The modular protein Alix is a central node in endosomal-lysosomal trafficking and the budding of HIV-1. The Gag p6 protein of HIV-1 contains a LYPX n LXXL motif that is required for Alixmediated budding and binds to a region of Alix spanning residues 360-702. The structure of this fragment of Alix has the shape of the letter "V" and is termed the "V domain". The V domain has a topologically complex arrangement of eleven α-helices, with connecting loops that cross three times between the two arms of the "V". The conserved residue Phe676 is at the center of a large hydrophobic pocket and is critical for binding to a peptide model of HIV-1 p6. Overexpression of the V domain inhibits HIV-1 release from cells. This inhibition of release is reversed by mutations that block binding of the Alix V domain to p6.The budding and release of nascent virus particles from the infected host cell is an essential phase of the retroviral replication cycle. The budding process requires the trafficking of the Gag polyprotein precursor from its site of synthesis in the cytosol to the appropriate cellular membrane at which virus assembly occurs. Concomitant with particle release from the host cell the viral protease (PR) cleaves the Gag precursor to generate the mature Gag proteins matrix (MA), capsid (CA), and nucleocapsid (NC). In the case of HIV-1 and equine infectious anemia virus (EIAV), additional domains known as p6 and p9, respectively, are located at the C-terminus of the Gag precursor 1 . p6 and p9, and functionally analogous Gag proteins of other retroviruses, encode the so-called "late" domains that promote the release of virions from the infected cell 2-4 . Late domains function by co-opting the host cell endosomal sorting machinery via direct interactions with defined components of the cellular protein trafficking machinery 2-4 . Three retroviral late domains have been characterized to date, containing the short sequence motifs P(T/S)AP, PPPY, and LYPX n LXXL, respectively. P(T/S)AP, located within p6, promotes release by interacting with Tsg101, a component of the cellular ESCRT-I protein complex 5-10 . The PPPY motif, found in the Gag proteins of a number of retroviruses including murine leukemia virus (MLV) 11 , functions by interacting with Nedd4-like ubiquitin ligases 12-14 . The human protein Alix was first discovered and named AIP1 or Alix for its association with the calcium binding protein ALG-2 (apoptosis-linked gene 2) 15,16 ; here we use the term "Alix" to differentiate it from other unrelated proteins named "AIP1". EIAV and HIV-1 contain LYPX n LXXL motifs that promote release via a direct interaction with Alix 9,17-19 . The central domain (residues 360-716) is the least-studied region of Alix. Its best defined function is to bind viral LYPX n LXXL motifs 17,24 . The characterization of this domain is of great interest given that its overexpression strongly inhibits HIV-1 budding in cell culture 24 . To better understand Alix and the mechanism of its interaction with viral LYPX n LXXL motifs, we have ...
During the late phase of HIV-1 replication, newly synthesized retroviral Gag proteins are targeted to lipid raft regions of specific cellular membranes, where they assemble and bud to form new virus particles. Gag binds preferentially to the plasma membrane (PM) of most hematopoietic cell types, a process mediated by interactions between the cellular PM marker phosphatidylinositol-(4,5)-bisphosphate (PI(4,5)P 2 ) and Gag's N-terminally-myristoylated matrix (MA) domain. We recently demonstrated that PI(4,5)P 2 binds to a conserved cleft on MA and promotes myristate exposure, suggesting a role as both a direct membrane anchor and myristyl switch trigger. Here we show that PI(4,5)P 2 is also capable of binding to MA proteins containing point mutations that inhibit membrane binding in vitro, and in vivo, including V7R, L8A and L8I. However, these mutants do not exhibit PI(4,5)P 2 -or concentration-dependent myristate exposure. NMR studies of V7R and L8A MA reveal minor structural changes that appear to be responsible for stabilizing the myristate-sequestered (myr (s)) species and inhibiting exposure. Unexpectedly, the myristyl group of a revertant mutant with normal PM targeting properties (V7R,L21K) is also tightly sequestered and insensitive to PI(4,5) P 2 binding. This mutant binds PI(4,5)P 2 with two-fold higher affinity compared with the native protein, suggesting a potential compensatory mechanism for membrane binding. KeywordsHuman immunodeficiency virus type-1 (HIV-1); Gag; myristyl (myr); matrix (MA); phosphatidylinositol-4,5-bisphosphate (PI(4,5)P 2 ); analytical ultracentrifugation (AU); nuclear magnetic resonance (NMR)
Human immunodeficiency virus type 1 (HIV-1) particle production, a process driven by the Gag polyprotein precursor, occurs on the plasma membrane in most cell types. The plasma membrane contains cholesterolenriched microdomains termed lipid rafts, which can be isolated as detergent-resistant membrane (DRM). Previously, we and others demonstrated that HIV-1 Gag is associated with DRM and that disruption of Gag-raft interactions impairs HIV-1 particle production. However, the determinants of Gag-raft association remain undefined. In this study, we developed a novel epitope-based Gag multimerization assay to examine whether Gag assembly is essential for its association with lipid rafts. We observed that membrane-associated, full-length Gag is poorly detected by immunoprecipitation relative to non-membrane-bound Gag. This poor detection is due to assembly-driven masking of Gag epitopes, as denaturation greatly improves immunoprecipitation. Gag mutants lacking the Gag-Gag interaction domain located in the N terminus of the nucleocapsid (NC) were efficiently immunoprecipitated without denaturation, indicating that the epitope masking is caused by higher-order Gag multimerization. We used this assay to examine the relationship between Gag assembly and Gag binding to total cellular membrane and DRM. Importantly, a multimerization-defective NC mutant displayed wild-type levels of membrane binding and DRM association, indicating that NC-mediated Gag multimerization is dispensable for association of Gag with membrane or DRM. We also demonstrate that different properties of sucrose and iodixanol membrane flotation gradients may explain some discrepancies regarding Gag-raft interactions. This report offers new insights into the association of HIV-1 Gag with membrane and with lipid rafts.
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