Disulfide Bond That Constrains the HIV-1 gp120 V3 Domain Is Cleaved by Thioredoxin

Iman Azimi,Lisa J Matthias,Rob J Center,Jason W.H Wong,Philip J Hogg

doi:10.1074/jbc.m110.185371

Iman Azimi, Lisa J Matthias + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m110.185371

Copy DOI

Abstract

A functional disulfide bond in both the HIV envelope glycoprotein, gp120, and its immune cell receptor, CD4, is involved in viral entry, and compounds that block cleavage of the disulfide bond in these proteins inhibit HIV entry and infection. The disulfide bonds in both proteins are cleaved at the cell surface by the small redox protein, thioredoxin. The target gp120 disulfide and its mechanism of cleavage were determined using a thioredoxin kinetic trapping mutant and mass spectrometry. A single disulfide bond was cleaved in isolated and cell surface gp120, but not the gp160 precursor, and the extent of the reaction was enhanced when gp120 was bound to CD4. The Cys(32) sulfur ion of thioredoxin attacks the Cys(296) sulfur ion of the gp120 V3 domain Cys(296)-Cys(331) disulfide bond, cleaving the bond. Considering that V3 sequences largely determine the chemokine receptor preference of HIV, we propose that cleavage of the V3 domain disulfide, which is facilitated by CD4 binding, regulates chemokine receptor binding. There are 20 possible disulfide bond configurations, and, notably, the V3 domain disulfide has the same unusual -RHStaple configuration as the functional disulfide bond cleaved in CD4.

Highlights

Research Council of Australia and the Australian Research Council. 1 To whom correspondence should be addressed: Level 2, Lowy Cancer Research Centre, University of New South Wales, Sydney, NSW 2052, Australia
Cleavage of the gp120 disulfide bond is critical for env-mediated cell-cell fusion and human immunodeficiency virus (HIV) entry and infection as these processes are blocked by both small molecule and protein inhibitors of cell surface oxidoreductases [3,4,5,6]
The disulfide bond or bonds cleaved in gp120 by oxidoreductases during fusion of the viral and cell membranes was determined using a thioredoxin trapping mutant, in which the active site cysteine (Cys35) that normally resolves the mixed disulfide has been mutated to serine [9, 17]

Summary

Introduction

Research Council of Australia and the Australian Research Council. 1 To whom correspondence should be addressed: Level 2, Lowy Cancer Research Centre, University of New South Wales, Sydney, NSW 2052, Australia. Either one or two of the nine disulfide bonds in gp120 is cleaved during viral and target cell membrane fusion [3,4,5,6, 13].

Results

Conclusion