Systematic, genome-wide identification of host genes affecting replication of a positive-strand RNA virus

Abstract

Brome mosaic virus (BMV), a member of the alphavirus-like superfamily of human-, animal-, and plant-infecting (ϩ)RNA viruses, has been studied as a model for viral RNA replication, encapsidation, recombination, and other processes (3). BMV has three genomic RNAs. RNAs 1 and 2 encode the interacting, multifunctional 1a helicase-like and 2a polymerase RNA replication factors (4, 5), which form endoplasmic reticulum (ER) membrane-associated RNA replication complexes with functional similarities to the replicative cores of retrovirus and double-strand (ds)RNA virus virions (6). RNA3 encodes protein 3a that enables infection spread between cells in natural hosts. The negative-strand [(Ϫ) RNA]3 replication intermediate also serves as a template for synthesis of a subgenomic (sg) mRNA, RNA4, which encodes the viral coat protein (Fig. 1A).The yeast Saccharomyces cerevisiae has proven a valuable model for normal and disease processes in human and other cells. The unusual ability of BMV to direct its genomic RNA replication, gene expression, encapsidation, and other processes in this yeast (7,8) has allowed traditional yeast mutagenic analyses that have identified host genes involved in multiple steps of BMV RNA replication and gene expression. Such host genes encode a wide variety of functions and contribute to diverse replication steps, including supporting and regulating viral translation, selecting and recruiting viral RNAs as replication templates, activating the RNA replication complex through chaperones, and providing a lipid profile compatible with membrane-associated viral RNA replication (9-14; reviewed in refs. 2 and 15).Here, we sought to develop a more rapid, global method to systematically identify yeast host factors with effects on BMV RNA replication by using an ordered array of yeast deletion strains (16) to assay virus replication in the absence of each of Ϸ4,500 yeast factors, which is Ϸ80% of the yeast genome. We describe screening this deletion array by using a whole-cell assay based on BMV-directed Renilla luciferase (Rluc) expression by pathways dependent on viral RNA replication and viral RNAdirected sg mRNA synthesis. The assay identified nearly 100 host genes whose absence repressed or enhanced BMV-directed Rluc expression by 3-to 25-fold. The results provide a significantly expanded view of virus-host interactions and should advance understanding of virus and cell pathways. Materials and MethodsYeast. YMI04 and ded1i yeast were described (11). Strains BY4743 (WT; ref. 17) and the homozygous diploid deletion series (BY4743 strain background; ref. 16) were from Research Genetics (Huntsville, AL). Standard yeast techniques were used (18), except for 96-well transformations, which were based on a one-step procedure (19). Briefly, yeast were grown to saturation overnight at 30°C in 96-well plates (1.2 ml per well), pelleted, suspended in 100 l of transformation mix (0.18 M LiAc, pH 5.5, 36% polyethylene glycol-3350, 90 mM DTT, 0.5 mg͞ml sheared salmon sperm DNA, and 20 g͞ml of each plasmid), incubate...

Yeast genome-wide screen reveals dissimilar sets of host genes affecting replication of RNA viruses

“…The remaining 4,548 YKO strains were tested individually (four samples per strain) for their ability to support DI-72 RNA replication by measuring the accumulation level of DI-72 RNA in total yeast RNA extracts, as described in Materials and Methods. Measurements of DI-72 RNA replication products in samples should give easily interpretable data on replication when compared with more complex virus-based reporter-protein assays, which depend not only on replication but on translation of the reporter gene (20). We found that 90 YKO strains (Ͻ2% of total genes) supported the accumulation of DI-72 RNA replicon at Ͻ50% of the level observed in the parental yeast strain (Table 1 and Fig.…”

Selective Repression of Translation by the Brome Mosaic Virus 1a RNA Replication Protein

“…Several yeast proteins have been identified to regulate BMV replication and translation, such as Lsm1p-7p/Pat1p deadenylation-dependent mRNA-decapping factors and DED1. Mutations in these host proteins affected viral replication in yeast at the levels of both viral replication and gene expression (1,17,25,26,30,32,34,46). If 1a represses translation through affecting cellular proteins, then these proteins are prime candidates for the B box binding proteins.…”

Hepatitis C virus RNA replication is regulated by host geranylgeranylation and fatty acids

“…These data strongly suggest that HCV may directly affect one or more steps in cholesterol and͞or fatty acid biosynthesis. This is not surprising because cholesterol, fatty acids, and lipid rafts have been demonstrated to be critical for efficient replication and͞or infection of RNA and many DNA viruses (18)(19)(20)(21)(22)(23)(24)(25)(26)(27).Genes encoding enzymes involved in cholesterol and fatty acid biosynthesis are transcriptionally regulated by sterol regulatory element-binding proteins (SREBPs), which are members of a family of the basic helix-loop-helix leucine-zipper family of transcription factors (reviewed in ref. 28).…”