Assembly-hub Function of ER-Localized SNARE Proteins in Biogenesis of Tombusvirus Replication Compartment

Authors

Zsuzsanna Sasvari, University of KentuckyFollow
Nikolay Kovalev, University of KentuckyFollow
Paulina Alatriste Gonzalez, University of KentuckyFollow
Kai Xu, University of KentuckyFollow
Peter D. Nagy, University of KentuckyFollow

Abstract

Positive-strand RNA viruses assemble numerous membrane-bound viral replicase complexes within large replication compartments to support their replication in infected cells. Yet the detailed mechanism of how given subcellular compartments are subverted by viruses is incompletely understood. Although, Tomato bushy stunt virus (TBSV) uses peroxisomal membranes for replication, in this paper, we show evidence that the ER-resident SNARE (soluble NSF attachment protein receptor) proteins play critical roles in the formation of active replicase complexes in yeast model host and in plants. Depletion of the syntaxin 18-like Ufe1 and Use1, which are components of the ER SNARE complex in the ERAS (ER arrival site) subdomain, in yeast resulted in greatly reduced tombusvirus accumulation. Over-expression of a dominant-negative mutant of either the yeast Ufe1 or the orthologous plant Syp81 syntaxin greatly interferes with tombusvirus replication in yeast and plants, thus further supporting the role of this host protein in tombusvirus replication. Moreover, tombusvirus RNA replication was low in cell-free extracts from yeast with repressed Ufe1 or Use1 expression. We also present evidence for the mislocalization of the tombusviral p33 replication protein to the ER membrane in Ufe1p-depleted yeast cells. The viral p33 replication protein interacts with both Ufe1p and Use1p and co-opts them into the TBSV replication compartment in yeast and plant cells. The co-opted Ufe1 affects the virus-driven membrane contact site formation, sterol-enrichment at replication sites, recruitment of several pro-viral host factors and subversion of the Rab5-positive PE-rich endosomes needed for robust TBSV replication. In summary, we demonstrate a critical role for Ufe1 and Use1 SNARE proteins in TBSV replication and propose that the pro-viral functions of Ufe1 and Use1 are to serve as assembly hubs for the formation of the extensive TBSV replication compartments in cells. Altogether, these findings point clearly at the ERAS subdomain of ER as a critical site for the biogenesis of the TBSV replication compartment.

Document Type

Article

Publication Date

5-10-2018

Notes/Citation Information

Published in PLOS Pathogens, v. 14, 5, e1007028, p. 1-31.

© 2018 Sasvari et al.

This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Digital Object Identifier (DOI)

https://doi.org/10.1371/journal.ppat.1007028

Funding Information

This work was supported by the National Science Foundation (grant#: MCB1517751). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Related Content

Data Availability: All relevant data are within the paper and its Supporting Information files.

S1 Fig. Proximal localization of peroxisomes with Ufe1-positive subdomain of ER.

https://doi.org/10.1371/journal.ppat.1007028.s001 (TIF)

S2 Fig. Repression of the expression of Sec20p ER SNARE protein inhibits TBSV repRNA accumulation in yeast.

https://doi.org/10.1371/journal.ppat.1007028.s002 (TIF)

S3 Fig. Deletions of Sey1p and Yop1p proteins do not affect TBSV repRNA accumulation in yeast.

https://doi.org/10.1371/journal.ppat.1007028.s003 (TIF)

S4 Fig. Knockdown of DSL1 gene inhibits TBSV RNA replication in N. benthamiana plants.

https://doi.org/10.1371/journal.ppat.1007028.s004 (TIF)

S1 Text. Experimental procedures.

https://doi.org/10.1371/journal.ppat.1007028.s005 (DOCX)

S1 Table. The names and sequences of oligo primers used in this study.

https://doi.org/10.1371/journal.ppat.1007028.s006 (DOCX)

Repository Citation

Sasvari, Zsuzsanna; Kovalev, Nikolay; Gonzalez, Paulina Alatriste; Xu, Kai; and Nagy, Peter D., "Assembly-hub Function of ER-Localized SNARE Proteins in Biogenesis of Tombusvirus Replication Compartment" (2018). Plant Pathology Faculty Publications. 80.
https://uknowledge.uky.edu/plantpath_facpub/80