Title: HIV-1 Rev multimerization: potential target for antiretroviral therapy?
Other Titles: HIV-1 Rev-multimerisatie: potentieel doelwit voor antiretrovirale therapie?
Authors: Vercruysse, Thomas
Issue Date: 6-Jun-2012
Abstract: Currently, 26 antivirals have been approved by the US Food and Drug Administration (FDA) for the treatment of HIV/AIDS. These antiviral drugs target the viral proteins reverse transcriptase (NRTIs and NNRTIs), protease, integrase, gp41 (TM) and the cellular co-receptor CCR5. Despite this broad range of antiviral drug classes, resistance development and long-term toxicity remain a major issue. Moreover, the presence of latent HIV reservoirs, which can be dormant for several decades without detectable viral load in the bloodstream, impede total clearance of the virus from infected patients. Therefore, it remains necessary to search for new antiviral targets and to develop new treatment strategies. The function of the HIV-1 Rev protein is crucial for viral replication. In the absence of Rev, intron-containing unspliced viral mRNA is, after its transcription in the nucleus, either spliced or degraded. Rev multimerizes on these intron-containing mRNAs via a secondary stem-loop structure, the Rev responsive element (RRE), followed by interaction with the cellular transport factor CRM1. Thus, Rev bridges RRE-containing viral mRNAs to a cellular nucleocytoplasmic transport pathway, resulting in translocation of this viral mRNA to the cytoplasm of the infected cell. Subsequently, viral proteins are expressed in the cytoplasm and assembled into new viral particles in which the viral genomic RNA is packaged. Although the Rev protein has been studied intensively for more than twenty years, many aspects of its function remain unclear. There are more and more indications that Rev is involved in processes other than viral mRNA export only, like e.g. translation and packaging. Moreover, dozens of cellular proteins have been associated with the Rev/RRE function, but their precise role still needs to be unraveled. Nevertheless, the fact that Rev plays a central role in viral replication and that it interacts with several cellular partners renders it an interesting target for the development of new antiviral strategies and provides a multitude of possible interaction sites. Considerable efforts have been conducted to develop Rev inhibitors, but none of these turned out to be a promising lead for further development as antiviral therapeutic. However, these attempts were mainly directed to the Rev-RRE and the Rev-CRM1 interaction, while a third crucial aspect of Rev function, being its multimerization on the RRE, has often been overlooked. Therefore, the goal of our research was to inhibit the HIV-1 Rev multimerization and validate this process as a potential novel target for antiviral therapy. The search for HIV-1 Rev multimerization inhibitors has been hampered by the lack of a way to rapidly measure Rev-Rev interactions in vitro. Therefore, we first developed a fast high-throughput in vitro Rev multimerization assay based on fluorescence resonance energy transfer (FRET). In this test, Rev-Rev interactions can be monitored both in solution and on the RRE. The assay was validated by the use of Rev multimerization mutants. The inhibition of protein-protein interactions requires a more rational approach than other targets like e.g. enzymes. Therefore, we utilized the distinctive features of small single-domain antibodies (nanobodies), which are derived from the heavy-chain antibodies (hcAb) found in llamas. After immunization of a llama with recombinant Rev protein, we could derive a dozen of Rev-binding nanobodies from its peripheral blood lymphocytes. These nanobodies were evaluated in the in vitro Rev multimerization assay and at least one of them (Nb190) was found to potently inhibit Rev multimerization. Gel-mobility shift assay experiments revealed that Nb190 inhibits Rev-Rev interactions on the RRE without disrupting the Rev-RRE interactions. In cells, Nb190 changes the subcellular localization of Rev from predominantly nucleolar to a more cytoplasmic localization and it prevents the expression of Rev-dependent viral mRNAs. Furthermore, this nanobody is able to drastically decrease the production of new HIV-1 particles in a Rev-specific way. Taken together, these results validate Rev multimerization as a potential target for the development of new antiviral therapies. To use the Nb190-Rev interaction surface as platform for the future development of small molecule inhibitors of Rev-Rev interactions, we have mapped the Nb190 paratope and the Rev epitope and performed docking experiments to delineate the Nb190-Rev interaction site. Residues K20 and Y23 in Rev are most crucial for epitope recognition, while residues T33, F100, Y105 and D107 constitute the Rev binding surface of Nb190. Further unraveling of the Rev export process will require the implementation of new high-resolution fluorescence techniques that allow single particle tracking of Rev export complexes. To master these techniques, as a first step, we visualized different parts of isolated viral particles with the use of photoactivatable localization microscopy (PALM) and direct stochastic optical reconstruction microscopy (dSTORM) combined with total internal reflection fluorescence microscopy (TIRFM).In summary, we applied a nanobody-based approach to discover an HIV-1 Rev multimerization inhibitor that abbrogates viral replication. This way, we identified and validated Rev multimerization as a potential target for the development of novel antiviral strategies.
Publication status: published
KU Leuven publication type: TH
Appears in Collections:Laboratory of Virology and Chemotherapy (Rega Institute)

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