Summary It has been known for over 15 years that the in vitro assembly of HIV-1 particles can be stimulated under particular conditions by the addition of inositol hexakisphosphate (IP6), but the mechanism and biological relevance of this finding was never resolved. Recently, it was determined that IP6 binds to a six-helix bundle that is formed by HIV-1 structural protein (Gag) and stimulates bundle formation. In crystal structures, IP6 binds in the center of the six-helix bundle and forms a density that precisely matches an unidentified density that had been reported in authentic HIV-1 particles. To explore the importance of IP6 in HIV-1 replication, the Johnson lab generated two clonal knockout cell lines using CRISPR that each lack a gene (IPPK and IPMK) required for the synthesis of IP6 in cells. These cell lines both displayed a severe defect in their ability to produce infectious HIV-1 particles. However, many questions remain about how IP6 affects HIV-1 replication. The specific aims of this proposal are: 1. Determine what step in replication is inhibited in the IP6 deficient cells. 2. Quantitate the levels of IP6 in cells and virus, and determine the level required for HIV-1 replication. 3. Determine what retroviruses depend on IP6 for replication. 4. Determine if HIV-1 can adapt to IP6 deficient conditions.