Genome-Wide Analysis of Host Factors in Nodavirus RNA Replication

Linhui Hao,Billy Dye,Xiaofeng Wang,David Kushner,Paul Ahlquist,Qiuling He,Michael Newton,Brett Lindenbach

doi:10.1371/journal.pone.0095799

Linhui Hao, Billy Dye + Show 6 more

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Flock House virus (FHV), the best studied of the animal nodaviruses, has been used as a model for positive-strand RNA virus research. As one approach to identify host genes that affect FHV RNA replication, we performed a genome-wide analysis using a yeast single gene deletion library and a modified, reporter gene-expressing FHV derivative. A total of 4,491 yeast deletion mutants were tested for their ability to support FHV replication. Candidates for host genes modulating FHV replication were selected based on the initial genome-wide reporter gene assay and validated in repeated Northern blot assays for their ability to support wild type FHV RNA1 replication. Overall, 65 deletion strains were confirmed to show significant changes in the replication of both FHV genomic RNA1 and sub-genomic RNA3 with a false discovery rate of 5%. Among them, eight genes support FHV replication, since their deletion significantly reduced viral RNA accumulation, while 57 genes limit FHV replication, since their deletion increased FHV RNA accumulation. Of the gene products implicated in affecting FHV replication, three are localized to mitochondria, where FHV RNA replication occurs, 16 normally reside in the nucleus and may have indirect roles in FHV replication, and the remaining 46 are in the cytoplasm, with functions enriched in translation, RNA processing and trafficking.

Highlights

All viruses depend on host cell functions for multiple replication steps, and modulate host pathways to make infected cells a better environment for virus replication
The first change addressed the complication that wt Flock House virus (FHV) RNA1 is both the mRNA that expresses RNA replication protein A and an RNA replication template that is multiplied by protein A (Figure 1A)
Any host gene deletion in the yeast knockout (YKO) library that interfered with FHV RNA replication would have its effects further amplified by the resulting secondary inhibition of protein A expression

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Introduction

All viruses depend on host cell functions for multiple replication steps, and modulate host pathways to make infected cells a better environment for virus replication. High throughput assays of FHV RNAdependent RNA replication in the ordered YKO strain library, we generated plasmid pF1R, which uses the galactose-inducible GAL1 promoter to express an FHV genomic RNA1 derivative with two engineered changes (Figure 1B).

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