The Role of Cellular Factors in Promoting HIV Budding

Program in Gene Function and Expression, University of Massachusetts Medical School, Worcester, MA 01605, USA.
Journal of Molecular Biology (Impact Factor: 4.33). 07/2011; 410(4):525-33. DOI: 10.1016/j.jmb.2011.04.055
Source: PubMed


Human immunodeficiency virus type 1 (HIV-1) becomes enveloped while budding through the plasma membrane, and the release of nascent virions requires a membrane fission event that separates the viral envelope from the cell surface. To facilitate this crucial step in its life cycle, HIV-1 exploits a complex cellular membrane remodeling and fission machinery known as the endosomal sorting complex required for transport (ESCRT) pathway. HIV-1 Gag directly interacts with early-acting components of this pathway, which ultimately triggers the assembly of the ESCRT-III membrane fission complex at viral budding sites. Surprisingly, HIV-1 requires only a subset of ESCRT-III components, indicating that the membrane fission reaction that occurs during HIV-1 budding differs in crucial aspects from topologically related cellular abscission events.

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Available from: Heinrich G Gottlinger, Oct 09, 2015
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    • "The myristylation of Gly2 in MA targets the Gag protein to the plasma membrane [1] [20], and mutation of this glycine myristylation site prevents virus budding and leads to accumulation of Gag within the host cell [21]. The structure of MA has been determined by both X-ray crystallography and NMR, and comprises 5 alpha helices and a three strand mixed beta sheet [22] [23] [24] which forms a trimer in the virion shell in the mature HIV-1 particle. "
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    ABSTRACT: The HIV-1 Gag precursor protein, Pr55Gag, is a multi-domain polyprotein that drives HIV-1 assembly. The morphological features of HIV-1 suggested Pr55Gag assumes a variety of different conformations during virion assembly and maturation, yet structural determination of HIV-1 Pr55Gag has not been possible due to an inability to express and to isolate large amounts of full-length recombinant Pr55Gag for biophysical and biochemical analyses. This challenge is further complicated by HIV-1 Gag’s natural propensity to multimerize for the formation of viral particle (with ∼2500 Gag molecules per virion), and this has led Pr55Gag to aggregate and be expressed as inclusion bodies in a number of in vitro protein expression systems. This study reported the production of a recombinant form of HIV-1 Pr55Gag using a bacterial heterologous expression system. Recombinant HIV-1 Pr55Gag was expressed with a C-terminal Hisx6 tag, and purified using a combination of immobilized metal affinity chromatography and size exclusion chromatography. This procedure resulted in the production of milligram quantities of high purity HIV-1 Pr55Gag that has a mobility that resembles a trimer in solution using size exclusion chromatography analysis. The high quantity and purity of the full length HIV Gag will be suitable for structural and functional studies to further understand the process of viral assembly, maturation and the development of inhibitors to interfere with the process.
    Protein Expression and Purification 08/2014; 100. DOI:10.1016/j.pep.2014.04.013 · 1.70 Impact Factor
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    • "These pathways involve direct interaction between members of or adapters of cellular ESCRT complexes and budding determinants in the virus encoded structural precursor polyprotein, Gag. Typically, budding of wild-type (WT) Gag virus-like particles (VLPs) or of authentic virus particles requires the binding of Tsg101, a component of ESCRT-I, to the P7TAP motif in the C-terminal p6 region of Gag (ESCRT = endosomal sorting complex required for transport; ([5-7]; reviewed in [8-10]). The motif is designated as Late (L) domain-1. "
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    ABSTRACT: 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. 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. The S40F-mediated release defects were exacerbated when the viral-encoded protease (PR) was inactivated or when L domain-1 function was disrupted or when budding was almost completely obliterated by the disruption of both L domain-1 and -2. S40F mutation also resulted in stronger Gag-Alix interaction, as detected by yeast 2-hybrid assay. Reducing Alix binding by mutational disruption of contact residues restored single particle release, implicating the perturbed Gag-Alix interaction in the aberrant budding events. Interestingly, introduction of S40F partially rescued the negative effects on budding of CA NTD mutations EE75,76AA and P99A, which both prevent membrane curvature and therefore block budding at an early stage. The results indicate that the S40 residue is a novel determinant of HIV-1 egress that is most likely involved in regulation of a critical assembly event required for budding in the Tsg101-, Alix-, Nedd4- and CA N-terminal domain affected pathways.
    Retrovirology 11/2013; 10(1):143. DOI:10.1186/1742-4690-10-143 · 4.19 Impact Factor
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    • "depends on the cellular compartments and organelles that the virions associate with during assembly, and on the abundance of particular proteins in these compartments. The assembly process is primarily driven by HIV-1 Gag (Ono, 2009; Accola et al., 2000) and exploits the endosomal sorting complex required for transport (ESCRT) pathway for the cellular membrane remodeling and budding of the virions ((Demirov et al., 2002; Garrus et al., 2001; von Schwedler et al., 2003; Martin-Serrano et al., 2003; Martin-Serrano and Neil, 2011; Weiss and Gottlinger, 2011), for a recent review see Meng and Lever (2013)). Whereas analysis of the virion assembly in the T lymphocytes and model epithelial cells and fibroblasts indicated plasma membrane as a primary cellular site of HIV particle assembly and budding (Perez-Caballero et al., 2004; Ono and Freed, 2004; Finzi et al., 2007), the electron microscopy and immunofluoresent microscopic studies of infected macrophages revealed accumulation of virions within intracellular compartments—the late endosomes or multivesicular bodies (MVB) (Pelchen-Matthews et al., 2003; Raposo et al., 2002; Jouve et al., 2007). "
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    ABSTRACT: Macrophages and CD4-positive T lymphocytes are the major targets and producers of HIV-1. While the molecular details underlying HIV replication in macrophages and T cells become better understood, it remains unclear whether viruses produced by these target cells differ in their biological properties. Recent reports suggest that HIV virions incorporate a large number of producer cell proteins and lipids which have an effect on subsequent viral replication in newly infected cells. The identity and abundance of these incorporated factors varies between different types of producer cells, suggesting that they may influence the replication capacity and pathogenic activity of the virions produced by T cells and macrophages.
    Virology 06/2013; 443(2). DOI:10.1016/j.virol.2013.05.023 · 3.32 Impact Factor
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