Technical Reference #1744

1744.

Glycoprotein-Dependent Acidification of Vesicular Stomatitis Virus Enhances Release of Matrix Protein Chad E. Mire; Derek Dube; Sue E. Delos; Judith M. White; Michael A. Whitt, University of Tennessee Health Science Center, Journal of Virology, 83(1744), (2009)
Link To Paper

Abstract:
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" 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.

Materials & Methods:
Plasmid design and construction. pBS-M-Lumio was constructed using pBluescript- SK (Stratagene) by annealing two overlapping oligonucleotides (CEM6 and CEM7) that encoded the C-terminal tetracysteine (in boldface) Lumio tag (AEAAAREACCRECCARA). The oligonucleotides were phosphorylated with T4 polynucleotide kinase annealed to form a linker with NheI and EagI overhangs and ligated to pBS-M-Flag-C that was digested with NheI and EagI.

Microscopic Technique
Confocal Microscopy

Cell Type(s)
BHK-21 cells