INI1/hSNF5/SMARCB1/BAF47 is an HIV-specific integrase (IN)-binding protein, which influences HIV-1 transcription and particle production. INI1 binds to SAP18 (Sin3a associated protein 18 kDa) and both INI1 and SAP18 are incorporated into HIV-1 virions. To determine the significance of INI1 and the INI1-SAP18 interaction during HIV-1 replication, we isolated a panel of SAP18-Interaction-Defective (SID) INI1 mutants using a yeast reverse two-hybrid screen. SID-INI1 mutants, which retained the ability to bind to IN, cMYC and INI1, but impaired for binding to SAP18, were tested for their effect on HIV-1 particle production. SID-INI1 dramatically reduced intracellular Gag/Gag-Pol protein levels and additionally decreased viral particle production. SID-INI1-mediated effects were less dramatic in trans-complementation assays using IN deleted viruses with Vpr-RT-IN. SID-INI1 did not inhibit LTR-mediated transcription, but marginally decreased steady state gag RNA levels, suggesting a post-transcriptional effect. Pulse chase analysis indicated that in SID-INI1 expressing cells the pr55Gag levels rapidly decreased. RNA interference analysis indicated that shRNA-mediated knockdown of INI1 reduced intracellular Gag/Gag-Pol levels, and further inhibited HIV-1 particle production. These results suggest that SID-INI1 mutants inhibit multiple stages of post-transcriptional events of HIV-1 replication, including intracellular Gag/Gag-Pol RNA and protein levels, which in turn inhibit assembly and particle production. Interfering with INI1 leads to a decrease in particle production and Gag/GagPol protein levels. Understanding the role of INI1 and SAP18 in HIV-1 replication is likely to provide novel insight into the stability of Gag/Gag-Pol, which may lead to the development of novel therapeutic strategies to inhibit HIV-1 late events.
Significant gaps exist in our current understanding of the mechanism and host factors that influence HIV-1 post-transcriptional events including gag RNA levels, Gag/Gag-Pol protein levels, assembly and particle production. Our previous studies suggested that IN-binding host factor INI1 plays a role in HIV-1 assembly. An ectopically expressed minimal IN-binding domain of INI1, S6, potently and selectively inhibited HIV-1 Gag/Gag-Pol trafficking and particle production. However, whether or not endogenous INI1 and its interacting partners, such as SAP18, are required for late events was unknown. Here we report that endogenous INI1 as well as its interaction with SAP18 are necessary to maintain intracellular levels of Gag/Gag-Pol and for particle production. Interfering with INI1 or INI1-SAP18 interaction leads to the impairment of these processes, suggesting a novel strategy for inhibiting post-transcriptional events of HIV-1 replication.