Human ESCRT-II Complex and Its Role in Human Immunodeficiency Virus Type 1 Release

ABSTRACT The budding of many enveloped RNA viruses, including human immunodeficiency virus type 1 (HIV-1), requires some of the same cellular machinery as vesicle formation at the multivesicular body (MVB). In Saccharomyces cerevisiae, the ESCRT-II complex performs a central role in MVB protein sorting and vesicle formation, as it is recruited by the upstream ESCRT-I complex and nucleates assembly of the downstream ESCRT-III complex. Here, we report that the three subunits of human ESCRT-II, EAP20, EAP30, and EAP45, have a number of properties in common with their yeast orthologs. Specifically, EAP45 bound ubiquitin via its N-terminal GRAM-like ubiquitin-binding in EAP45 (GLUE) domain, both EAP45 and EAP30 bound the C-terminal domain of TSG101/ESCRT-I, and EAP20 bound the N-terminal half of CHMP6/ESCRT-III. Consistent with its expected role in MVB vesicle formation, (i) human ESCRT-II localized to endosomal membranes in a VPS4-dependent fashion and (ii) depletion of EAP20/ESCRT-II and CHMP6/ESCRT-III inhibited lysosomal targeting and downregulation of the epidermal growth factor receptor, albeit to a lesser extent than depletion of TSG101/ESCRT-I. Nevertheless, HIV-1 release and infectivity were not reduced by efficient small interfering RNA depletion of EAP20/ESCRT-II or CHMP6/ESCRT-III. These observations indicate that there are probably multiple pathways for protein sorting/MVB vesicle formation in human cells and that HIV-1 does not utilize an ESCRT-II-dependent pathway to leave the cell.

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Expression of Human Immunodeficiency Virus Type 1 Gag Modulates Ligand-Induced Downregulation of EGF Receptor

Journal of Virology ◽

10.1128/jvi.78.22.12386-12394.2004 ◽

2004 ◽

Vol 78 (22) ◽

pp. 12386-12394 ◽

Cited By ~ 13

Author(s):

Rajeshwari R. Valiathan ◽

Marilyn D. Resh

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Egf Receptor ◽

Protein Sorting ◽

Vacuolar Protein Sorting ◽

Immunodeficiency Virus ◽

Vacuolar Protein ◽

Hiv 1

ABSTRACT Many enveloped viruses use the ESCRT proteins of the cellular vacuolar protein sorting pathway for efficient egress from the cell. Recruitment of the ESCRT proteins by human immunodeficiency virus type 1 (HIV-1) Gag is required for HIV-1 particle budding and egress. ESCRT proteins normally function at endosomal membranes, where they facilitate the downregulation of mitogen-activated receptors such as EGF receptor (EGFR) through multivesicular body biogenesis. It is not known whether the Gag-mediated recruitment of ESCRT proteins functionally depletes the pool of these molecules that is available for the downregulation of EGFR. Here we show that the expression of HIV-1 Gag decreases the rate of EGFR downregulation, as assessed by decreases in the rates of 125I-EGF and EGFR degradation. The effect of Gag was dependent on the presence of the TSG101 binding motif (PTAP) within the Gag C-terminal p6 domain. Cells expressing HIV-1 Gag retained more EGFR in late endosomes. This effect occurred when Gag was expressed alone from a heterologous promoter and when Gag expression was driven by the HIV-1 long terminal repeat within pHXB2ΔBalD25S, a noninfectious lentiviral vector. Gag-expressing cells exhibited higher levels of activated mitogen-activated protein kinase for longer times after EGF addition than did cells that did not express HIV-1 Gag. These results indicate that HIV-1 Gag can impinge upon the functioning of the cellular vacuolar protein sorting pathway and reveal yet another facet of the intricate effects of HIV-1 infection on host cell physiology.

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Cell-Type-Dependent Targeting of Human Immunodeficiency Virus Type 1 Assembly to the Plasma Membrane and the Multivesicular Body

Journal of Virology ◽

10.1128/jvi.78.3.1552-1563.2004 ◽

2004 ◽

Vol 78 (3) ◽

pp. 1552-1563 ◽

Cited By ~ 196

Author(s):

Akira Ono ◽

Eric O. Freed

Keyword(s):

Human Immunodeficiency Virus ◽

Plasma Membrane ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Multivesicular Body ◽

Cell Type ◽

Gag Proteins ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT The human immunodeficiency virus type 1 (HIV-1) assembly-and-release pathway begins with the targeting of the Gag precursor to the site of virus assembly. The molecular mechanism by which Gag is targeted to the appropriate subcellular location remains poorly understood. Based on the analysis of mutant Gag proteins, we and others have previously demonstrated that a highly basic patch in the matrix (MA) domain of Gag is a major determinant of Gag transport to the plasma membrane. In this study, we determined that in HeLa and T cells, the MA mutant Gag proteins that are defective in plasma membrane targeting form virus particles in a CD63-positive compartment, defined as the late endosome or multivesicular body (MVB). Interestingly, we find that in primary human macrophages, both wild-type (WT) and MA mutant Gag proteins are targeted specifically to the MVB. Despite the fact that particle assembly in macrophages occurs at an intracellular site rather than at the plasma membrane, we observe that WT Gag expressed in this cell type is released as extracellular virions with high efficiency. These results demonstrate that Gag targeting to and assembly in the MVB are physiologically important steps in HIV-1 virus particle production in macrophages and that particle release in this cell type may follow an exosomal pathway. To determine whether Gag targeting to the MVB is the result of an interaction between the late domain in p6Gag and the MVB sorting machinery (e.g., TSG101), we examined the targeting and assembly of Gag mutants lacking p6. Significantly, the MVB localization of Gag was still observed in the absence of p6, suggesting that an interaction between Gag and TSG101 is not required for Gag targeting to the MVB. These data are consistent with a model for Gag targeting that postulates two different cellular binding partners for Gag, one on the plasma membrane and the other in the MVB.

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Productive Human Immunodeficiency Virus Type 1 Assembly Takes Place at the Plasma Membrane

Journal of Virology ◽

10.1128/jvi.00308-07 ◽

2007 ◽

Vol 81 (14) ◽

pp. 7476-7490 ◽

Cited By ~ 75

Author(s):

Andrés Finzi ◽

Alexandre Orthwein ◽

Johanne Mercier ◽

Éric A. Cohen

Keyword(s):

Human Immunodeficiency Virus ◽

Plasma Membrane ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Multivesicular Body ◽

Subcellular Fractionation ◽

Particle Assembly ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT Gag proteins are necessary and sufficient to direct human immunodeficiency virus type 1 (HIV-1) particle assembly and budding. Recent evidence suggests that Gag targeting to late endosomal/multivesicular body (LE/MVB) compartments occurs prior to viral particle budding at the plasma membrane (PM). However, the route that Gag follows before reaching its steady-state destinations still remains a subject of debate. Using a subcellular fractionation method that separates PM from LE/MVB combined with pulse-chase labeling, we analyzed Gag trafficking in HIV-1-producing HEK 293T cells. Our results reveal that the majority of newly synthesized Gag is primarily targeted to the PM. While PM-targeted Gag was efficiently released, a significant fraction of the remaining cell surface-associated Gag was found to be subsequently internalized to LE/MVB, where it accumulated, thus accounting for the majority of LE/MVB-associated Gag. Importantly, this accumulation of Gag in LE/MVB was found to be cholesterol dependent since it was sensitive to the sterol-binding drugs filipin and methyl-β-cyclodextrin. These results point towards the PM as being the primary site of productive HIV-1 assembly in cells that also support Gag accumulation in intracellular compartments.

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Proteomic and Biochemical Analysis of Purified Human Immunodeficiency Virus Type 1 Produced from Infected Monocyte-Derived Macrophages

Journal of Virology ◽

10.1128/jvi.01013-06 ◽

2006 ◽

Vol 80 (18) ◽

pp. 9039-9052 ◽

Cited By ~ 303

Author(s):

Elena Chertova ◽

Oleg Chertov ◽

Lori V. Coren ◽

James D. Roser ◽

Charles M. Trubey ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Multivesicular Body ◽

Human Monocyte ◽

Cell Types ◽

Rab Proteins ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) infects CD4+ T lymphocytes and monocytes/macrophages, incorporating host proteins in the process of assembly and budding. Analysis of the host cell proteins incorporated into virions can provide insights into viral biology. We characterized proteins in highly purified HIV-1 virions produced from human monocyte-derived macrophages (MDM), within which virus buds predominantly into intracytoplasmic vesicles, in contrast to the plasmalemmal budding of HIV-1 typically seen with infected T cells. Liquid chromatography-linked tandem mass spectrometry of highly purified virions identified many cellular proteins, including 33 previously described proteins in HIV-1 preparations from other cell types. Proteins involved in many different cellular structures and functions were present, including those from the cytoskeleton, adhesion, signaling, intracellular trafficking, chaperone, metabolic, ubiquitin/proteasomal, and immune response systems. We also identified annexins, annexin-binding proteins, Rab proteins, and other proteins involved in membrane organization, vesicular trafficking, and late endosomal function, as well as apolipoprotein E, which participates in cholesterol transport, immunoregulation, and modulation of cell growth and differentiation. Several tetraspanins, markers of the late endosomal compartment, were also identified. MDM-derived HIV contained 26 of 37 proteins previously found in exosomes, consistent with the idea that HIV uses the late endosome/multivesicular body pathway during virion budding from macrophages.

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