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

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 ␣-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.Nuclear export of viral mRNA is critical for the life cycle of the human immunodeficiency virus (HIV).2 Fully spliced viral mRNA is exported to the cytoplasm of the infected cell through cellular mechanisms. However, in contrast to cellular systems, HIV uses a sophisticated molecular machinery to transport unspliced and partially spliced forms of its viral mRNA. The nuclear export of these late viral messengers is required for both the expression of late viral genes and packaging of genomic RNA and is mediated by the HIV-encoded regulatory protein Rev (1). 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 (2-4) and exploits the CRM1-mediated cellular machinery to transport these RNAs from the nucleus to the cytoplasm (5-7).Rev is a small protein of 116 amino acid residues. Under steady state conditions, Rev localizes mainly to the nucleoli of cells (8). However, different functional elements cause Rev to continuously shuttle between the nucleus and the cytoplasm (9 -11). 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). This arginine-rich region is flanked on both sides by sequences that contribute to the oligomerization of Rev on the RRE. Its strong tendency to oligomerize has hampered the structure determination of Rev by x-ray crystallography or NMR spectroscopy. However, circular dichroism, nuclear magnetic resonance, and Raman spectroscopy studies strongly suggest that the oligomerization domains and the nuclear localization signal are components of a helix-turn-helix motif (12-14). A...

Nuclear Export Signal Masking Regulates HIV-1 Rev Trafficking and Viral RNA Nuclear Export

“…Conversely, it is worth noting that NLS peptides have also been found to be masked in both cellular and viral protein contexts (85,(91)(92)(93). To our knowledge, this study is the first to directly implicate NES masking as a regulatory feature of Rev trafficking and the HIV-1 life cycle and may be relevant to previous observations from Daelemans and coworkers wherein targeted disruption of Rev multimerization caused reduced steady-state levels of Rev in the nucleolus (94,95). Interestingly, Gu et al have also provided evidence for cell-type-specific Rev NLS masking regulated by a cellular cofactor MDFIC (also known as the human I-mfa domain-containing protein or HIC) (96).…”

Functional inhibition of β-catenin-mediatedWnt signaling by intracellular VHHantibodies

“…These observations validated the selection of llama VHH domains for use as intracellular antibodies in this study and were in accordance with previous reports of VHHs as intracellular antibodies. [39][40][41][42][43][44] Mutation of 3 residues in CDR3 of LL3 was sufficient to disrupt antigen binding; this was in line with another intracellular domain antibody study where mutation of 4 residues in CDR1 was sufficient to disrupt intracellular function. 29 A number of studies have investigated the effect of intracellular antibody pI on intracellular aggregation, 45 and concluded that an overall negative charge at pH7.4 was beneficial to intracellular antibody solubility.…”

Single Domain Antibodies and Bioengineering Drugs on Their Basis: New Opportunities for Diagnostics and Therapy

“…Было получено наноантитело Nb190, которое способно ингибировать белок-белковое взаимодействие, не только затрудняя мультимеризацию Rev, но также способствуя разборке уже существующих мультимеров. В итоге было показано, что экс-прессия Nb190 в цитоплазме способна дозоза-висимо ингибировать продукцию ВИЧ [123]. В 2007 году Управлением по контролю качества пищевых продуктов и лекарственных препара-тов США (FDA), а также Европейской комисси-ей был утвержден препарат Maraviroc (Selzentry, или Celsentri) -антагонист рецептора CCR5 -белка, способствующего проникновению ВИЧ в клетку.…”