An intrabody based on a llama single-domain antibody targeting the N-terminal alpha-helical multimerization domain of HIV-1 rev prevents viral production.

TitleAn intrabody based on a llama single-domain antibody targeting the N-terminal alpha-helical multimerization domain of HIV-1 rev prevents viral production.
Publication TypeJournal Article
Year of Publication2010
AuthorsVercruysse, T., E. Pardon, E. Vanstreels, J. Steyaert, and D. Daelemans
JournalJ Biol Chem
Volume285
Issue28
Pagination21768-80
Date Published2010 Jul 9
ISSN1083-351X
KeywordsActive Transport, Cell Nucleus, Amino Acid Sequence, Animals, Anti-HIV Agents, Antibodies, Camelids, New World, Dimerization, Fluorescence Resonance Energy Transfer, Gene Products, rev, HeLa Cells, HIV Infections, HIV-1, Humans, Lysine, Molecular Sequence Data, Protein Interaction Mapping, Protein Structure, Secondary, Protein Structure, Tertiary, Tyrosine
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.

DOI10.1074/jbc.M110.112490
Alternate JournalJ. Biol. Chem.
PubMed ID20406803
PubMed Central IDPMC2898381