Abstract
HIV-1 Rev is the key protein in the nucleocytoplasmic export and expression of the late viral mRNAs. An important aspect for its function is its ability to multimerize on these mRNAs. We have recently identified a llama single-domain antibody (Nb190) as the first inhibitor targeting the Rev multimerization function in cells. This nanobody is a potent intracellular antibody that efficiently inhibits HIV-1 viral production. In order to gain insight into the Nb190-Rev interaction interface, we performed mutational and docking studies to map the interface between the nanobody paratope and the Rev epitope. Alanine mutants of the hyper-variable domains of Nb190 and the Rev multimerization domains were evaluated in different assays measuring Nb190-Rev interaction or viral production. Seven residues within Nb190 and five Rev residues are demonstrated to be crucial for epitope recognition. These experimental data were used to perform docking experiments and map the Nb190-Rev structural interface. This Nb190-Rev interaction model can guide further studies of the Nb190 effect on HIV-1 Rev function and could serve as starting point for the rational development of smaller entities binding to the Nb190 epitope, aimed at interfering with protein-protein interactions of the Rev N-terminal domain.
Highlights
Nuclear export of viral mRNAs is a crucial step in the HIV-1 replication cycle [1]
The Rev-GFP fusion protein expressed from a transfected plasmid localizes mainly to the nucleoli (Fig. 2A), while wild-type Nb190 fused to monomeric Kusabira Orange (mKO) is found both in the cytoplasm and the nucleus, but is excluded from the nucleoli (Fig. 2B)
When these two proteins are co-expressed in the same cell, Rev-GFP and Nb190-mKO colocalize in the cytoplasm (Fig. 2C), implying interaction between these two proteins
Summary
Nuclear export of viral mRNAs is a crucial step in the HIV-1 replication cycle [1]. Fully spliced mRNA expressing the ‘early genes’ is exported through the cellular host mechanism. For the transport of unspliced and incompletely spliced ‘late’ mRNA species that encode structural viral proteins and serve as viral RNA genome, HIV-1 uses a complex mechanism These late viral RNA species all contain a secondary structured RNA element (Rev responsive element or RRE) on which a multimeric Rev export complex is formed [2,3] that employs the CRM1mediated cellular pathway for nuclear export [4,5,6]. The N-terminal helix-turn-helix loop [7] contains a basic argininerich stretch that interacts with the RRE and serves as a nucleolar localization signal (NoLS) [2] This NoLS is flanked by two hydrophobic regions responsible for Rev multimerization [8]. Under steady state conditions Rev localizes mainly to the nucleoli, it shuttles continuously between the cytoplasm and the nucleus [11,12]
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