Reconstituted antiviral defense pathway in surrogate host yeast is used as an intracellular probe to further our understanding of virus-host interactions and the role of co-opted host factors in formation of membrane-bound viral replicase complexes in protection of the viral RNA against ribonucleases. The inhibitory effect of the RNA interference (RNAi) machinery of S. castellii, which only consists of the two-component DCR1 and AGO1 genes, was measured against tomato bushy stunt virus (TBSV) in wild type and mutant yeasts. We show that deletion of the co-opted ESCRT-I (endosomal sorting complexes required for transport I) or ESCRT-III factors makes TBSV replication more sensitive to the RNAi machinery in yeast. Moreover, the lack of these pro-viral cellular factors in cell-free extracts (CFEs) used for in vitro assembly of the TBSV replicase results in destruction of dsRNA replication intermediate by a ribonuclease at the 60 min time point when the CFE from wt yeast has provided protection for dsRNA. In addition, we demonstrate that co-opted oxysterol-binding proteins and membrane contact sites, which are involved in enrichment of sterols within the tombusvirus replication compartment, are required for protection of viral dsRNA. We also show that phosphatidylethanolamine level influences the formation of RNAi-resistant replication compartment. In the absence of peroxisomes in pex3Δ yeast, TBSV subverts the ER membranes, which provide as good protection for TBSV dsRNA against RNAi or ribonucleases as the peroxisomal membranes in wt yeast. Altogether, these results demonstrate that co-opted protein factors and usurped lipids are exploited by tombusviruses to build protective subcellular environment against the RNAi machinery and possibly other cellular ribonucleases.

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Published in PLOS Pathogens, v. 13, 7, e1006520, p. 1-26.

© 2017 Kovalev 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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This work was supported by the National Science Foundation US, MCB-1122039 awarded to PDN.

journal.ppat.1006520.s001.tif (1178 kB)
S1 Fig.

journal.ppat.1006520.s002.tif (2123 kB)
S2 Fig. Reduced ergosterol enrichment in the viral replication compartment increases the sensitivity of replicating tombusvirus RNA to the reconstituted RNAi in yeast.

journal.ppat.1006520.s003.tif (1649 kB)
S3 Fig. Reduced phospholipid biosynthesis in yeast enhances the sensitivity of replicating tombusvirus RNA to the reconstituted RNAi.

journal.ppat.1006520.s004.doc (70 kB)
S1 Text. Small interfering RNA (vsiRNA) detection from yeast cells.