Cross-talk between Rac1 and Cdc42 GTPases regulates formation of filopodia required for dengue virus type-2 entry into HMEC-1 cells

Abstract

Infection with dengue virus type-2 (DENV-2) begins with virus adherence to cell surface receptors. In endothelial cells (HMEC-1), a cell model for DENV-2 infection, a5b3 integrin has been identified as a putative receptor for the virus. Previous work had suggested that the actin cytoskeleton of HMEC-1 cells plays an important role in virus entry and infection. In the present work, fixed and living HMEC-1 cells expressing enhanced green fluorescent protein-actin were monitored for actin reorganization after virus inoculation, utilizing fluorescence and time lapse microscopy. Cell infection and production of infective viruses were quantified using an anti-E protein antibody and by measuring the p.f.u. ml . Specific drugs that antagonize actin organization and regulate actin-signalling pathways were tested in viral adhesion and infection assays, as were the expression of dominant-negative Rac1 and Cdc42 proteins. Disorganization of actin precluded infection, while microtubule depolymerization had no effect. Activation of Rac1 and Cdc42 signalling, which occurs upon virus binding, induced reorganization of actin to form filopodia in the cellular periphery. Formation of filopodia was a requirement for virus entry and further cell infection. Expression of the dominant-negative proteins Rac1 and Cdc42 confirmed the role of these GTPases in the actin reorganization that is required to form filopodia. In addition, inhibition of the ATPase activity of myosin II greatly decreased infection, suggesting its participation in filopodial stability. We show here, for the first time, that internalization of DENV-2 into endothelial cells requires viral induction of dynamic filopodia regulated by Rac1 and Cdc42 cross-talk and myosin II motor activities.

Rho’ing in and out of cells

“…The mammalian homolog of diaphanous (mDia) is another important RhoA effector mediating actin nucleation. 38,39 Rho GTPases also play a crucial role in another viral pre-entry process. Virion entry requires interactions with specific receptors and coreceptors that are not always readily accessible.…”

Squalamine as a broad-spectrum systemic antiviral agent with therapeutic potential

“…We chose this model for several reasons. Dengue virus, which infects the microvascular and hepatic endothelium in the human disease (37), has been recently shown to engage a Rac1-dependent pathway in the endothelial cell during its entry phase (22,38); the early fusion process between the virus and the endosomal membrane is believed to involve electrostatic interactions between the viral E protein and anionic phospholipids (39). Our hypothesis predicts that squalamine should interrupt this infection.…”

“…The HMEC-1 was inoculated with dengue virus Den V2 using a published procedure (22). At a squalamine concentration of 40 μg/mL, dengue infection was inhibited by about 60%, with complete inhibition observed at 100 μg/mL (Table 1).…”

“…that squalamine might have, as a consequence, antiviral properties. Many viruses enter cells through engagement of the ρ-GTPases, such as Rac1, to influence the actin cytoskeleton (20)(21)(22)(23). Displacement of key proteins anchored through electrostatic forces (of host or viral origin) from the cytoplasmic face of the plasma membrane might interfere with entry (20,21,24), protein synthesis (25), virion assembly (26,27), virion budding (28), or other steps in the viral replication cycle (29).…”

Molecular Signaling and Cellular Pathways for Virus Entry

“…e actin cytoskeleton of HMEC-1 cells plays an important role in virus entry and infection. Internalization of DENV-2 into endothelial cells requires viral induction of dynamic �lopodia regulated by Rac1 and Cdc42 cross-talk and myosin II motor activities [88]. Adenoviruses are a signi�cant cause of acute infections in humans.…”