Investigating the Role of F-Actin in Human Immunodeficiency Virus Assembly by Live-Cell Microscopy

Rahman, Sheikh Abdul; Koch, Peter J.; Weichsel, Julian; Godinez, William J.; Schwarz, Ulrich S.; Rohr, Karl; Lamb, Don C.; Kräusslich, Hans‐Georg; Müller, Bárbara

doi:10.1128/jvi.00431-14

Cited by 25 publications

(27 citation statements)

References 45 publications

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“…Moreover, it was shown previously by immunoprecipitation and fractionation that actin interacts with the NC domain of Gag (32,33), and another study suggested that actin-enriched structures could be localized underneath viral assembly sites (34). However, these results are discussed in the scientific community, because in another cell type, it was recently observed by live-cell imaging microscopy that F-actin seems not to be recruited at the HIV Gag assembly site (35). Nonetheless, the actin cytoskeleton might play an important role in virus assembly and production, since its drug-mediated inhibition changes the intracellular localization of Gag and diminishes virus production in T cells (36)(37)(38).…”

contrasting

confidence: 40%

“…However, a recent study failed to detect F-actin at the HIV-1 Gag assembly sites by live-cell imaging microscopy (35). Of note, F-actin was labeled through Life-actin-mCherry, with the uncertainty of the incorporation of such a molecule in filamentous actin, and this was done in adherent HeLa cells (35). Nonetheless, by using Jurkat T cells or PBLs, this study, as well as others (36)(37)(38), clearly showed that drug-mediated or siRNA-mediated inhibition of actin cytoskeleton dynamics changed the intracellular Gag membrane localization and impaired virus production.…”

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Involvement of the Rac1-IRSp53-Wave2-Arp2/3 Signaling Pathway in HIV-1 Gag Particle Release in CD4 T Cells

Thomas

Mariani-Floderer

López-Huertas

et al. 2015

J Virol

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During HIV-1 assembly, the Gag viral proteins are targeted and assemble at the inner leaflet of the cell plasma membrane. This process could modulate the cortical actin cytoskeleton, located underneath the plasma membrane, since actin dynamics are able to promote localized membrane reorganization. In addition, activated small Rho GTPases are known for regulating actin dynamics and membrane remodeling. Therefore, the modulation of such Rho GTPase activity and of F-actin by the Gag protein during virus particle formation was considered. Here, we studied the implication of the main Rac1, Cdc42, and RhoA small GTPases, and some of their effectors, in this process. The effect of small interfering RNA (siRNA)-mediated Rho GTPases and silencing of their effectors on Gag localization, Gag membrane attachment, and virus-like particle production was analyzed by immunofluorescence coupled to confocal microscopy, membrane flotation assays, and immunoblot assays, respectively. In parallel, the effect of Gag expression on the Rac1 activation level was monitored by G-LISA, and the intracellular F-actin content in T cells was monitored by flow cytometry and fluorescence microscopy. Our results revealed the involvement of activated Rac1 and of the IRSp53-Wave2-Arp2/3 signaling pathway in HIV-1 Gag membrane localization and particle release in T cells as well as a role for actin branching and polymerization, and this was solely dependent on the Gag viral protein. In conclusion, our results highlight a new role for the Rac1-IRSp53-Wave2-Arp2/3 signaling pathway in the late steps of HIV-1 replication in CD4 T lymphocytes. IMPORTANCEDuring HIV-1 assembly, the Gag proteins are targeted and assembled at the inner leaflet of the host cell plasma membrane. Gag interacts with specific membrane phospholipids that can also modulate the regulation of cortical actin cytoskeleton dynamics. Actin dynamics can promote localized membrane reorganization and thus can be involved in facilitating Gag assembly and particle formation. Activated small Rho GTPases and effectors are regulators of actin dynamics and membrane remodeling. We thus studied the effects of the Rac1, Cdc42, and RhoA GTPases and their specific effectors on HIV-1 Gag membrane localization and viral particle release in T cells. Our results show that activated Rac1 and the IRSp53-Wave2-Arp2/3 signaling pathway are involved in Gag plasma membrane localization and viral particle production. This work uncovers a role for cortical actin through the activation of Rac1 and the IRSp53/Wave2 signaling pathway in HIV-1 particle formation in CD4 T lymphocytes.

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contrasting

confidence: 40%

Section: Discussionmentioning

confidence: 99%

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Involvement of the Rac1-IRSp53-Wave2-Arp2/3 Signaling Pathway in HIV-1 Gag Particle Release in CD4 T Cells

Thomas

Mariani-Floderer

López-Huertas

et al. 2015

J Virol

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“…However, cells are also flat at the posterior end, where the actin meshwork is much less dense. The results of the companion paper (43) indicate that disruption of the F-actin network does not affect HIV-1 assembly rates, and the present study shows that NC-actin interactions are not needed for assembly or release and that actin is not enriched in the virion. The combined results argue against an important role for F-actin in HIV-1 assembly and show that the putative NCactin interaction is not required for assembly.…”

Section: Discussionmentioning

confidence: 62%

“…Our results suggest that these processes are independent of NC, and the reported NC-actin interaction does not appear to play a role in HIV-1 assembly. Furthermore, actin was not enriched in HIV-1 particles compared to producer cells, and results reported in the companion article (43) suggest that disruption of the F-actin network does not influence HIV-1 assembly rates. The combined results indicate that actin and its putative interaction with the NC domain do not have a major function in HIV-1 assembly.…”

Section: Discussionmentioning

confidence: 82%

The Nucleocapsid Domain of Gag Is Dispensable for Actin Incorporation into HIV-1 and for Association of Viral Budding Sites with Cortical F-Actin

Stauffer

Rahman

Marco

et al. 2014

J Virol

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Actin and actin-binding proteins are incorporated into HIV-1 particles, and F-actin has been suggested to bind the NC domain in HIV-1 Gag. Furthermore, F-actin has been frequently observed in the vicinity of HIV-1 budding sites by cryo-electron tomography (cET). Filamentous structures emanating from viral buds and suggested to correspond to actin filaments have been observed by atomic force microscopy. To determine whether the NC domain of Gag is required for actin association with viral buds and for actin incorporation into HIV-1, we performed comparative analyses of virus-like particles (VLPs) obtained by expression of wild-type HIV-1 Gag or a Gag variant where the entire NC domain had been replaced by a dimerizing leucine zipper [Gag(LZ)]. The latter protein yielded efficient production of VLPs with near-wild-type assembly kinetics and size and exhibited a regular immature Gag lattice. Typical HIV-1 budding sites were detected by using cET in cells expressing either Gag or Gag(LZ), and no difference was observed regarding the association of buds with the F-actin network. Furthermore, actin was equally incorporated into wild-type HIV-1 and Gag-or Gag(LZ)-derived VLPs, with less actin per particle observed than had been reported previously. Incorporation appeared to correlate with the relative intracellular actin concentration, suggesting an uptake of cytosol rather than a specific recruitment of actin. Thus, the NC domain in HIV-1 Gag does not appear to have a role in actin recruitment or actin incorporation into HIV-1 particles. IMPORTANCEHIV-1 particles bud from the plasma membrane, which is lined by a network of actin filaments. Actin was found to interact with the nucleocapsid domain of the viral structural protein Gag and is incorporated in significant amounts into HIV-1 particles, suggesting that it may play an active role in virus release. Using electron microscopy techniques, we previously observed bundles of actin filaments near HIV-1 buds, often seemingly in contact with the Gag layer. Here, we show that this spatial association is observed independently of the proposed actin-binding domain of HIV-1. The absence of this domain also did not affect actin incorporation and had a minor effect on the viral assembly rate. Furthermore, actin was not enriched in the virus compared to the average levels in the respective producing cell. Our data argue against a specific recruitment of actin to HIV-1 budding sites by the nucleocapsid domain of Gag.

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Various Facets of Pathogenic Lipids in Infectious Diseases: Exploring Virulent Lipid-Host Interactome and Their Druggability

Dadhich

Kapoor

2020

J Membrane Biol

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Lipids form an integral, structural, and functional part of all life forms. They play a significant role in various cellular processes such as membrane fusion, fission, endocytosis, protein trafficking, and protein functions. Interestingly, recent studies have revealed their more impactful and critical involvement in infectious diseases, starting with the manipulation of the host membrane to facilitate pathogenic entry. Thereafter, pathogens recruit specific host lipids for the maintenance of favorable intracellular niche to augment their survival and proliferation. In this review, we showcase the lipid-mediated host pathogen interplay in context of life-threatening viral and bacterial diseases including the recent SARS-CoV-2 infection. We evaluate the emergent lipid-centric approaches adopted by these pathogens, while delineating the alterations in the composition and organization of the cell membrane within the host, as well as the pathogen. Lastly, crucial nexus points in their interaction landscape for therapeutic interventions are identified.

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Investigating the Role of F-Actin in Human Immunodeficiency Virus Assembly by Live-Cell Microscopy

Cited by 25 publications

References 45 publications

Involvement of the Rac1-IRSp53-Wave2-Arp2/3 Signaling Pathway in HIV-1 Gag Particle Release in CD4 T Cells

Involvement of the Rac1-IRSp53-Wave2-Arp2/3 Signaling Pathway in HIV-1 Gag Particle Release in CD4 T Cells

The Nucleocapsid Domain of Gag Is Dispensable for Actin Incorporation into HIV-1 and for Association of Viral Budding Sites with Cortical F-Actin

Various Facets of Pathogenic Lipids in Infectious Diseases: Exploring Virulent Lipid-Host Interactome and Their Druggability

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