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Journal of Virology, December 2009, p. 12139-12150, Vol. 83, No. 23
0022-538X/09/$08.00+0     doi:10.1128/JVI.00955-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Glycoprotein-Dependent Acidification of Vesicular Stomatitis Virus Enhances Release of Matrix Protein{triangledown}

Chad E. Mire,1 Derek Dube,2 Sue E. Delos,2 Judith M. White,2 and Michael A. Whitt1*

Department of Molecular Sciences, 858 Madison Avenue, University of Tennessee Health Science Center, Memphis, Tennessee 38163,1 Department of Cell Biology, University of Virginia, 1340 Jefferson Park Avenue, Charlottesville, Virginia 22908-07322

Received 13 May 2009/ Accepted 7 September 2009

To study vesicular stomatitis virus (VSV) entry and uncoating, we generated a recombinant VSV encoding a matrix (M) protein containing a C-terminal tetracysteine Lumio tag (rVSV-ML) that could be fluorescently labeled using biarsenical compounds. Quantitative confocal microscopy showed that there is a transient loss of fluorescence at early times after the initiation of endocytosis of rVSV-ML-Green (rVSV-MLG) virions, which did not occur when cells were treated with bafilomycin A1. The reduction in fluorescence occurred 5 to 10 min postentry, followed by a steady increase in fluorescence intensity from 15 to 60 min postentry. A similar loss of fluorescence was observed in vitro when virions were exposed to acidic pH. The reduction in fluorescence required G protein since "bald" {Delta}G-MLG particles did not show a similar loss of fluorescence at low pH. Based on the pH-dependent fluorescence properties of Lumio Green, we hypothesize that the loss of fluorescence of rVSV-MLG virions during virus entry is due to a G ectodomain-dependent acidification of the virion interior. Biochemical analysis indicated that low pH also resulted in an enhancement of M protein dissociation from partially permeabilized, but otherwise intact, wild-type virions. From these data we propose that low-pH conformational changes in G protein promote acidification of the virus interior, which facilitates the release of M from ribonucleoprotein particles during uncoating.

* Corresponding author. Mailing address: Department of Molecular Sciences, 858 Madison Ave., University of Tennessee Health Science Center, Memphis, TN 38163. Phone: (901) 448-4634. Fax: (901) 448-8462. E-mail: mwhitt{at}utmem.edu

{triangledown} Published ahead of print on 23 September 2009.

Journal of Virology, December 2009, p. 12139-12150, Vol. 83, No. 23
0022-538X/09/$08.00+0     doi:10.1128/JVI.00955-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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