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

UNLABELLED: 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. IMPORTANCE: During 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.

Retroviral matrix and lipids, the intimate interaction.

Retroviruses are enveloped viruses that assemble on the inner leaflet of cellular membranes. Improving biophysical techniques has recently unveiled many molecular aspects of the interaction between the retroviral structural protein Gag and the cellular membrane lipids. This interaction is driven by the N-terminal matrix domain of the protein, which probably undergoes important structural modifications during this process, and could induce membrane lipid distribution changes as well. This review aims at describing the molecular events occurring during MA-membrane interaction, and pointing out their consequences in terms of viral assembly. The striking conservation of the matrix membrane binding mode among retroviruses indicates that this particular step is most probably a relevant target for antiviral research.

The absence of p53 aggravates polyploidy and centrosome number abnormality induced by Aurora-C overexpression.

Aurora-C is the third member of the aurora serine/threonine kinase family and was found only in mammals. Because Aurora-C is overexpressed in many different types of cancer cells we decided to analyze the consequences of Aurora-C overexpression in human cells. We first investigated the subcellular localization of overexpressed GFP-Aurora-C in mitosis and interphase in HeLa cells. As expected, during mitosis, we found that Aurora-C mimics Aurora-B. Surprisingly, in few interphase cells, we found that Aurora-C localized to the centrosome, like Aurora-A. We then examined the phenotype generated by Aurora-C overexpression. Basically it looked similar to the phenotypes observed after overexpression of the other Aurora kinases. We observed an augmentation of polyploid cells containing more than two centrosomes. More interestingly this phenotype was aggravated in the absence of a functional p53. Although the physiological function of Aurora-C in somatic cells remains to be clarified, our results, just like for the two other Aurora kinases, raised the question of a role of Aurora-C in the development and progression of cancer especially in the presence of mutated p53.