The role of co-opted ESCRT proteins and lipid factors in protection of tombusviral double-stranded RNA replication intermediate against reconstituted RNAi in yeast.

Nikolay Kovalev,Zhenghe Li,Peter D Nagy,Jun-Ichi Inaba,Shou-Wei Ding

doi:10.1371/journal.ppat.1006520

Nikolay Kovalev, Zhenghe Li + Show 3 more

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https://doi.org/10.1371/journal.ppat.1006520

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Abstract

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.

Highlights

One of the hallmark features of positive-strand (+)RNA viruses, including tomato bushy stunt virus (TBSV), is to assemble numerous membrane-bound viral replicase complexes (VRCs) that leads to replication of the viral genomic RNA inside the infected cells
The reconstituted RNA interference (RNAi) machinery from S. castellii in S. cerevisiae was used as an intracellular probe to study the effect of various co-opted cellular proteins and lipids on the formation of RNAi-insensitive replication compartment
Constitutive co-expression of S. castellii DCR1 and AGO1 from TEF1 promoter led to complete inhibition of TBSV repRNA accumulation in wt yeast (S1A Fig), whereas separate expression of DCR1 and AGO1 did not interfere with TBSV repRNA accumulation, suggesting that co-expression of the two components is required for the RNAi machinery

Summary

Introduction

One of the hallmark features of positive-strand (+)RNA viruses, including tomato bushy stunt virus (TBSV), is to assemble numerous membrane-bound viral replicase complexes (VRCs) that leads to replication of the viral genomic RNA inside the infected cells. These viruses coopt subcellular membranes and alter lipid metabolism in addition to usurping host proteins to form replication compartment or organelle. Regardless of the structure of these replication organelles, it has been proposed that these elaborate membranous structures serve as platforms to assemble VRCs and to concentrate viral and host components for more efficient viral RNA synthesis. VRCs might hide the viral RNAs from recognition by the antiviral surveillance system and protect against degradation by cytosolic ribonucleases

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