HIV-1 assembly is driven by the viral Gag polyprotein, with host factors contributing essential activities.1-3 We and others have shown that host machinery of the Endosomal Sorting Complexes Required for Transport (ESCRT) pathway mediates the membrane fission reaction that releases HIV-1 virions (termed ?budding?)4-10, that the HECT ubiquitin E3 ligase NEDD4L stimulates release of HIV-1 constructs that cannot recruit ESCRT factors directly11-13, and that members of the Angiomotin (AMOT) family of NEDD4L-binding proteins promote progression of the assembling Gag lattices from hemispheres to membrane-enveloped spheres (termed ?envelopment?).14 Most recently, we have discovered that New World monkeys carry truncated, retrotransposed copies of a CHMP3 (ESCRT-III) protein that can potently inhibit release of HIV-1 and other retroviruses without undue cellular toxicity. We now propose to build on these observations by pursuing complementary structural, biochemical, imaging, and functional approaches to address three central questions in HIV-1 biogenesis: 1) How do assembling virions become wrapped in membranes? 2) How does the ESCRT machinery catalyze the membrane fission reaction of virus budding? 3) How can primates protect themselves against ESCRT-dependent viruses? In addition to their relevance for HIV, each of these processes has analogs in other viral and/or cellular systems, which should extend the impact of our studies. Specifically, we propose to characterize: how AMOT-NEDD4L complexes contribute to HIV virion envelopment (Aim 1); how late-acting ESCRT-III filaments and VPS4 ATPases collaborate to promote membrane fission (Aim 2); and how retrotransposed CHMP3 proteins (retroCHMP3) can inhibit retroviral budding without inducing cellular toxicity (Aim 3).
These Aims are buttressed by structural studies showing: how the AMOT PPXY1 and NEDD4L WW3 domains form a high affinity complex (Aim 1); how ESCRT-III proteins form soluble, monomeric proteins and membrane-binding filaments (Aims 2 and 3); and how VPS4 ATPases bind these filaments and remove ESCRT-III subunits (Aim 2).
Each Aim will also be supported by biochemical assays designed to elucidate: how the AMOT-NEDD4L complex remodels membranes, stabilizes F-actin, and activates NEDD4L ubiquitin E3 ligase activity (Aim 1); how the essential ESCRT-III subunits CHMP2 and CHMP4 co-assemble and are then disassembled by VPS4 (Aim 2); and whether retroCHMP3 has evolved to lose its ability to bind and sequester CHMP4 and/or CHMP2. Finally, all of the Aims will be facilitated by functional and imaging assays that will allow us to perform genetic analyses of the roles of AMOT, NEDD4L, CHMP2, CHMP4, VPS4 and retroCHMP3 variants in virus budding (and cell toxicity), and determine when these factors appear and how they function at sites of virus assembly. Our goal is to use these complementary approaches to generate and test mechanistic models for three fundamental processes in HIV-1 biogenesis.
Enveloped viruses like HIV-1 spread infection by usurping host pathways to facilitate their release from cells. The goals of our research are to understand precisely how HIV-1 utilizes these host pathways, and how innate immune systems can inhibit these processes without inducing cell toxicity. These studies should help reveal how viruses and cells remodel membranes and identify virus-specific interactions that are attractive targets for therapeutic intervention.
|McCullough, John; Frost, Adam; Sundquist, Wesley I (2018) Structures, Functions, and Dynamics of ESCRT-III/Vps4 Membrane Remodeling and Fission Complexes. Annu Rev Cell Dev Biol 34:85-109|
|Han, Han; Monroe, Nicole; Sundquist, Wesley I et al. (2017) The AAA ATPase Vps4 binds ESCRT-III substrates through a repeating array of dipeptide-binding pockets. Elife 6:|
|Schaller, Michael D; McDowell, Gary; Porter, André et al. (2017) What's in a name? Elife 6:|
|Votteler, Jörg; Sundquist, Wesley I (2013) Virus budding and the ESCRT pathway. Cell Host Microbe 14:232-41|
|Morita, Eiji; Arii, Jun; Christensen, Devin et al. (2012) Attenuated protein expression vectors for use in siRNA rescue experiments. Biotechniques 0:1-5|
|Skalicky, Jack J; Arii, Jun; Wenzel, Dawn M et al. (2012) Interactions of the human LIP5 regulatory protein with endosomal sorting complexes required for transport. J Biol Chem 287:43910-26|
|Zhai, Qianting; Landesman, Michael B; Robinson, Howard et al. (2011) Structure of the Bro1 domain protein BROX and functional analyses of the ALIX Bro1 domain in HIV-1 budding. PLoS One 6:e27466|
|Morita, Eiji; Sandrin, Virginie; McCullough, John et al. (2011) ESCRT-III protein requirements for HIV-1 budding. Cell Host Microbe 9:235-242|
|Morita, Eiji; Colf, Leremy A; Karren, Mary Anne et al. (2010) Human ESCRT-III and VPS4 proteins are required for centrosome and spindle maintenance. Proc Natl Acad Sci U S A 107:12889-94|
|Bajorek, Monika; Morita, Eiji; Skalicky, Jack J et al. (2009) Biochemical analyses of human IST1 and its function in cytokinesis. Mol Biol Cell 20:1360-73|