Structural basis of the HIV-1 and HIV-2 protease inhibition by a monoclonal antibody.

PavlÍna Řezáčová^a, JULIEN Lescar^b, JiřÍ Brynda^a, MILAN Fábry^a, MAGDA Hořejší^a, RENATA Štouračová^a, GRAHAM Bentley^a and JURAJ Sedláček^a

^aDepartment of Gene Manipulation, Institute of Molecular Genetics, Academy of Sciences, 166 37 Prague, Czech Republic, ^bESRF, BP220, F-38043 Grenoble, France ^cUnité d´Immunologie Structurale, Dept. d´Immunologie, Institut Pasteur, 75724 Paris, France

Abstract:

The protease of HIV (HIV PR) plays a critical role in the maturation of the infectious particles of the virus. The enzyme has therefore been extensively studied with the objective of developing therapeutics that inhibit viral proliferation.

We have produced murine monoclonal antibodies (mAb) raised against recombinant HIV-1 protease and screened them for inhibition of proteolytic activity of the enzyme. Our objective has been to use these antibodies as tools to probe proteolytic activity and as eventual aids in the design of inhibitors. The murine monoclonal antibody (mAb) 1696, produced by immunisation with the HIV-1 protease, inhibits the catalytic activity of the enzyme of both the HIV-1 and HIV-2 isolates, with inhibition constants in the low nanomolar range [1].

This antibody cross-reacts with peptides that include the N-terminus of the enzyme (residues 1-7), a region which is highly conserved in sequence among different viral strains and which, furthermore, is crucial for homodimerization to the active enzymatic form.

We report here two crystal structures of a recombinant single-chain Fv fragment of mAb 1696, expressed in E. coli, as a complex with a cross-reactive peptides from the HIV-1 PR and the HIV-2 PR at 2.7 Å resolution [2] and 1.9 Å resolution respectively.

On the basis of the interactions seen in the complex three-dimensional structures, the cross-reactivity between mAb 1696 with the HIV-1 and HIV-2 protease and their N-terminal peptides can be explained. In addition, a candidate mechanism of HIV PR inhibition by mAb 1696 is proposed which may help the design of alternative HIV protease inhibitors, aimed at dissociating the homodimeric viral enzyme.

[1] Lescar J., Brynda J., Rezacova P., Stouracova R., Riottot M-M., Chitarra V., Fabry M., Horejsi M., Sedlacek J., Bentley G.A Protein Sci. 8 (1999) 2686

[2] Rezacova P, Lescar J, Brynda J, Fabry M, Horejsi M, Sedlacek J, Bentley G.A.. Structure 9 (2001) 887