Selective Targeting of Virus Replication by Proton Pump Inhibitors.

Two proton pump inhibitors, tenatoprazole and esomeprazole, were previously shown to inhibit HIV-1 egress by blocking the interaction between Tsg101, a member of the ESCRT-I complex, and ubiquitin. Here, we deepen our understanding of prazole budding inhibition by studying a range of viruses in the presence of tenatoprazole. Furthermore, we investigate the relationship between the chemistry of prodrug activation and HIV-1 inhibition for diverse prazoles currently on the market.

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.

Tsg101 regulates PI(4,5)P2/Ca(2+) signaling for HIV-1 Gag assembly.

Our previous studies identified the 1,4,5-inositol trisphosphate receptor (IP3R), a channel mediating release of Ca(2+) from ER stores, as a cellular factor differentially associated with HIV-1 Gag that might facilitate ESCRT function in virus budding. Channel opening requires activation that is initiated by binding of 1,4,5-triphosphate (IP3), a product of phospholipase C (PLC)-mediated PI(4,5)P2 hydrolysis. The store emptying that follows stimulates store refilling which requires intact PI(4,5)P2.

The S40 residue in HIV-1 Gag p6 impacts local and distal budding determinants, revealing additional late domain activities.

Background: HIV-1 budding is directed primarily by two motifs in Gag p6 designated as late domain-1 and -2 that recruit ESCRT machinery by binding Tsg101 and Alix, respectively, and by poorly characterized determinants in the capsid (CA) domain. Here, we report that a conserved Gag p6 residue, S40, impacts budding mediated by all of these determinants.

Results: Whereas budding normally results in formation of single spherical particles ~100 nm in diameter and containing a characteristic electron-dense conical core, the substitution of Phe for S40, a change that does not alter the amino acids encoded in the overlapping pol reading frame, resulted in defective CA-SP1 cleavage, formation of strings of tethered particles or filopodia-like membrane protrusions containing Gag, and diminished infectious particle formation.

HIV Assembly and Budding: Ca(2+) Signaling and Non-ESCRT Proteins Set the Stage.

More than a decade has elapsed since the link between the endosomal sorting complex required for transport (ESCRT) machinery and HIV-1 protein trafficking and budding was first identified. L domains in HIV-1 Gag mediate recruitment of ESCRT which function in bud abscission releasing the viral particle from the host cell. Beyond virus budding, the ESCRT machinery is also involved in the endocytic pathway, cytokinesis, and autophagy.

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.

Sprouty2 regulates PI(4,5)P2/Ca2+ signaling and HIV-1 Gag release.

We reported recently that activation of the inositol 1,4,5-triphosphate receptor (IP3R) is required for efficient HIV-1 Gag trafficking and viral particle release. IP3R activation requires phospholipase C (PLC)-catalyzed hydrolysis of PI(4,5)P(2) to IP3 and diacylglycerol. We show that Sprouty2 (Spry2), which binds PI(4,5)P(2) and PLCγ, interfered with PI(4,5)P(2) in a manner similar to that of U73122, an inhibitor of PI(4,5)P(2) hydrolysis, suggesting that Spry2 negatively regulates IP3R by preventing formation of its activating ligand, IP3.

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.

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.

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).

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.