APOBECSG (A3G) and APOBECSF (A3F) are involved in various anti-viral protein and nucleic acid transactions. Important primary interactions include interprotein interactions, binding single-stranded nucleic acids (RNA and ssDNA), and neutralization by HIV Vif. The full elucidation of these intermolecular interactions is necessary to understand the mechanism by which these cellular proteins inhibit HIV replication. The role of oligomerization in their activity is an issue of great importance. These are interactions at the nanoscale level, and AFM is the method of choice capable to addressing problems listed above. AFM currently reached a level of development that enables this unique instrument to provide a wealth of important information to the characterization of biomolecular complexes at nanoscale. The topographic analysis of molecular system is the initial area of the AFM application for characterization of the structure and specificity of protein-DNA systems of a broad complexity. In addition to imaging, AFM is capable of direct measurements of molecular interaction within the system and research during this decade led to a dramatic progress in this area of the AFM applications. Our Preliminary Studies have already enabled us to provide the first images of A3G bound to ssDNA substrates. We further anticipate that the use of all modalities of AFM technology will help us detail these complexes, dissect the protein and chemical requirements, and extend key observations to ASF. To help achieve these overarching objectives, we will pursue the following three Specific Aims:
Aim 1. Image A3 complexes at nanoscale resolution using AFM (Program Objectives 1 &2);
Aim 2. Measure interactions within A3-ssDNA complexes using force spectroscopy (Program Objectives 1 &2) and Aim 3. Characterize A3-Vif interactions using AFM imaging and probing modalities (Program Objectives 3). The studies under this project are an integral component of our overall Program Project aiming to provide a comprehensive understanding of A3-AS3 A3-nucleic acid, and A3-Vif interactions. The nanoscale studies will provide a platform for evaluating any future therapeutics that function by leveraging the AS-Vif interaction.
HIV-1 Vif and human APOBEC3 proteins are exceptional targets for the development of new AIDS therapeutics. A3G and AS3 are involved in various intermolecular interactions and their elucidation will provide us with the mechanism of their HIV inhibition activity and will ultimately set a foundation for designing and testing novel HIV/AIDS therapies.
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