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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, 53 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.

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2009-12-01
2019-11-12
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The association between DENV-2 DiD and HMEC-1 expressing EGFP–actin. The sequence shows live cell activity during a 20 min sequence that corresponds to images in Fig. 3(b) of the main paper. The sequence is reproduced at 1 frame per min and each frame was captured with an exposure time of 2 s. [Video S1](3.4 MB)

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HMEC-1 expressing EGFP–actin. (a) HMEC-1 cells maintained in culture under non-stimulation conditions. (b) HMEC-1 expressing EGFP–actin, inoculated with DENV-2 and then transferred to 37 °C. The 20 min sequence shows live cell activity and is reproduced as for Supplementary Video S1. Selected frames are shown in Fig. 4(a, b) of the main paper. [Video S2](2.4 MB)

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[Video S3](2.5 MB)

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[Video S4](2.7 MB)

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[Video S5](2.5 MB)

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