Inositol hexakisphosphate (IP6) and inositol pentakisphosphate (IP5) are required for viral particle release of retroviruses belonging to the primate lentivirus genus

Abstract Inositol hexakisphosphate (IP6) potently stimulates HIV-1 particle assembly in vitro and infectious particle production in vivo. However, knockout cells lacking the enzyme inositol-pentakisphosphate 2-kinase (IPPK-KO), which adds the final phosphate to inositol pentakisphosphate (IP5) to produce IP6, were still able to produce infectious HIV-1 particles at a greatly reduced rate. HIV-1 in vitro assembly can also be stimulated to a lesser extent with IP5, but it was not known if IP5 could also function in promoting assembly in vivo. IPPK-KO cells expressed no detectable IP6 but elevated IP5 levels and displayed a 20-100-fold reduction in infectious particle production, correlating with lost virus release. Transient transfection of an IPPK expression vector stimulated infectious particle production and release in IPPK-KOs but not in wildtype cells. Several attempts to make an IP6 and IP5 deficient stable cell line were not successful, but transient expression of multiple inositol polyphosphate phosphatase-1 (MINPP1) into IPPK-KOs resulted in the near ablation of IP6 and IP5. Under these conditions, HIV-1 infectious particle production and virus release were essentially abolished (1000-fold reduction). However, other retroviruses including a Gammaretrovirus, a Betaretrovirus, and two non-primate Lentiviruses displayed only a modest (3-fold) reduction in infectious particle production from IPPK-KOs and were not significantly altered by expression of IPPK or MINPP1. The only other retrovirus found that showed a clear IP6/IP5 dependence was the primate (macaque) Lentivirus Simian Immunodeficiency Virus (SIV-mac), which displayed similar sensitivity to IP6/IP5 levels as HIV-1. Finally, we found that loss of IP6/IP5 in viral target cells had no effect on permissiveness to HIV-1 infection. However, because it was not possible to generate viral particles devoid of IP6 and IP5, we were not able to determine if IP6 or IP5 derived from the virus producer cells is required at additional steps beyond assembly. Author Summary Inositol hexakisphosphate (IP6) is a co-factor required for efficient production of infectious HIV-1 particles. The HIV-1 structural protein Gag forms a hexagonal lattice structure. The negatively charged IP6 sits in the middle of the hexamer and stabilizes a ring of positively charged lysines. Previously described results show that depletion of IP6 reduces, but does not eliminate, infectious virus production. This depletion was achieved through knock-out of inositol-pentakisphosphate 2-kinase (IPPK-KO), the enzyme responsible for adding the sixth and final phosphate to the molecule. Whether IP6 is required, another inositol phosphate can substitute, or IP6 is simply acting as an enhancer for virus production was unknown. Here, we show that loss of IP6 and inositol pentakisphosphate (IP5) abolishes infectious HIV-1 production from cells. We do this through a cell-based system using transiently expressed proteins to restore or deplete IP6 and IP5 in the IPPK-KO cell line. We further show that the IP6 and IP5 requirement is a feature of primate lentiviruses, but not all retroviruses, and that IP6 and IP5 is required in the producer but not the target cell for HIV-1 infection.