Plus-stranded RNA viruses have limited coding capacity and have to co-opt numerous pro-viral host factors to support their replication. Many of the co-opted host factors support the biogenesis of the viral replication compartments and the formation of viral replicase complexes on subverted subcellular membrane surfaces. Tomato bushy stunt virus (TBSV) exploits peroxisomal membranes, whereas the closely-related carnation Italian ringspot virus (CIRV) hijacks the outer membranes of mitochondria. How these organellar membranes can be recruited into pro-viral roles is not completely understood. Here, we show that the highly conserved Fis1 mitochondrial fission protein is co-opted by both TBSV and CIRV via direct interactions with the p33/p36 replication proteins. Deletion of FIS1 in yeast or knockdown of the homologous Fis1 in plants inhibits tombusvirus replication. Instead of the canonical function in mitochondrial fission and peroxisome division, the tethering function of Fis1 is exploited by tombusviruses to facilitate the subversion of membrane contact site (MCS) proteins and peroxisomal/mitochondrial membranes for the biogenesis of the replication compartment. We propose that the dynamic interactions of Fis1 with MCS proteins, such as the ER resident VAP tethering proteins, Sac1 PI4P phosphatase and the cytosolic OSBP-like oxysterol-binding proteins, promote the formation and facilitate the stabilization of virus-induced vMCSs, which enrich sterols within the replication compartment. We show that this novel function of Fis1 is exploited by tombusviruses to build nuclease-insensitive viral replication compartment.

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Published in PLOS Pathogens, v. 17, issue 3, e1009423.

© 2021 Lin 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.

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PDN received the grant award IOS-1922895 from the National Science Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

ppat.1009423.s001.tif (1056 kB)
S1 Fig. Fis1p mitochondrial fission 1 protein is an essential host factor for tombusvirus and alfanodavirus replication in yeast. https://doi.org/10.1371/journal.ppat.1009423.s001

ppat.1009423.s002.tif (178 kB)
S2 Fig. Over-expression of the N-terminal deletion mutants of Fis1p shows the lack of pro-viral function in fis1Δ yeast. https://doi.org/10.1371/journal.ppat.1009423.s002

ppat.1009423.s003.tif (165 kB)
S3 Fig. Confocal laser microscopy images show the partial co-localization of the YFP-tagged Fis1p protein and Pex13-RFP (peroxisomal marker) in WT yeast cells in the absence of viral components. https://doi.org/10.1371/journal.ppat.1009423.s003

ppat.1009423.s004.tif (1728 kB)
S4 Fig. Confocal laser microscopy images show the partial co-localization of the RFP-tagged AtFis1A and RFP-AtFis1B proteins and GFP-SKL (peroxisomal marker) in N. benthamiana in the absence or presence of TBSV infection. https://doi.org/10.1371/journal.ppat.1009423.s004

ppat.1009423.s005.tif (888 kB)
S5 Fig. Confocal laser microscopy images show the partial co-localization of the RFP-tagged AtFis1A and RFP-AtFis1B proteins and GFP-Tim21 (mitochondrial marker) in N. benthamiana in the absence of TBSV infection. https://doi.org/10.1371/journal.ppat.1009423.s005

ppat.1009423.s006.tif (568 kB)
S6 Fig. Co-localization of the viral (+)repRNA replication products with AtFis1B in N. benthamiana leaves infected with CNV. https://doi.org/10.1371/journal.ppat.1009423.s006

ppat.1009423.s007.tif (897 kB)
S7 Fig. Control experiments for the BiFC assays. https://doi.org/10.1371/journal.ppat.1009423.s007

ppat.1009423.s008.tif (647 kB)
S8 Fig. Additional control experiments for the BiFC assays. https://doi.org/10.1371/journal.ppat.1009423.s008

ppat.1009423.s009.tif (2305 kB)
S9 Fig. Control experiments for the BiFC assays in the absence of TBSV replication. https://doi.org/10.1371/journal.ppat.1009423.s009

ppat.1009423.s010.tif (2282 kB)
S10 Fig. Co-localization of AtFis1A/B with MCS proteins in N. benthamiana leaves. https://doi.org/10.1371/journal.ppat.1009423.s010

ppat.1009423.s011.tif (1366 kB)
S11 Fig. BiFC assays in Fis1-silenced plants supporting TBSV replication. https://doi.org/10.1371/journal.ppat.1009423.s011

ppat.1009423.s012.tif (413 kB)
S12 Fig. Analysis of vsiRNA accumulation in Fis1-silenced plants supporting TBSV replication. https://doi.org/10.1371/journal.ppat.1009423.s012

ppat.1009423.s013.docx (67 kB)
S1 Text. Supplementary material and methods. https://doi.org/10.1371/journal.ppat.1009423.s013

ppat.1009423.s014.docx (24 kB)
S1 Table. List of plasmids constructed in this study. https://doi.org/10.1371/journal.ppat.1009423.s014

ppat.1009423.s015.docx (19 kB)
S2 Table. List of plasmids described in previous studies. https://doi.org/10.1371/journal.ppat.1009423.s015

ppat.1009423.s016.docx (19 kB)
S3 Table. List of primers used in this study. https://doi.org/10.1371/journal.ppat.1009423.s016