An intrabody based on a llama single-domain antibody targeting the N-terminal alpha-helical multimerization domain of HIV-1 rev prevents viral production.
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.
Vercruysse
Thomas
T
Rega Institute for Medical Research, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium.
Pardon
Els
E
Vanstreels
Els
E
Steyaert
Jan
J
Daelemans
Dirk
D
D021581:Active Transport, Cell Nucleus; D000595:Amino Acid Sequence; D000818:Animals; D019380:Anti-HIV Agents / Q000494:pharmacology; D000906:Antibodies / Q000737:chemistry; D002161:Camelids, New World; D019281:Dimerization; D031541:Fluorescence Resonance Energy Transfer; D015694:Gene Products, rev / Q000737:chemistry* / Q000378:metabolism; D015658:HIV Infections / Q000188:drug therapy*; D015497:HIV-1 / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008239:Lysine / Q000737:chemistry; D008969:Molecular Sequence Data; D025941:Protein Interaction Mapping; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D014443:Tyrosine / Q000737:chemistry
D019380:Anti-HIV Agents; D000906:Antibodies; D015694:Gene Products, rev; D014443:Tyrosine; D008239:Lysine
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