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

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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

All Science Journal Classification (ASJC) codes

  • 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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