The alphavirus 6K protein activates endogenous ionic conductances when expressed in Xenopus oocytes.
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.
Antoine
Anne-Frédérique
AF
Equipe d'Accueil 4020, Institut Fédératif de la Recherche 147, Université de Lille, 59655 Villeneuve d'Ascq Cedex, France. anne-frederique.antoine@univ-lille1.fr
Montpellier
Claire
C
Cailliau
Katia
K
Browaeys-Poly
Edith
E
Vilain
Jean-Pierre
JP
Dubuisson
Jean
J
D000524:Alphavirus / Q000502:physiology*; D000818:Animals; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000378:metabolism*; D018118:Chloride Channels / Q000378:metabolism; D002712:Chlorides / Q000378:metabolism; D005260:Female; D015967:Gene Expression Regulation, Viral / Q000502:physiology*; D009865:Oocytes / Q000378:metabolism*; D014759:Viral Envelope Proteins / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D014981:Xenopus; D014982:Xenopus laevis
C092026:6K protein, Sindbis virus; D015220:Calcium Channels; D018118:Chloride Channels; D002712:Chlorides; D014759:Viral Envelope Proteins; D002118:Calcium