title: A spatio-temporal analysis of matrix protein and nucleocapsid trafficking during vesicular stomatitis virus uncoating.
abstract: To study VSV entry and the fate of incoming matrix (M) protein during virus uncoating we used recombinant viruses encoding M proteins with a C-terminal tetracysteine tag that could be fluorescently labeled using biarsenical (Lumio) compounds. We found that uncoating occurs early in the endocytic pathway and is inhibited by expression of dominant-negative (DN) Rab5, but is not inhibited by DN-Rab7 or DN-Rab11. Uncoating, as defined by the separation of nucleocapsids from M protein, occurred between 15 and 20 minutes post-entry and did not require microtubules or an intact actin cytoskeleton. Unexpectedly, the bulk of M protein remained associated with endosomal membranes after uncoating and was eventually trafficked to recycling endosomes. Another small, but significant fraction of M distributed to nuclear pore complexes, which was also not dependent on microtubules or polymerized actin. Quantification of fluorescence from high-resolution confocal micrographs indicated that after membrane fusion, M protein diffuses across the endosomal membrane with a concomitant increase in fluorescence from the Lumio label which occurred soon after the release of RNPs into the cytoplasm. These data support a new model for VSV uncoating in which RNPs are released from M which remains bound to the endosomal membrane rather than the dissociation of M protein from RNPs after release of the complex into the cytoplasm following membrane fusion.
authors.affiliation: Department of Molecular Sciences, University of Tennessee Health Science Center, Memphis, Tennessee, United States of America.
authors.forename: Chad E
authors.initials: CE
authors.lastname: Mire
authors.forename: Judith M
authors.initials: JM
authors.lastname: White
authors.forename: Michael A
authors.initials: MA
authors.lastname: Whitt
mesh_terms: D011992:Endosomes; D006023:Glycoproteins / Q000378:metabolism; D008561:Membrane Fusion; D015335:Molecular Probes; D019251:Nucleocapsid / Q000378:metabolism*; D021381:Protein Transport; D012261:Ribonucleoproteins; D018116:Vesiculovirus / Q000502:physiology*; D014763:Viral Matrix Proteins / Q000378:metabolism*; D053586:Virus Internalization; D018406:Virus Physiological Phenomena
chemical_list: D006023:Glycoproteins; D015335:Molecular Probes; D012261:Ribonucleoproteins; D014763:Viral Matrix Proteins; C023587:protein M (glycoprotein)