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

Finzi, Andrés; Orthwein, Alexandre; Mercier, Johanne; Cohen, Éric A.

doi:10.1128/jvi.00308-07

Cited by 101 publications

(100 citation statements)

References 59 publications

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“…First, Gag can initiate and complete assembly at the plasma membrane. This is thought to occur predominantly in T lymphocytes, and this process is supported by several lines of evidences: (i) disruption of endosomal trafficking with drugs does not prevent viral production (26,27); (ii) ESCRT complexes can be recruited at the plasma membrane, at sites where Gag accumulates (28 -30); (iii) Gag can be seen multimerizing and budding from the plasma membrane in live cells (31). Second, Gag could initiate assembly in endosomes, and then traffic to the cell surface to be released.…”

mentioning

confidence: 70%

Endosomal Trafficking of HIV-1 Gag and Genomic RNAs Regulates Viral Egress

Molle

Segura-Morales

Camus

et al. 2009

Journal of Biological Chemistry

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HIV-1 Gag can assemble and generate virions at the plasma membrane, but it is also present in endosomes where its role remains incompletely characterized. Here, we show that HIV-1 RNAs and Gag are transported on endosomal vesicles positive for TiVamp, a v-SNARE involved in fusion events with the plasma membrane. Inhibition of endosomal traffic did not prevent viral release. However, inhibiting lysosomal degradation induced an accumulation of Gag in endosomes and increased viral production 7-fold, indicating that transport of Gag to lysosomes negatively regulates budding. This also suggested that endosomal Gag-RNA complexes could access retrograde pathways to the cell surface and indeed, depleting cells of TiVampreduced viral production. Moreover, inhibition of endosomal transport prevented the accumulation of Gag at sites of cellular contact. HIV-1 Gag could thus generate virions using two pathways, either directly from the plasma membrane or through an endosome-dependent route. Endosomal Gag-RNA complexes may be delivered at specific sites to facilitate cell-to-cell viral transmission.

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mentioning

confidence: 70%

Endosomal Trafficking of HIV-1 Gag and Genomic RNAs Regulates Viral Egress

Molle

Segura-Morales

Camus

et al. 2009

Journal of Biological Chemistry

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“…We have previously observed a population of VLPs that move rapidly in and out the field adjacent to the plasma membrane and we have demonstrated that these are also labeled with markers for internal endocytic compartments including clathrin and CD63 (18) Several lines of evidence indicate that these endosome-localized particles are not productive preassembly intermediates (15,16,28).…”

Section: Dynamics Of Hiv-1 Rna Molecules At the Plasma Membrane Of Cellsmentioning

confidence: 99%

“…The productive site of HIV-1 assembly is the plasma membrane, both in cultured cell lines and primary cells (15)(16)(17). This makes the assembly of HIV-1 accessible to imaging with totalinternal-reflection (TIR) fluorescent microscopy, which selectively excites fluorophores in close proximity (ϷϽ70 nm) to the coverslip.…”

Section: Rna Packaging ͉ Virion Morphogenesismentioning

confidence: 99%

Imaging the interaction of HIV-1 genomes and Gag during assembly of individual viral particles

Jouvenet

Simon

Bieniasz

2009

Proc. Natl. Acad. Sci. U.S.A.

245

314

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The incorporation of viral genomes into particles has never previously been imaged in live infected cells. Thus, for many viruses it is unknown how the recruitment and packaging of genomes into virions is temporally and spatially related to particle assembly. Here, we devised approaches to simultaneously image HIV-1 genomes, as well as the major HIV-1 structural protein, Gag, to reveal their dynamics and functional interactions during the assembly of individual viral particles. In the absence of Gag, HIV-1 RNA was highly dynamic, moving in and out of the proximity of the plasma membrane. Conversely, in the presence of Gag, RNA molecules docked at the membrane where their lateral movement slowed and then ceased as Gag assembled around them and they became irreversibly anchored. Viral genomes were not retained at the membrane when their packaging signals were mutated, nor when expressed with a Gag mutant that was not myristoylated. In the presence of a Gag mutant that retained membrane-and RNAbinding activities but could not assemble into particles, the viral RNA docked at the membrane but continued to drift laterally and then often dissociated from the membrane. These results, which provide visualization of the recruitment and packaging of genomes into individual virus particles, demonstrate that a small number of Gag molecules recruit viral genomes to the plasma membrane where they nucleate the assembly of complete virions.

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“…HIV-1 Gag targeting to the plasma membrane (PM) 2 is critical for proper and efficient assembly to produce progeny virions (1,(3)(4)(5)(6)(7)(8)(9). During virus maturation, Gag is cleaved into myristoylated matrix (myr(ϩ)MA), capsid, and nucleocapsid proteins, inducing major morphological reorganization of the virus (1,2,4,5,10).…”

mentioning

confidence: 99%

Binding of Calmodulin to the HIV-1 Matrix Protein Triggers Myristate Exposure

Ghanam

Fernandez

Fledderman

et al. 2010

Journal of Biological Chemistry

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Steady progress has been made in defining both the viral and cellular determinants of retroviral assembly and release. Although it is widely accepted that targeting of the Gag polypeptide to the plasma membrane is critical for proper assembly of HIV-1, the intracellular interactions and trafficking of Gag to its assembly sites in the infected cell are poorly understood. HIV-1 Gag was shown to interact and co-localize with calmodulin (CaM), a ubiquitous and highly conserved Ca 2؉ -binding protein expressed in all eukaryotic cells, and is implicated in a variety of cellular functions. Binding of HIV-1 Gag to CaM is dependent on calcium and is mediated by the N-terminally myristoylated matrix (myr(؉)MA) domain. Herein, we demonstrate that CaM binds to myr(؉)MA with a dissociation constant (K d ) of ϳ2 M and 1:1 stoichiometry. Strikingly, our data revealed that CaM binding to MA induces the extrusion of the myr group. However, in contrast to all known examples of CaM-binding myristoylated proteins, our data show that the myr group is exposed to solvent and not involved in CaM binding. The interactions between CaM and myr(؉)MA are endothermic and entropically driven, suggesting that hydrophobic contacts are critical for binding. As revealed by NMR data, both CaM and MA appear to engage substantial regions and/or undergo significant conformational changes upon binding. We believe that our findings will provide new insights on how Gag may interact with CaM during the HIV replication cycle.Gag is the major structural protein encoded by HIV-1 and contains all of the viral elements required to drive virus assembly (1-3). HIV-1 Gag targeting to the plasma membrane (PM) 2 is critical for proper and efficient assembly to produce progeny virions (1, 3-9). During virus maturation, Gag is cleaved into myristoylated matrix (myr(ϩ)MA), capsid, and nucleocapsid proteins, inducing major morphological reorganization of the virus (1, 2, 4, 5, 10). In many cell types, HIV-1 Gag budding and assembly has been shown to occur predominantly on the PM (4 -9, 11-18). Gag binding to the PM is mediated by the MA domain and enhanced by multimerization. Proper assembly and efficient binding of Gag to the PM requires a myristyl (myr) group as a membrane anchor and a cluster of basic residues localized within the N-terminal domain to facilitate interactions with acidic phospholipids (1,2,19,20).Steady progress has been made in defining both the viral and cellular determinants of HIV-1 assembly and release (6). However, the trafficking pathway used by Gag to reach assembly sites in the infected cell is poorly understood. Studies by Freed, Ono, and co-workers (21-23) demonstrated that the ultimate localization of HIV-1 Gag at virus assembly sites is dependent on phosphatidylinositol-(4,5)-bisphosphate (PI(4,5)P 2 ), a cellular factor localized at the inner leaflet of the PM (24 -26). Our structural studies revealed that PI(4,5)P 2 binds directly to HIV-1 MA, inducing a conformational change that triggers myr exposure (27). In addition to PI(4,5)P 2 ...

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

Cited by 101 publications

References 59 publications

Endosomal Trafficking of HIV-1 Gag and Genomic RNAs Regulates Viral Egress

Endosomal Trafficking of HIV-1 Gag and Genomic RNAs Regulates Viral Egress

Imaging the interaction of HIV-1 genomes and Gag during assembly of individual viral particles

Binding of Calmodulin to the HIV-1 Matrix Protein Triggers Myristate Exposure

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