An Intrabody Based on a Llama Single-domain Antibody Targeting the N-terminal α-Helical Multimerization Domain of HIV-1 Rev Prevents Viral Production

Thomas Vercruysse,Els Pardon,Els Vanstreels,Jan Steyaert,Dirk Daelemans

doi:10.1074/jbc.m110.112490

Abstract

The human immunodeficiency virus, type 1 (HIV-1)-encoded Rev protein is essential for the expression of late viral mRNAs. Rev forms a large organized multimeric protein-protein complex on the Rev response element of these viral mRNA species and transports them from the nucleus to the cytoplasm, exploiting the CRM1-mediated cellular machinery. Here we report the selection of a nanobody, derived from a llama heavy-chain only antibody, that efficiently blocks the assembly of Rev multimers. The nanobody inhibits HIV-1 replication in cells and specifically suppresses the Rev-dependent expression of partially spliced and unspliced HIV-1 RNA. In HIV-susceptible cells, this nanobody thus has potential as an effective anti-HIV agent using genetic immunization strategies. Its binding site was mapped to Rev residues Lys-20 and Tyr-23 located in the N-terminal alpha-helical multimerization domain. In the presence of this nanobody, we observed an accumulation of dimeric Rev species, supporting a head-to-head/tail-to-tail molecular model for Rev assembly. The results indicate that the oligomeric assembly of Rev follows an ordered stepwise process and identify a new epitope within Rev that could guide strategies for the development of novel HIV inhibitors.

Highlights

The viral Rev protein forms a multimeric complex on a secondary structured RNA element (the Rev response element (RRE)) present in all unspliced and partially spliced mRNAs [2,3,4] and exploits the CRM1-mediated cellular machinery to transport these RNAs from the nucleus to the cytoplasm [5,6,7]
A short stretch of basic amino acids characterized by 10 arginine residues serves both as a nuclear localization signal for the nuclear import of Rev and as an RNA-binding domain during the export of RNA-Rev complexes [4]
From a dozen nanomolar affinity Rev binders, we identified one nanobody that blocks the multimerization of Rev and inhibits human immunodeficiency virus (HIV)-1 replication

Summary

Introduction

Gel shift experiments of the respective alanine mutant Rev binding to the high affinity IIB hairpin of RRE were performed in the presence of Nb190 (Fig. 4E). Nb190 Inhibits the Rev-dependent Expression of RRE-containing RNA—To evaluate whether Nb190 influences the Rev function in cells, we first analyzed its effect on the Revdependent nucleocytoplasmic RNA export using the system developed by Hope et al [40].

Results

Conclusion