In uninfected cells, RNA is transcribed from DNA, processed then transported out of the nucleus and translated into protein. In cells infected with HIV-1, the viral RNA genomes must be exported out of the nucleus without being processed so they can be packaged into new viral particles. To do this it must bind its own RNA genome from among the host RNA in the nucleus. This is achieved using the HIV-1 Rev protein that recognizes a Rev response element (RRE) in the viral RNA. Once bound to RRE, Rev self-associates and binds other host proteins forming a multiprotein-RNA complex which is exported from the nucleus. Our current studies are directed at describing the molecular details of this complex. Apart from fundamental information on the mechanism of viral replication, these studies may highlight points of vulnerability that may be suitable targets for therapeutic intervention including Rev itself. A picture of how Rev binds to RRE has come from our previous structural studies of both Rev and the RRE. The structure of the N-terminal half of dimeric Rev (region involved in RNA interaction) was solved for first time by using an antibody fragment (Fab) as a crystallization chaperone. The RRE RNA forms an A shape with one leg shorter than the other. The legs are about 55 Angstrom part and position the known binding sites for Rev on either arm of the A. The higher affinity binding site is on the lower part of the short arm and the lower affinity site is on the lower part of the longer arm, placing them about 55 Angstrom from each other. This spacing matches the interaction domains of the Rev dimer that are also about 55 Angstrom apart. Once bound to RRE, the Rev oligomerizes forming a complex that engages with the host nuclear export machinery. The oligomerization of Rev on RRE is essential for formation of an active nuclear export complex. To study this protein association, filaments were generated from the soluble Rev dimers and in collaboration with Laboratory of Structural Biology (NIAMS), their structure was determined by high resolution electron microscopy incorporating X-ray data from the N-terminal domain of Rev dimers. Our data revealed a third interface between the Rev which offers an explanation for how the arrangement of Rev subunits adapts to the A-shaped architecture of the RRE in the export-active complexes. Also, the structures contained additional density indicating that C-terminal domains (CTD) become partially ordered in the context of filaments. This is the first time structural information on the Rev CTD has been acquired as this domain is disordered in the crystals used for X-ray determinations (study published in Structure 2016). Further studies are required to determine in more detail the structure of the export-competent ensemble to expand our understanding of HIV-1 Rev's key role in the nuclear export of viral mRNA. These studies are ongoing and include preparation of Rev complexed with RNA and other proteins required to mediate nuclear export including CRM1 and RanGTP. All these proteins have been expressed and characterized and crystallization trials are underway. The antibody fragment (Fab) used for stabilizing Rev for structural studies was derived from a phage display antibody library. This antibody expressed in bacteria was humanized and was effective by binding to Rev with a very high affinity thereby preventing its oligomerization, which as mentioned above, is required for its function. In previous work (from 2014) we showed that this antibody had anti-HIV-1 activity. We also showed that cyclic peptides (up to 12 amino acids long) from the antibody variable regions (CDRs) could bind to Rev but we have not yet shown whether they also have anti-HIV-1 activity; this is ongoing research. However, we are attempting to co-crystallize the peptides with Rev in order to obtain a high-resolution structure of the complex, which may help design or model low-molecular weight analogous with improved (stronger) binding to Rev. In a parallel approach to targeting Rev, we are again using the fact that polymerization or self-association of Rev is required for function and hence is a model for drug screening. As a first step we are developing assays which can be used to measure Rev self-association and then applied to high-throughput screening where compounds that block Rev-Rev interactions can be rapidly identified. For this we have used light scattering with some limited success but in order to develop a more robust assay we have engineered Rev to include site-specific cysteine residues for introducing fluorescent probes, which will allow sensitive monitoring. HIV protease, a homodimeric protein, is essential in the viral life cycle and a major anti-HIV drug target. We have expressed and purified a number of wild type and drug resistant forms of the protease that have been used in structural studies. Novel drugs that bind to the protease have been studied by co-crystallization and by examining the crystal structures used to rationalize and optimize drug binding. Structural details of interactions between drug moieties and protein have led to the synthesis of new compounds with higher anti-HIV potency.
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