Protease cleavage sites in HIV-1 gp120 recognized by antigen processing enzymes are conserved and located at receptor binding sites

Abstract

The identification of vaccine immunogens able to elicit broadly neutralizing antibodies (bNAbs) is a major goal in HIV vaccine research. Although it has been possible to produce recombinant envelope glycoproteins able to adsorb bNAbs from HIV-positive sera, immunization with these proteins has failed to elicit antibody responses effective against clinical isolates of HIV-1. Thus, the epitopes recognized by bNAbs are present on recombinant proteins, but they are not immunogenic. These results led us to consider the possibility that changes in the pattern of antigen processing might alter the immune response to the envelope glycoprotein to better elicit protective immunity. In these studies, we have defined protease cleavage sites on HIV gp120 recognized by three major human proteases (cathepsins L, S, and D) important for antigen processing and presentation. Remarkably, six of the eight sites identified in gp120 were highly conserved and clustered in regions of the molecule associated with receptor binding and/or the binding of neutralizing antibodies. These results suggested that HIV may have evolved to take advantage of major histocompatibility complex (MHC) class II antigen processing enzymes in order to evade or direct the antiviral immune response.

FIG. 1.

Cathepsin digestion of MN-rgp120. MN-rgp120 was digested with either cathepsin L (A), cathepsin S (B), or cathepsin D (C). At the indicated times, samples were removed, the digestion was stopped by the addition of liquid nitrogen, and samples were prepared for SDS-PAGE analysis. Bands were visualized by Coomassie blue staining. The mobilities of the fragments identified are shown to the left of each panel. The mobility values of prestained molecular mass markers are shown to the right of panel C.

FIG. 2.

Diagram of MN-rgp120 fragments generated by cathepsin digestion. MN-rgp120 was digested with cathepsin L, S, or D, and the resulting fragments were purified and analyzed by Edman sequence degradation. Solid lines indicate peptides that were resolved by polyacrylamide gel electrophoresis and characterized by N-terminal sequence analysis. Dashed lines indicate the locations of peptides that were deduced from mobility and sequence analyses but were not recovered.

FIG. 3.

Locations of cathepsin L, S, and D cleavage sites on MN-gp120 disulfide-bonded schematic. Cathepsin L, S, and D cleavage sites in MN-rgp120 were identified by Edman sequence degradation and located on the disulfide-bonded structure of gp120 determined by Leonard et al. (58). Cathepsin L sites are indicated by closed arrows, cathepsin S sites are indicated by thin-line arrows, and cathepsin D sites are indicated by open arrows. The locations of amino acid residues reported to be important for the binding of CD4, chemokine receptors, and the α4β7 integrin are indicated by residues shaded red, dark blue, and light blue, respectively. The locations of amino acids known to be important for binding of the broadly neutralizing antibody b12 and neutralizing antibodies to the V3 domain are indicated by amino acids shaded green and purple, respectively. Residues with two or more colors indicate amino acids involved in the binding of two or more receptors or neutralizing monoclonal antibodies. The figure was created based on results from Kwong et al. (55), Zhou et al. (105), Rizzuto et al. (79), Decker et al. (25), and Arthos et al. (2). The numbering provided is based on the sequence of gp120 from the MN_GNE isolate of HIV (71).

FIG. 4.

Locations of cathepsin L, S, and D cleavage sites on three-dimensional structure of gp120 bound to CD4. The locations of cathepsin cleavage sites were located on the three-dimensional structure of gp120 based on the structure of a gp120 fragment complexed with CD4, described by Huang et al. (47). Cathepsin L sites are indicated in green, cathepsin S sites are in red, and cathepsin D sites are in blue. The structure of CD4 is shown in yellow. Numbering is based on the sequence of MN-rgp120.

FIG. 5.

Antibody binding to cathepsin L- and D-treated gp120. Purified MN-rgp120 was treated with either cathepsin L or cathepsin D and captured on the surfaces of microtiter plates coated with a polyclonal antibody, D7324, directed to the C terminus of gp120. Monoclonal antibodies were incubated with the gp120-coated microtiter plates, and binding was determined by ELISA. Closed symbols indicate binding to untreated gp120, open circles represent binding to cathepsin L-treated gp120, and open squares indicate binding to cathepsin D-treated gp120.