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.
The HIV epidemic has claimed over 35 million lives to date, however ? thanks to the advances of HAART ? the number of people dying from HIV continues to drop; yet because new infectious continue to occur the number of people living with HIV-1 (36.7 million in 2015 according to the World Health Organization) continues to increase. To date, all of the FDA approved therapeutics against HIV-1 target virus that has already exited the infected cell and thus none of these therapeutics affect processes that occur within the virus-producing cell. Because elimination of latent virus producing cells would be required to terminate a viral infection, activities such as viral assembly that occur within virus producing cells are appealing targets for small molecular inhibitors, therefore this application proposes to characterize a small molecule that was recently discovered as an essential co-factor for HIV-1 assembly.
