Assembly and Replication of HIV-1 in T Cells with Low Levels of Phosphatidylinositol-(4,5)-Bisphosphate

Particle formation of retroviruses, including HIV-1, is driven by the precursor polyprotein Gag. HIV-1 Gag consists of four major domains, matrix (MA), capsid (CA), nucleocapsid (NC), and p6, as well as two spacer peptides, SP1 and SP2 (1). MA is required for Gag targeting and binding to the plasma membrane (PM). CA and NC are essential for Gag multimerization. p6 recruits the cellular ESCRT complexes that facilitate release of virions. These domains give rise to individual mature Gag proteins upon proteolytic cleavage mediated by viral protease, which occurs during or immediately after virus particle release. MA is composed of five major α-helices and a three-stranded β-sheet, which form a single globular domain (34). The N terminus of MA is modified by myristoylation, which is essential for membrane binding (7, 24, 27, 63). The myristoyl moiety is normally sequestered in the MA globular domain. Nuclear magnetic resonance (NMR) studies suggest that upon Gag multimerization or MA-phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P2] binding, the myristoyl moiety is exposed and mediates binding of Gag to the membrane (60, 65). In addition, the MA highly basic region (HBR), which is comprised of residues 17 to 31 in MA, interacts with acidic phospholipids in the inner leaflet of the membrane and is required for targeting of Gag to the PM in HeLa and T cells (33, 36, 53, 56, 71). Several amino acids within the MA globular domain are also involved in incorporation of the viral envelope glycoprotein into virions and the postentry process (5, 6, 8, 10, 15, 16, 21-23, 31, 32, 37, 38, 45, 48, 49, 55, 69, 70). Interaction of MA with the cytoplasmic tail of Env also regulates fusogenicity of Env (47, 68). Accumulating evidence suggests that the MA domain of HIV-1 Gag, in particular the HBR, interacts with a PM-specific acidic phospholipid, PI(4,5)P2, thereby facilitating proper localization of Gag to the PM and efficient Gag-membrane binding. Overexpression of polyphosphoinositide 5-phosphatase IV (5ptaseIV), which removes the phosphate group at the D5 position of the inositol ring of PI(4,5)P2, significantly reduces HIV-1 release from HeLa and HEK293T cells (11, 51). This virus release defect is due to Gag mislocalization to intracellular compartments and reduced membrane binding (13, 51). Similarly, 5ptaseIV expression inhibits release of HIV-2, murine leukemia virus (MLV), and Mason-Pfizer monkey virus (MPMV) but not equine infectious anemia virus (EIAV) from cell lines including HeLa, HEK293, and 293T cells (11, 29, 59, 64). In the case of EIAV, PI(3)P and PI(3,5)P2 are required for virus release (19). Consistent with the possibility that HIV-1 MA mediates Gag-PI(4,5)P2 interactions, HIV-1 particles are enriched in PI(4,5)P2 in an MA-dependent manner (11). These findings indicate that PI(4,5)P2 interacts with MA and plays an important role in virus particle production at least in adherent cell lines, such as HeLa cells. However, the role of PI(4,5)P2 in HIV-1 assembly in T cells, a natural host cell type for HIV-1, still remains to be determined. Several groups have examined interactions between PI(4,5)P2 and retroviral MA and/or Gag using various in vitro assays (2, 4, 12-14, 29, 59, 61, 62). NMR, surface plasmon resonance, and protein footprinting studies of HIV-1 MA/Gag-PI(4,5)P2 complexes revealed that the basic residues in HBR associate with water-soluble derivatives of PI(4,5)P2 (4, 61, 62). Interaction of HIV-1 Gag and MA with membrane-associated PI(4,5)P2 in its natural cellular form was demonstrated using liposomes or a lipid monolayer system (3, 13, 14). Furthermore, site-directed mutagenesis studies of the HBR showed that the basic residues in the HBR are important for binding to PI(4,5)P2 in liposomes as well (13, 14). Altogether, these reports strongly suggest that basic residues in the MA HBR play a key role in MA-PI(4,5)P2 binding. However, it remains to be determined whether residues outside of the HBR are also required for PI(4,5)P2 dependence of Gag. In this study, we found that Gag membrane binding, Gag localization to the PM, and virus release efficiency were impaired in T cells expressing 5ptaseIV, demonstrating that PI(4,5)P2 facilitates HIV-1 assembly in T cells. Using T cell clones that express 5ptaseIV upon HIV-1 infection, we obtained a mutant virus adapted to low PI(4,5)P2 conditions. The adaptive Leu-to-Arg mutation at MA residue 74 impaired virus release in T cells with normal PI(4,5)P2 levels but allowed virus production at efficiencies comparable to that of wild type (WT) HIV-1 under low-PI(4,5)P2 conditions, suggesting that Leu74 is involved in PI(4,5)P2 dependence. Unexpectedly, this mutation increased infectivity of progeny virions, allowing HIV-1 to replicate with faster kinetics than the WT in cell clones expressing 5ptaseIV. These results demonstrate that HIV-1 can overcome the disadvantage imposed by the lack of PI(4,5)P2 through enhancement of virus infectivity in T cells.