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The Alphavirus 6K Protein Activates Endogenous Ionic Conductances when Expressed in Xenopus Oocytes

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Abstract

The Alphavirus Sindbis 6K protein is involved in several functions. It contributes to the processing and membrane insertion of E1 and PE2 viral envelope glycoproteins and to virus budding. It also permeabilizes Escherichia coli and mammalian cells. These viroporin-like properties have been proposed to help virus budding by modifying membrane permeabilities. We expressed Sindbis virus 6K cRNA in Xenopus oocytes to further characterize the effect of 6K on membrane conductances and permeabilization. Although no intrinsic channel properties were seen, cell shrinkage was observed within 24 h. Voltage-clamp experiments showed that 6K upregulated endogenous currents: a hyperpolarization-activated inward current (I in) and a calcium-dependent chloride current (I Cl). 6K was located at both the plasma and the endoplasmic reticulum membranes. The plasma membrane current upregulation likely results from disruption of the calcium homeostasis of the cell at the endoplasmic reticulum level. Indeed, 6K cRNA expression induced reticular calcium store depletion and capacitative calcium entry activation. By experimental modifications of the incubation medium, we showed that downstream of these events cell shrinkage resulted from a 6K -induced KCl efflux (I Cl upregulation leads to chloride efflux, which itself electrically drives potassium efflux), which was responsible for an osmotic water efflux. Our data confirm that 6K specifically triggers a sequential cascade of events that leads to cytoplasmic calcium elevation and cell permeabilization, which likely play a role in the Sindbis virus life cycle.

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Acknowledgement

We gratefully appreciate the help provided by A. Lescuyer and S. Ung. We are grateful to Charles M. Rice (Rockefeller University, New York, NY) for providing plasmid pToto1101. This work was supported by a microbiology program from the French Ministry of Research, the CNRS (project ACIM-1–4) and the Nord Pas de Calais Region. J. D. is an international scholar of the Howard Hughes Medical Institute.

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  1. Equipe d’Accueil 4020, Institut Fédératif de la Recherche 147, Université de Lille, 59655, Villeneuve d’Ascq Cedex, France

    Anne-Frédérique Antoine, Katia Cailliau, Edith Browaeys-Poly & Jean-Pierre Vilain

  2. Centre National de la Recherche Scientifique—Unité Propre de Recherche 2511, Institut de Biologie de Lille-Institut Pasteur de Lille, 59021, Lille Cedex, France

    Claire Montpellier & Jean Dubuisson

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  1. Anne-Frédérique Antoine
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Correspondence to Anne-Frédérique Antoine.

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Antoine, AF., Montpellier, C., Cailliau, K. et al. The Alphavirus 6K Protein Activates Endogenous Ionic Conductances when Expressed in Xenopus Oocytes. J Membrane Biol 215, 37–48 (2007). https://doi.org/10.1007/s00232-007-9003-6

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  • DOI: https://doi.org/10.1007/s00232-007-9003-6

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