Role of the Nucleocapsid Domain in HIV-1 Gag Oligomerization and Trafficking to the Plasma Membrane: A Fluorescence Lifetime Imaging Microscopy Investigation

Meshri, Salah Edin El; Dujardin, Denis; Godet, Julien; Richert, Ludovic; Boudier, Christian; Darlix, Jean Luc; Didier, Pascal; Mély, Yves; Rocquigny, Hugues de

doi:10.1016/j.jmb.2015.01.015

Cited by 54 publications

(65 citation statements)

References 74 publications

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“…The discrepancy between the theoretical and experimental values is likely due to Gag-mCH tendency to multimerize and interact in the cytosol with cellular factors. This conclusion was further strengthened by the D value of 10.4 ± 2.1 µm 2 /s measured for the cytoplasmic diffusion of eGFP trimers (Mw = 81 kDa) (in good agreement with 9.5 µm²/s reported by (74)) that have approximately the same size as Gag-mCH and are not supposed to bind to any cellular components (44,76,77). In line with a previous study (77), mutations affecting the myristoylation site do not affect Gag mobility (DGagG2A = 1.3 ± 0.6 µm²/s).…”

Section: The Comparison Between the Expected And Experimental D Valuesupporting

confidence: 81%

“…On the other hand, the deletion of a single ZF delayed the gRNA accumulation at the assembly sites, as it took about 73.5 ± 3.7 min and 94.5 ± 4.7 min to accumulate gRNA at the PM in the case of Gag∆ZF1 and Gag∆ZF2, respectively ( Figure 4B). Data from the literature showed that the NC domain and the Cterminal p6 domain in Gag are both involved in the budding cellular machinery, since deletions of the NC domain or its two ZFs were found to interfere with virus release by impairing the recruitment of Tsg101 ESCRT-I proteins and their co-factors, such as ALIX (44,85,86) .…”

Section: Discussionmentioning

confidence: 99%

“…This increased diffusion could be explained by the impacted capacity of Gag to multimerize and to bind to cellular factors when its NC domain is deleted (44,76).…”

Section: Discussionmentioning

confidence: 99%

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The two zinc fingers in the nucleocapsid domain of the HIV-1 Gag precursor are equivalent for the interaction with the genomic RNA in the cytoplasm, but not for the recruitment of the complexes at the plasma membrane

Bernacchi

Boutant

Bonzi

et al. 2020

Preprint

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The HIV-1 Gag precursor specifically selects the unspliced viral genomic RNA (gRNA) from the bulk of cellular and spliced viral RNAs via its nucleocapsid (NC) domain and drives gRNA encapsidation at the plasma membrane (PM). To further identify the determinants governing the intracellular trafficking of Gag-gRNA complexes and their accumulation at the PM, we compared, in living and fixed cells, the interactions between gRNA and wild-type (WT) Gag or Gag mutants carrying deletions in NC zinc fingers (ZFs), or a non-myristoylated version of Gag. Our data showed that the deletion of both ZFs simultaneously or the complete NC domain completely abolished intracytoplasmic Gag-gRNA interactions. Deletion of either ZF delayed the delivery of gRNA to the PM but did not prevent Gag-gRNA interactions in the cytoplasm,

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Section: The Comparison Between the Expected And Experimental D Valuesupporting

confidence: 81%

Section: Discussionmentioning

confidence: 99%

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The two zinc fingers in the nucleocapsid domain of the HIV-1 Gag precursor are equivalent for the interaction with the genomic RNA in the cytoplasm, but not for the recruitment of the complexes at the plasma membrane

Bernacchi

Boutant

Bonzi

et al. 2020

Preprint

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“…The p6 C-terminus truncated recombinant Gag protein (Pr50 GagΔp6 ), with the addition of nucleic acid and lipid-mimicking inositol phosphate, has previously been shown to assemble in vitro , forming virus-like particles (VLPs) in the absence of other viral proteins [7, 8]. Cellular imaging and immunoprecipitation studies with virion producing cells have suggested that nucleic acid initially binds to cytosolic Pr55 Gag , therefore promoting the formation of low order oligomers of Pr55 Gag in the cytosol; higher order Pr55 Gag oligomers only occur upon Pr55 Gag binding to the plasma membrane [10–13]. However, cytoplasmic HIV-1 RNA has also been shown to traffic to the membrane by passive diffusion independent of Pr55 Gag , suggesting that the viral RNA genome may bind to Pr55 Gag molecules on the plasma membrane where a high local concentration of Pr55 Gag has already been achieved for oligomerization [14].…”

Section: Introductionmentioning

confidence: 99%

The thermodynamics of Pr55Gag-RNA interaction regulate the assembly of HIV

et al. 2017

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The interactions that occur during HIV Pr55Gag oligomerization and genomic RNA packaging are essential elements that facilitate HIV assembly. However, mechanistic details of these interactions are not clearly defined. Here, we overcome previous limitations in producing large quantities of full-length recombinant Pr55Gag that is required for isothermal titration calorimetry (ITC) studies, and we have revealed the thermodynamic properties of HIV assembly for the first time. Thermodynamic analysis showed that the binding between RNA and HIV Pr55Gag is an energetically favourable reaction (ΔG<0) that is further enhanced by the oligomerization of Pr55Gag. The change in enthalpy (ΔH) widens sequentially from: (1) Pr55Gag-Psi RNA binding during HIV genome selection; to (2) Pr55Gag-Guanosine Uridine (GU)-containing RNA binding in cytoplasm/plasma membrane; and then to (3) Pr55Gag-Adenosine(A)-containing RNA binding in immature HIV. These data imply the stepwise increments of heat being released during HIV biogenesis may help to facilitate the process of viral assembly. By mimicking the interactions between A-containing RNA and oligomeric Pr55Gag in immature HIV, it was noted that a p6 domain truncated Pr50Gag Δp6 is less efficient than full-length Pr55Gag in this thermodynamic process. These data suggest a potential unknown role of p6 in Pr55Gag-Pr55Gag oligomerization and/or Pr55Gag-RNA interaction during HIV assembly. Our data provide direct evidence on how nucleic acid sequences and the oligomeric state of Pr55Gag regulate HIV assembly.

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“…As depicted in Fig. 8.14, at 12 h post transfection the distribution of τ 1 value was centred at 1.45 ns (corresponding to a FRET efficiency of 41 %) and homogeneously distributed within the cell as shown by the regular green staining [34]. These data indicated that Gag /Pr55 Gag-eGFP / Pr55…”

Section: /Pr55mentioning

confidence: 73%

Monitoring HIV-1 Protein Oligomerization by FLIM FRET Microscopy

Richert

Didier

Rocquigny

2015

Springer Series in Chemical Physics

Self Cite

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The majority of the human immunodeficiency virus type 1 (HIV-1) proteins are able to self assemble into oligomers. Since these oligomers generally exhibit functions that differ from those of their monomeric counterpart, the regulation of the monomer-oligomer equilibria plays a central role in the viral cycle. To characterize the oligomerization of these proteins in live cells, the combination of fluorescence lifetime imaging microscopy (FLIM) with Förster resonance energy transfer (FRET) has proven to be very powerful. In this review, we illustrate the application of FRET-FLIM on the characterization of the oligomerization of the Vpr, Vif and Pr55Gag proteins of HIV-1 in fusion with eGFP and mCherry. For Vpr and Pr55Gag proteins, very high levels of FRET leading to strong decreases in eGFP fluorescence lifetime are obtained, as a consequence of the rather small size of the viral proteins, the strong packing of the protomers and the presence of multiple acceptors for one donor. Analyzing the time-resolved decays by a two-component analysis further provides the possibility to discriminate monomers from oligomers and to monitor the spatiotemporal evolution of both populations in the cells. Though FRET-FLIM unambiguously reveals the oligomerization of a given protein, it hardly discloses the oligomer stoichiometry (number of protomers per oligomers). This parameter can be obtained by fluorescence correlation spectroscopy, which allows further interpreting the FRET-FLIM data. FRET-FLIM is also highly useful to identify the determinants of the oligomerization process and to investigate its regulation by other HIV-1 proteins and host proteins.

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Role of the Nucleocapsid Domain in HIV-1 Gag Oligomerization and Trafficking to the Plasma Membrane: A Fluorescence Lifetime Imaging Microscopy Investigation

Cited by 54 publications

References 74 publications

The two zinc fingers in the nucleocapsid domain of the HIV-1 Gag precursor are equivalent for the interaction with the genomic RNA in the cytoplasm, but not for the recruitment of the complexes at the plasma membrane

The two zinc fingers in the nucleocapsid domain of the HIV-1 Gag precursor are equivalent for the interaction with the genomic RNA in the cytoplasm, but not for the recruitment of the complexes at the plasma membrane

The thermodynamics of Pr55Gag-RNA interaction regulate the assembly of HIV

Monitoring HIV-1 Protein Oligomerization by FLIM FRET Microscopy

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