A spatio-temporal analysis of matrix protein and nucleocapsid trafficking during vesicular stomatitis virus uncoating
Chad Mire, Judith M. White, Michael A. Whitt
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 dominantnegative (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 highresolution 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.
Virus Uncoating
Spatio-Temporal Analysis
Vesicular Stomatitis
Nucleocapsid Proteins
Nuclear Pore
Membrane Fusion
Endosomes
Actin Cytoskeleton
Mire, C., White, J. M., & Whitt, M. A. (2010). A spatio-temporal analysis of matrix protein and nucleocapsid trafficking during vesicular stomatitis virus uncoating. PLoS Pathogens, 6(7), 1-15. [e1000994]. https://doi.org/10.1371/journal.ppat.1000994
A spatio-temporal analysis of matrix protein and nucleocapsid trafficking during vesicular stomatitis virus uncoating. / Mire, Chad; White, Judith M.; Whitt, Michael A.
In: PLoS Pathogens, Vol. 6, No. 7, e1000994, 07.2010, p. 1-15.
Mire, C, White, JM & Whitt, MA 2010, 'A spatio-temporal analysis of matrix protein and nucleocapsid trafficking during vesicular stomatitis virus uncoating', PLoS Pathogens, vol. 6, no. 7, e1000994, pp. 1-15. https://doi.org/10.1371/journal.ppat.1000994
Mire C, White JM, Whitt MA. A spatio-temporal analysis of matrix protein and nucleocapsid trafficking during vesicular stomatitis virus uncoating. PLoS Pathogens. 2010 Jul;6(7):1-15. e1000994. https://doi.org/10.1371/journal.ppat.1000994
Mire, Chad ; White, Judith M. ; Whitt, Michael A. / A spatio-temporal analysis of matrix protein and nucleocapsid trafficking during vesicular stomatitis virus uncoating. In: PLoS Pathogens. 2010 ; Vol. 6, No. 7. pp. 1-15.
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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 dominantnegative (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 highresolution 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.",
author = "Chad Mire and White, {Judith M.} and Whitt, {Michael A.}",
doi = "10.1371/journal.ppat.1000994",
journal = "PLoS Pathogens",
T1 - A spatio-temporal analysis of matrix protein and nucleocapsid trafficking during vesicular stomatitis virus uncoating
AU - Mire, Chad
AU - White, Judith M.
AU - Whitt, Michael A.
N2 - 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 dominantnegative (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 highresolution 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.
AB - 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 dominantnegative (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 highresolution 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.
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DO - 10.1371/journal.ppat.1000994
JO - PLoS Pathogens
JF - PLoS Pathogens
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10.1371/journal.ppat.1000994