Positive-strand RNA viruses build extensive membranous replication compartments to support replication and protect the virus from antiviral responses by the host. These viruses require host factors and various lipids to form viral replication complexes (VRCs). The VRCs built by Tomato bushy stunt virus (TBSV) are enriched with phosphatidylethanolamine (PE) through a previously unknown pathway. To unravel the mechanism of PE enrichment within the TBSV replication compartment, in this paper, the authors demonstrate that TBSV co-opts the guanosine triphosphate (GTP)-bound active form of the endosomal Rab5 small GTPase via direct interaction with the viral replication protein. Deletion of Rab5 orthologs in a yeast model host or expression of dominant negative mutants of plant Rab5 greatly decreases TBSV replication and prevents the redistribution of PE to the sites of viral replication. We also show that enrichment of PE in the viral replication compartment is assisted by actin filaments. Interestingly, the closely related Carnation Italian ringspot virus, which replicates on the boundary membrane of mitochondria, uses a similar strategy to the peroxisomal TBSV to hijack the Rab5-positive endosomes into the viral replication compartments. Altogether, usurping the GTP-Rab5–positive endosomes allows TBSV to build a PE-enriched viral replication compartment, which is needed to support peak-level replication. Thus, the Rab family of small GTPases includes critical host factors assisting VRC assembly and genesis of the viral replication compartment.
Digital Object Identifier (DOI)
National Science Foundation (grant number 1517751). Awarded to PDN.
Xu, Kai and Nagy, Peter D., "Enrichment of Phosphatidylethanolamine in Viral Replication Compartments via Co-opting the Endosomal Rab5 Small GTPase by a Positive-Strand RNA Virus" (2016). Plant Pathology Faculty Publications. 60.
S1 Fig. Complementation of tombusvirus replication by Vps21 in yeast lacking the three Rab5 orthologous genes.
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S2 Fig. Lack of complementation of tombusvirus replication by various yeast Rab GTPases in yeast lacking the three Rab5 orthologous genes.
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S3 Fig. Lack of PC enrichment within tombusvirus replication compartment in plant cells.
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S4 Fig. Decreased stability of TBSV replication proteins in yeast lacking the three Rab5 orthologous genes.
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S5 Fig. Lack of enrichment of PE at TBSV replication sites in vps21Δypt52Δypt53Δ yeast.
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S6 Fig. Measuring PE enrichment at TBSV replication sites in N. benthamiana cells expressing dominant negative mutants of AtRab5 proteins.
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S7 Fig. Measuring Rab5 colocalization with PE enriched TBSV replication compartment in N. benthamiana cells.
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S8 Fig. Measuring PE-richness of Rab5-positive endosomes in the absence of tombusviruses in yeast and N. benthamiana cells.
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S9 Fig. PI3P is partly co-localized with the tombusvirus replication compartment in plant cells.
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S10 Fig. Altered TBSV replication in PE synthesis pathway deletion yeast strains.
journal.pbio.2000128.s011.DOCX (59 kB)
S1 Text. Materials and Methods.
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S1 Data. Data file.
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S1 Video. Recruitment of Arabidopsis Rab5B into the tombusvirus replication compartment in N. benthamiana.
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S2 Video. Recruitment of Arabidopsis Rab5-positive endosomes into the TBSV replication compartment through actin filaments in N. benthamiana.
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S3 Video. Recruitment of Arabidopsis Rab5-positive endosomes into the CIRV replication compartment through actin filaments in N. benthamiana.