Production Of HIV And HIV Related Proteins For Structural Studies

National Institute Of Arthritis And Musculoskeletal And Skin Diseases

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Abstract

In uninfected cells, RNA is transcribed from DNA, processed, and 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, the cell 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 that is exported from the nucleus. Our current studies are directed at describing the molecular details of this complex. In addition to contributing 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. The determination of RNA structures is difficult due to conformational flexibility and methodology for the study of RNA conformers was developed and applied to model HIV RRE. The A-shaped molecules confirm previous work and gives further insight into high affinity Rev binding (Nature 2025). Extending this work, we have used cryo-electron microscopy to determine high resolution structures of RRE and of the RRE- Rev complex. The structures are being refined and will be submitted to publicly accessible databases. The structures will be used to map potentially inhibitory molecules with ant-HIV activity. An antibody fragment (Fab) was used as a chaperone for stabilizing Rev in the initial structural studies. The Fab was derived from a phage display antibody library. This chimeric antibody (human framework and rabbit variable domains) was effective in binding to Rev with a very high affinity, thereby preventing its oligomerization. In previous work, 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. In addition, we are attempting to co-crystallize the peptides with Rev to obtain high-resolution structures of the complexes, which may help design or model low-molecular weight mimics with improved (stronger) binding to Rev. HIV-1 AIDS is associated with improper chromatid separation and aneuploidy. We have previously shown that Rev interacts strongly and stoichiometrically with tubulin to form double-ring-like complexes in vitro. We have also shown that treatment of tubulin with Cryptophycin-45 (cancer drug) forms highly stable single-rings composed of eight tubulin heterodimers, and that these bind Rev stoichiometrically to also form double-rings. The first structure of Hela tubulin in complex with cryptophycin was determined at 3.45 Angstrom resolution using cryo-EM techniques. We have also determined structure of Hela tubulin-cryptophycin rings in complex with HIV-Rev protein at 3.85 Angstrom resolution using cryo-EM techniques. The structure shows tubulin double rings formed in the presence of Rev. These findings are of interest to both the cancer and tubulin research communities. The double-rings formed when Rev is added will also provide further insights into Rev structure and function.