraries has successfully identified rheumatoid factor-specific mimotopes (22) and allergen mimotopes (19) and has mapped both linear and discontinuous viral epitopes recognized by antibodies specific for various infectious agents (5,10,11,16,20).…”

Mass Spectrometric Characterization of a Discontinuous Epitope of the HIV Envelope Protein HIV-gp120 Recognized by the Human Monoclonal Antibody 1331A

“…HIV-gp120 and the synthetic peptides had comparable affinity for the Ab. Because the peptides had a low percentage of ␣ helices in solution, a disordered conformation of the same sequence in the native HIV-gp120 molecule was proposed (13). However, the mAb used by Ferrer et al is of murine origin induced by immunization with recombinant HIV-1 gp120, and therefore the structural information obtained may not reflect the conformation of HIV-gp120 in the intact virus particle.…”

“…Furthermore, it can provide insights into the tertiary structure of HIV-gp120 (13), because x-ray analysis of only a truncated HIV-gp120 molecule is available (5). Several methods for the characterization of epitopes have been described, such as random phage epitope library screening, x-ray analysis, proteolytic footprinting, and epitope excision/extraction techniques.…”

Identification and Characterization of Peptides Binding to Newcastle Disease Virus by Phage Display

“…It allows rapid identification and amplification of the peptide ligands for a certain target molecule by the affinity of their specific-binding [3,4,16,18]. Recently, it has been widely used in many fields, such as for identification and characterization of a peptide mimicking the epitope of HIV [5] and HIV-infected cells [10], and identification of enzyme inhibitors [8], immunodepressants [2], and many antigens. This technique is superior to the methods with antibodies, which have been often used to detect such binding peptides, since a peptide displayed on the surface of filamentous bacteriophage is able to bind to the regions in a molecule that the immune system can not recognize.…”