Multiplex discovery of synthetic host-protein combinations that inhibit HIV

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PROJECT SUMMARY Decades of HIV research have yielded a panel of potent HIV drugs, but we need the help of new approaches to eliminate the global HIV epidemic. Modern biotechnologies have opened new avenues toward developing engineered proteins that target the remaining vulnerabilities in the virus life-cycle. Novel protein combinations linking host-derived HIV binding peptides with effector domains that can mark the HIV protein for degradation may potently interfere with infection without causing major cellular toxicity. Unfortunately, there are hundreds to thousands of combinations that need to be tested, which is nearly impossible using traditional, one-by-one approaches. We propose to apply cutting edge synthetic biology tools for protein and cell engineering, harnessing multiplex, library-based assays to identify the most promising designs. In the first aim, we will perform large-scale pairwise fusions of known HIV binding peptides with different cellular ubiquitin ligase effector domains, to identify the combinations that have the best therapeutic potentials. The second aim uses an unbiased, genome-wide transposon insertional mutagenesis approach to uncover novel binders capable of inhibiting HIV replication when fused to a ubiquitin ligase domain. Both approaches will harness novel combinations of protein sequences already found and expressed in humans, avoiding immune responses to non-self peptide antigens. This work will yield new lead therapeutics, advance rational protein design, and provide biological insight into how the stoichiometries of HIV protein targets and the multimerization of the engineered proteasomal degradation machinery provides the most accurate and specific antiviral responses.