A spatio-temporal analysis of matrix protein and nucleocapsid trafficking during vesicular stomatitis virus uncoating.

Chad E. Mire, Judith M. White, Michael A. Whitt

Research output: Contribution to journalArticle

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.

Original languageEnglish (US)
Article numbere1000994
JournalPLoS Pathogens
Volume6
Issue number7
StatePublished - 2010
Externally publishedYes

Fingerprint

Virus Uncoating
Spatio-Temporal Analysis
Vesicular Stomatitis
Protein Transport
Microtubules
Membranes
Cytoplasm
Proteins
Membrane Fusion Proteins
Fluorescence
Nucleocapsid Proteins
Nuclear Pore
Membrane Fusion
Endosomes
Actin Cytoskeleton
Actins
Viruses

ASJC Scopus subject areas

  • Microbiology
  • Parasitology
  • Virology
  • Immunology
  • Genetics
  • Molecular Biology

Cite this

A spatio-temporal analysis of matrix protein and nucleocapsid trafficking during vesicular stomatitis virus uncoating. / Mire, Chad E.; White, Judith M.; Whitt, Michael A.

In: PLoS Pathogens, Vol. 6, No. 7, e1000994, 2010.

Research output: Contribution to journalArticle

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