Molecular Design, Functional Characterization and Structural Basis of a Protein Inhibitor Against the HIV-1 Pathogenicity Factor Nef

Sebastian Breuer,Wulf Blankenfeldt,Matthias Geyer,Antje Schulte,Oliver T Fackler,Simone I Schievink,Johan K Sandberg

doi:10.1371/journal.pone.0020033

Abstract

Increased spread of HIV-1 and rapid emergence of drug resistance warrants development of novel antiviral strategies. Nef, a critical viral pathogenicity factor that interacts with host cell factors but lacks enzymatic activity, is not targeted by current antiviral measures. Here we inhibit Nef function by simultaneously blocking several highly conserved protein interaction surfaces. This strategy, referred to as “wrapping Nef”, is based on structure-function analyses that led to the identification of four target sites: (i) SH3 domain interaction, (ii) interference with protein transport processes, (iii) CD4 binding and (iv) targeting to lipid membranes. Screening combinations of Nef-interacting domains, we developed a series of small Nef interacting proteins (NIs) composed of an SH3 domain optimized for binding to Nef, fused to a sequence motif of the CD4 cytoplasmic tail and combined with a prenylation signal for membrane association. NIs bind to Nef in the low nM affinity range, associate with Nef in human cells and specifically interfere with key biological activities of Nef. Structure determination of the Nef-inhibitor complex reveals the molecular basis for binding specificity. These results establish Nef-NI interfaces as promising leads for the development of potent Nef inhibitors.

Highlights

Active antiretroviral therapy (HAART) currently employed to treat AIDS patients consists of a combination of drugs that target the HIV enzymes reverse transcriptase, protease and integrase as well as inhibitors of virus entry
Nef mutagenesis studies established that only simultaneous disruption of several independent protein interactions of Nef efficiently abrogates the complex array of its biological activities [8]
We hypothesized that a Nef protein inhibitor has to shield several of these interaction motifs at a time

Summary

Introduction

Active antiretroviral therapy (HAART) currently employed to treat AIDS patients consists of a combination of drugs that target the HIV enzymes reverse transcriptase, protease and integrase as well as inhibitors of virus entry. One of many strategies to improve this situation is the exploitation of additional drug targets that could be added to the current regiment. In infected patients or monkeys Nef is critical for high virus replication and disease progression. Defects in the nef gene lead to slowly progressing or even asymptomatic infections and transgenic mice expressing Nef as the only HIV-1 gene product develop AIDS-like disease [4,5,6,7]

Methods

Results

Conclusion