Nuclear trafficking of the Rabies virus interferon antagonist P-protein is regulated by an importin-binding nuclear localization sequence in the C-terminal domain

Caitlin L. Rowe, Kylie M. Wagstaff, Sibil Oksayan, Dominic J. Glover, David A. Jans, Gregory W. Moseley

Research output: Contribution to journalArticleResearchpeer-review

Abstract

Rabies virus P-protein is expressed as five isoforms (P1-P5) which undergo nucleocytoplasmic trafficking important to roles in immune evasion. Although nuclear import of P3 is known to be mediated by an importin (IMP)-recognised nuclear localization sequence in the N-terminal region (N-NLS), the mechanisms underlying nuclear import of other P isoforms in which the N-NLS is inactive or has been deleted have remained unresolved. Based on the previous observation that mutation of basic residues K214/R260 of the P-protein C-terminal domain (P-CTD) can result in nuclear exclusion of P3, we used live cell imaging, protein interaction analysis and in vitro nuclear transport assays to examine in detail the nuclear trafficking properties of this domain. We find that the effect of mutation of K214/R260 on P3 is largely dependent on nuclear export, suggesting that nuclear exclusion of mutated P3 involves the P-CTD-localized nuclear export sequence (C-NES). However, assays using cells in which nuclear export is pharmacologically inhibited indicate that these mutations significantly inhibit P3 nuclear accumulation and, importantly, prevent nuclear accumulation of P1, suggestive of effects on NLS-mediated import activity in these isoforms. Consistent with this, molecular binding and transport assays indicate that the P-CTD mediates IMPα2/IMPβ1-dependent nuclear import by conferring direct binding to the IMPα2/IMPβ1 heterodimer, as well as to a truncated form of IMPα2 lacking the IMPβ-binding autoinhibitory domain (ΔIBB-IMPα2), and IMPβ1 alone. These properties are all dependent on K214 and R260. This provides the first evidence that P-CTD contains a genuine IMP-binding NLS, and establishes the mechanism by which P-protein isoforms other than P3 can be imported to the nucleus. These data underpin a refined model for P-protein trafficking that involves the concerted action of multiple NESs and IMP-binding NLSs, and highlight the intricate regulation of P-protein subcellular localization, consistent with important roles in infection.
Original languageEnglish
Article number0150477
Number of pages18
JournalPLoS ONE
Volume11
Issue number3
DOIs
Publication statusPublished - 3 Mar 2016

Keywords

  • nuclear import
  • confocal laser microscopy
  • fluorescence imaging
  • cytoplasm
  • functional genomics
  • protein interactions
  • rabies virus
  • viral genomics

Cite this

@article{1614c009880342a4b6258e264379ecda,
title = "Nuclear trafficking of the Rabies virus interferon antagonist P-protein is regulated by an importin-binding nuclear localization sequence in the C-terminal domain",
abstract = "Rabies virus P-protein is expressed as five isoforms (P1-P5) which undergo nucleocytoplasmic trafficking important to roles in immune evasion. Although nuclear import of P3 is known to be mediated by an importin (IMP)-recognised nuclear localization sequence in the N-terminal region (N-NLS), the mechanisms underlying nuclear import of other P isoforms in which the N-NLS is inactive or has been deleted have remained unresolved. Based on the previous observation that mutation of basic residues K214/R260 of the P-protein C-terminal domain (P-CTD) can result in nuclear exclusion of P3, we used live cell imaging, protein interaction analysis and in vitro nuclear transport assays to examine in detail the nuclear trafficking properties of this domain. We find that the effect of mutation of K214/R260 on P3 is largely dependent on nuclear export, suggesting that nuclear exclusion of mutated P3 involves the P-CTD-localized nuclear export sequence (C-NES). However, assays using cells in which nuclear export is pharmacologically inhibited indicate that these mutations significantly inhibit P3 nuclear accumulation and, importantly, prevent nuclear accumulation of P1, suggestive of effects on NLS-mediated import activity in these isoforms. Consistent with this, molecular binding and transport assays indicate that the P-CTD mediates IMPα2/IMPβ1-dependent nuclear import by conferring direct binding to the IMPα2/IMPβ1 heterodimer, as well as to a truncated form of IMPα2 lacking the IMPβ-binding autoinhibitory domain (ΔIBB-IMPα2), and IMPβ1 alone. These properties are all dependent on K214 and R260. This provides the first evidence that P-CTD contains a genuine IMP-binding NLS, and establishes the mechanism by which P-protein isoforms other than P3 can be imported to the nucleus. These data underpin a refined model for P-protein trafficking that involves the concerted action of multiple NESs and IMP-binding NLSs, and highlight the intricate regulation of P-protein subcellular localization, consistent with important roles in infection.",
keywords = "nuclear import, confocal laser microscopy, fluorescence imaging, cytoplasm, functional genomics, protein interactions, rabies virus, viral genomics",
author = "Rowe, {Caitlin L.} and Wagstaff, {Kylie M.} and Sibil Oksayan and Glover, {Dominic J.} and Jans, {David A.} and Moseley, {Gregory W.}",
year = "2016",
month = "3",
day = "3",
doi = "10.1371/journal.pone.0150477",
language = "English",
volume = "11",
journal = "PLoS ONE",
issn = "1932-6203",
publisher = "Public Library of Science",
number = "3",

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Nuclear trafficking of the Rabies virus interferon antagonist P-protein is regulated by an importin-binding nuclear localization sequence in the C-terminal domain. / Rowe, Caitlin L.; Wagstaff, Kylie M.; Oksayan, Sibil; Glover, Dominic J.; Jans, David A.; Moseley, Gregory W.

In: PLoS ONE, Vol. 11, No. 3, 0150477, 03.03.2016.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - Nuclear trafficking of the Rabies virus interferon antagonist P-protein is regulated by an importin-binding nuclear localization sequence in the C-terminal domain

AU - Rowe, Caitlin L.

AU - Wagstaff, Kylie M.

AU - Oksayan, Sibil

AU - Glover, Dominic J.

AU - Jans, David A.

AU - Moseley, Gregory W.

PY - 2016/3/3

Y1 - 2016/3/3

N2 - Rabies virus P-protein is expressed as five isoforms (P1-P5) which undergo nucleocytoplasmic trafficking important to roles in immune evasion. Although nuclear import of P3 is known to be mediated by an importin (IMP)-recognised nuclear localization sequence in the N-terminal region (N-NLS), the mechanisms underlying nuclear import of other P isoforms in which the N-NLS is inactive or has been deleted have remained unresolved. Based on the previous observation that mutation of basic residues K214/R260 of the P-protein C-terminal domain (P-CTD) can result in nuclear exclusion of P3, we used live cell imaging, protein interaction analysis and in vitro nuclear transport assays to examine in detail the nuclear trafficking properties of this domain. We find that the effect of mutation of K214/R260 on P3 is largely dependent on nuclear export, suggesting that nuclear exclusion of mutated P3 involves the P-CTD-localized nuclear export sequence (C-NES). However, assays using cells in which nuclear export is pharmacologically inhibited indicate that these mutations significantly inhibit P3 nuclear accumulation and, importantly, prevent nuclear accumulation of P1, suggestive of effects on NLS-mediated import activity in these isoforms. Consistent with this, molecular binding and transport assays indicate that the P-CTD mediates IMPα2/IMPβ1-dependent nuclear import by conferring direct binding to the IMPα2/IMPβ1 heterodimer, as well as to a truncated form of IMPα2 lacking the IMPβ-binding autoinhibitory domain (ΔIBB-IMPα2), and IMPβ1 alone. These properties are all dependent on K214 and R260. This provides the first evidence that P-CTD contains a genuine IMP-binding NLS, and establishes the mechanism by which P-protein isoforms other than P3 can be imported to the nucleus. These data underpin a refined model for P-protein trafficking that involves the concerted action of multiple NESs and IMP-binding NLSs, and highlight the intricate regulation of P-protein subcellular localization, consistent with important roles in infection.

AB - Rabies virus P-protein is expressed as five isoforms (P1-P5) which undergo nucleocytoplasmic trafficking important to roles in immune evasion. Although nuclear import of P3 is known to be mediated by an importin (IMP)-recognised nuclear localization sequence in the N-terminal region (N-NLS), the mechanisms underlying nuclear import of other P isoforms in which the N-NLS is inactive or has been deleted have remained unresolved. Based on the previous observation that mutation of basic residues K214/R260 of the P-protein C-terminal domain (P-CTD) can result in nuclear exclusion of P3, we used live cell imaging, protein interaction analysis and in vitro nuclear transport assays to examine in detail the nuclear trafficking properties of this domain. We find that the effect of mutation of K214/R260 on P3 is largely dependent on nuclear export, suggesting that nuclear exclusion of mutated P3 involves the P-CTD-localized nuclear export sequence (C-NES). However, assays using cells in which nuclear export is pharmacologically inhibited indicate that these mutations significantly inhibit P3 nuclear accumulation and, importantly, prevent nuclear accumulation of P1, suggestive of effects on NLS-mediated import activity in these isoforms. Consistent with this, molecular binding and transport assays indicate that the P-CTD mediates IMPα2/IMPβ1-dependent nuclear import by conferring direct binding to the IMPα2/IMPβ1 heterodimer, as well as to a truncated form of IMPα2 lacking the IMPβ-binding autoinhibitory domain (ΔIBB-IMPα2), and IMPβ1 alone. These properties are all dependent on K214 and R260. This provides the first evidence that P-CTD contains a genuine IMP-binding NLS, and establishes the mechanism by which P-protein isoforms other than P3 can be imported to the nucleus. These data underpin a refined model for P-protein trafficking that involves the concerted action of multiple NESs and IMP-binding NLSs, and highlight the intricate regulation of P-protein subcellular localization, consistent with important roles in infection.

KW - nuclear import

KW - confocal laser microscopy

KW - fluorescence imaging

KW - cytoplasm

KW - functional genomics

KW - protein interactions

KW - rabies virus

KW - viral genomics

UR - http://www.ncbi.nlm.nih.gov/pubmed/26939125

U2 - 10.1371/journal.pone.0150477

DO - 10.1371/journal.pone.0150477

M3 - Article

VL - 11

JO - PLoS ONE

JF - PLoS ONE

SN - 1932-6203

IS - 3

M1 - 0150477

ER -