Tsg101 chaperone function revealed by HIV-1 assembly inhibitors.

HIV-1 replication requires Tsg101, a component of cellular endosomal sorting complex required for transport (ESCRT) machinery. Tsg101 possesses an ubiquitin (Ub) E2 variant (UEV) domain with a pocket that can bind PT/SAP motifs and another pocket that can bind Ub. The PTAP motif in the viral structural precursor polyprotein, Gag, allows the recruitment of Tsg101 and other ESCRTs to virus assembly sites where they mediate budding. It is not known how or even whether the UEV Ub binding function contributes to virus production. Here, we report that disruption of UEV Ub binding by commonly used drugs arrests assembly at an early step distinct from the late stage involving PTAP binding disruption. NMR reveals that the drugs form a covalent adduct near the Ub-binding pocket leading to the disruption of Ub, but not PTAP binding. We conclude that the Ub-binding pocket has a chaperone function involved in bud initiation.

ESCRT machinery potentiates HIV-1 utilization of the PI(4,5)P(2)-PLC-IP3R-Ca(2+) signaling cascade.

Human immunodeficiency virus type 1 (HIV-1) release efficiency is directed by late (L) domain motifs in the viral structural precursor polyprotein Gag, which serve as links to the ESCRT (endosomal sorting complex required for transport) machinery. Linkage is normally through binding of Tsg101, an ESCRT-1 component, to the P(7)TAP motif in the p6 region of Gag. In its absence, budding is directed by binding of Alix, an ESCRT adaptor protein, to the LY(36)PX(n)L motif in Gag. We recently showed that budding requires activation of the inositol 1,4,5-triphosphate receptor (IP3R), a protein that "gates" Ca(2+) release from intracellular stores, triggers Ca(2+) cell influx and thereby functions as a major regulator of Ca(2+) signaling. In the present study, we determined whether the L domain links Gag to Ca(2+) signaling machinery. Depletion of IP3R and inactivation of phospholipase C (PLC) inhibited budding whether or not Tsg101 was bound to Gag. PLC hydrolysis of phosphatidylinositol-(4,5)-bisphosphate generates inositol (1,4,5)-triphosphate, the ligand that activates IP3R. However, with Tsg101 bound, Gag release was independent of Gq-mediated activation of PLC, and budding was readily enhanced by pharmacological stimulation of PLC. Moreover, IP3R was redistributed to the cell periphery and cytosolic Ca(2+) was elevated, events indicative of induction of Ca(2+) signaling. The results suggest that L domain function, ESCRT machinery and Ca(2+) signaling are linked events in Gag release.

Phosphoinositides direct equine infectious anemia virus gag trafficking and release.

Phosphatidylinositol 4,5-biphosphate [PI(4,5)P(2) ], the predominant phosphoinositide (PI) on the plasma membrane, binds the matrix (MA) protein of human immunodeficiency virus type 1 (HIV-1) and equine infectious anemia virus (EIAV) with similar affinities in vitro. Interaction with PI(4,5)P(2) is critical for HIV-1 assembly on the plasma membrane. EIAV has been shown to localize in internal compartments; hence, the significance of its interaction with PI(4,5)P(2) is unclear. We therefore investigated the binding in vitro of other PIs to EIAV MA and whether intracellular association with compartments bearing these PIs was important for assembly and release of virus-like particles (VLPs) formed by Gag. In vitro, EIAV MA bound phosphatidylinositol 3-phosphate [PI(3)P] with higher affinity than PI(4,5)P(2) as revealed by nuclear magnetic resonance (NMR) spectra upon lipid titration. Gag was detected on the plasma membrane and in compartments enriched in phosphatidylinositol 3,5-biphosphate [PI(3,5)P(2) ]. Treatment of cells with YM201636, a kinase inhibitor that blocks production of PI(3,5)P(2) from PI(3)P, caused Gag to colocalize with aberrant compartments and inhibited VLP release. In contrast to HIV-1, release of EIAV VLPs was not significantly diminished by coexpression with 5-phosphatase IV, an enzyme that specifically depletes PI(4,5)P(2) from the plasma membrane. However, coexpression with synaptojanin 2, a phosphatase with broader specificity, diminished VLP production. PI-binding pocket mutations caused striking budding defects, as revealed by electron microscopy. One of the mutations also modified Gag-Gag interaction, as suggested by altered bimolecular fluorescence complementation. We conclude that PI-mediated targeting to peripheral and internal membranes is a critical factor in EIAV assembly and release.

Activation of the inositol (1,4,5)-triphosphate calcium gate receptor is required for HIV-1 Gag release.

The structural precursor polyprotein, Gag, encoded by all retroviruses, including the human immunodeficiency virus type 1 (HIV-1), is necessary and sufficient for the assembly and release of particles that morphologically resemble immature virus particles. Previous studies have shown that the addition of Ca(2+) to cells expressing Gag enhances virus particle production. However, no specific cellular factor has been implicated as mediator of Ca(2+) provision. The inositol (1,4,5)-triphosphate receptor (IP3R) gates intracellular Ca(2+) stores. Following activation by binding of its ligand, IP3, it releases Ca(2+) from the stores. We demonstrate here that IP3R function is required for efficient release of HIV-1 virus particles. Depletion of IP3R by small interfering RNA, sequestration of its activating ligand by expression of a mutated fragment of IP3R that binds IP3 with very high affinity, or blocking formation of the ligand by inhibiting phospholipase C-mediated hydrolysis of the precursor, phosphatidylinositol-4,5-biphosphate, inhibited Gag particle release. These disruptions, as well as interference with ligand-receptor interaction using antibody targeted to the ligand-binding site on IP3R, blocked plasma membrane accumulation of Gag. These findings identify IP3R as a new determinant in HIV-1 trafficking during Gag assembly and introduce IP3R-regulated Ca(2+) signaling as a potential novel cofactor in viral particle release.

Cell biology of HIV-1 infection of macrophages.

HIV infection of macrophages is a critically important component of viral pathogenesis and progression to AIDS. Although the virus follows the same life cycle in macrophages and T lymphocytes, several aspects of the virus-host relationship are unique to macrophage infection. Examples of these are the long-term persistence of productive infection, sustained by the absence of cell death, and the ability of progeny virus to bud into and accumulate in endocytic compartments designated multivesicular bodies (MVBs). Recently, the hypothesis that viral exploitation of the macrophage endocytic machinery is responsible for perpetuating the chronic state of infection unique to this cell type has been challenged in several independent studies employing a variety of experimental strategies. This review examines the evidence supporting and refuting the canonical hypothesis and highlights recently identified cellular factors that may contribute to the unique aspects of the HIV-macrophage interaction.

Tsg101 can replace Nedd4 function in ASV Gag release but not membrane targeting.

The Late (L) domain of the avian sarcoma virus (ASV) Gag protein binds Nedd4 ubiquitin ligase E3 family members and is the determinant of efficient virus release in avian and mammalian cells. We previously demonstrated that Nedd4 and Tsg101 constitutively interact raising the possibility that Nedd4 links ASV Gag to the ESCRT machinery. We now demonstrate that covalently linking Tsg101 to ASV Gag lacking the Nedd4 binding site (Deltap2b-Tsg101) ablates the requirement for Nedd4, but the rescue of budding occurs by use of a different budding mechanism than that used by wild type ASV Gag. The evidence that Tsg101 and Nedd4 direct release by different pathways is: (i) Release of the virus-like particles (VLPs) assembled from Gag in DF-1, an avian cell line, was resistant to dominant-negative interference by a Tsg101 mutant previously shown to inhibit release of both HIV and Mo-MLV. (ii) Release of VLPs from DF-1 cells was resistant to siRNA-mediated depletion of the endogenous pool of Tsg101 in these cells. (iii) VLPs assembled from wild-type ASV Gag exhibited highly efficient release from endosome-like membrane domains enriched in the tetraspanin protein CD63 or a fluorescent analogue of the phospholipid phosphatidylethanolamine. However, the VLPs assembled from the L domain mutant Deltap2b or a chimeric Deltap2b-Tsg101 Gag lacked these domain markers even though the chimeric Gag was released efficiently compared to the Deltap2b mutant. These results suggest that Tsg101 and Nedd4 facilitate Gag release through functionally exchangeable but independent routes and that Tsg101 can replace Nedd4 function in facilitating budding but not directing through the same membranes.