During the past three and one half years we (i) demonstrated that several nitroso-, disulfide- and azo-containing compounds with anti-HIV activity and chemotherapeutic potential function mechanistically by ejecting zinc from the HIV-1 NC zinc fingers, (ii) established an expression system for the preparation of recombinant nucleocapsid protein (NC) from HIV-1, (iii) determined the dynamical properties of HIV-1 NC from a complete NMR relaxation/dynamics analysis, (iv) prepared RNA fragments of the HIV-1 psi-packaging signal by T7 RNA polymerase methods, and (v) very recently obtained NMR data of outstanding quality for a complex formed between the HIV-1 NC protein and a 20-nucleotide psi-RNA stem-loop (SL3)that is essential for viral genome recognition and packaging (Kd = 80 nM). We have also cloned, expressed, purified, and initiated structural studies of the NC protein from mouse mammary tumor virus (MMTV), which appears to be related to a virus associated with breast cancer in humans, and discovered a novel zinc finger fold. We are now poised to determine the 3D structure of the HIV-1 NC-SL3 psi- RNA complex, which will provide details regarding the protein-RNA interactions that lead to viral RNA encapsidation. Thermodynamic and kinetic parameters associated with the HIV-1 NC-SL3 psi-RNA equilibrium will be evaluated by a combination of NMR, fluorimetric and gel-shift methods. Attention will then focus on three additional stem-loop structures located within the HIV-1 psi-RNA recognition site that are important for encapsidation. We will also complete the 3D structure determination of the MMTV NC protein, which contains a novel CCHC zinc finger structure, and extend studies of NC-RNA interactions to other retroviral systems, including human T-Cell leukemia virus (HTLV), Rous sarcoma virus (RSV) and MMTV. Studies of zinc-ejecting antiviral agents with chemotherapeutic potential be continued, and new studies of agents that appear to interfere with NC-RNA recognition will be initiated. The long term objective is to develop understanding of the molecular basis for retroviral genome recognition and packaging. Knowledge of the structural, dynamical, and intermolecular interactive properties of retroviral NC proteins and their interactions with RNA and potential therapeutic agents is essential for understanding molecular-level aspects of virus assembly and should facilitate the development of new approaches for the treatment of AIDS and cancer.
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