The proposed research will utilize a novel genetic platform to functionally profile the entire HIV genome at selected conditions with mutation at every single nucleotide position. The genetic interaction of HIV genome will be determined with selected host restriction factors. The genetic platform will quantitatively identify regions of the genome (and the corresponding coded amino acids) that are essential for the functional interaction, and represents a major step forward to identify host-pathogen interactions. Using the novel high-resolution genetic profiling approach that the research group has developed and successfully demonstrated, the study will quantitatively assess mutational impact for all possible nucleic acid base changes for every nucleotide position within the HIV-1 genome. The combination of saturation mutagenesis with next generation sequence will be applied to monitor mutations at all positions of the genome simultaneously. In accomplishing the research goals, the study will aim to establish the initial profile gauging the essentialness of each nucleotide position for HIV-1 replication, and subsequently identify nucleotide positions in the genome for resistance and compensatory effects to host restriction factor interferon-induced trans-membrane protein 3 (IFITM3). A well-characterized restriction factor apolipoprotein B mRNA-editing enzyme, catalytic polypeptide- like 3G (APOBEC3G) will be used as a comparison. More importantly, the proposed functional profiling method will demonstrate the ability to evaluate a large and diverse viral mutant population spanning an entire viral genome in all genomic space for the involvement in a replication process or pathogenic process on a single experimental platform, which will impact not just HIV-1 research, but the study of many other viral pathogens. From the obtained high resolution map of the entire HIV-1 genome detailing mutational tolerability at each nucleotide position for all possible base changes, which should prove highly beneficial for HIV/AIDS vaccine development. Identification of viral-cellular interactions provides a great wealth of new potentially useful targets for therapeutic drug design.
The study will apply newly developed functional profiling technology to quickly and efficiently map HIV-1 genomic regions implicated in cofactor interactions with no prior knowledge of where in the genome the interaction may be located. The identification of new host-HIV-1 protein-protein interactions will provide a wealth of new target sites for the rational design and development of novel HIV/AIDS therapeutics useful for clinical evaluation.
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|Olson, C Anders; Wu, Nicholas C; Sun, Ren (2014) A comprehensive biophysical description of pairwise epistasis throughout an entire protein domain. Curr Biol 24:2643-51|