Systematic, genome-wide identification of host genes affecting replication of a positive-strand RNA virus David B. Kushner*†, Brett D. Lindenbach*‡§, Valery Z. Grdzelishvili*, Amine O. Noueiry*, Scott M. Paul*¶, and Paul Ahlquist*‡ʈ *Institute for Molecular Virology and ‡Howard Hughes Medical Institute, University of Wisconsin, Madison, WI 53706 Contributed by Paul Ahlquist, October 23, 2003 Positive-strand RNA viruses are the largest virus class and include serves as a template for synthesis of a subgenomic (sg) mRNA, many pathogens such as hepatitis C virus and the severe acute RNA4, which encodes the viral coat protein (Fig. 1A). respiratory syndrome coronavirus (SARS). Brome mosaic virus The yeast Saccharomyces cerevisiae has proven a valuable (BMV) is a representative positive-strand RNA virus whose RNA model for normal and disease processes in human and other replication, gene expression, and encapsidation have been repro- cells. The unusual ability of BMV to direct its genomic RNA duced in the yeast Saccharomyces cerevisiae. By using traditional replication, gene expression, encapsidation, and other processes yeast genetics, host genes have been identified that function in in this yeast (7, 8) has allowed traditional yeast mutagenic controlling BMV translation, selecting BMV RNAs as replication analyses that have identified host genes involved in multiple steps templates, activating the replication complex, maintaining a lipid of BMV RNA replication and gene expression. Such host genes composition required for membrane-associated RNA replication, encode a wide variety of functions and contribute to diverse and other steps. To more globally and systematically identify such replication steps, including supporting and regulating viral trans- host factors, we used engineered BMV derivatives to assay viral lation, selecting and recruiting viral RNAs as replication RNA replication in each strain of an ordered, genome-wide set of templates, activating the RNA replication complex through yeast single-gene deletion mutants. Each deletion strain was chaperones, and providing a lipid profile compatible with transformed to express BMV replicase proteins and a BMV RNA membrane-associated viral RNA replication (9–14; reviewed in replication template with the capsid gene replaced by a luciferase refs. 2 and 15). reporter. Luciferase expression, which is dependent on viral RNA Here, we sought to develop a more rapid, global method to replication and RNA-dependent mRNA synthesis, was measured in systematically identify yeast host factors with effects on BMV intact yeast cells. Approximately 4,500 yeast deletion strains RNA replication by using an ordered array of yeast deletion Ϸ strains (16) to assay virus replication in the absence of each of ( 80% of yeast genes) were screened in duplicate and selected Ϸ Ϸ strains analyzed further. This functional genomics approach re- 4,500 yeast factors, which is 80% of the yeast genome. We vealed nearly 100 genes whose absence inhibited or stimulated describe screening this deletion array by using a whole-cell assay based on BMV-directed Renilla luciferase (Rluc) expression by BMV RNA replication and͞or gene expression by 3- to >25-fold. pathways dependent on viral RNA replication and viral RNA- Several of these genes were shown previously to function in BMV directed sg mRNA synthesis. The assay identified nearly 100 host replication, validating the approach. Newly identified genes in- genes whose absence repressed or enhanced BMV-directed Rluc clude some in RNA, protein, or membrane modification pathways expression by 3- to 25-fold. The results provide a significantly and genes of unknown function. The results further illuminate expanded view of virus–host interactions and should advance virus and cell pathways. Further refinement of virus screening understanding of virus and cell pathways. likely will reveal contributions from additional host genes. Materials and Methods any or most steps in virus infections involve interactions of Yeast. YMI04 and ded1i yeast were described (11). Strains Mviral and host factors. Such virus–host interactions thus BY4743 (WT; ref. 17) and the homozygous diploid deletion are crucial determinants of virus host range, replication, and series (BY4743 strain background; ref. 16) were from Research pathology. Studies of virus–host interactions have advanced Genetics (Huntsville, AL). Standard yeast techniques were used understanding of viral and cellular function and can provide (18), except for 96-well transformations, which were based on a targets for antiviral development. Positive-strand RNA one-step procedure (19). Briefly, yeast were grown to saturation [(ϩ)RNA] viruses are the largest genetic class of viruses and overnight at 30°C in 96-well plates (1.2 ml per well), pelleted, include significant human pathogens such as hepatitis C virus, suspended in 100 ␮l of transformation mix (0.18 M LiAc, pH 5.5, West Nile virus, and the coronavirus causing severe acute 36% polyethylene glycol-3350, 90 mM DTT, 0.5 mg͞ml sheared respiratory syndrome (SARS). Whereas recent studies show that salmon sperm DNA, and 20 ␮g͞ml of each plasmid), incubated host factors are critical for (ϩ)RNA virus genome replication, at 45°C for 60 min, and plated on solid minimal media. mRNA synthesis, and other steps (1, 2), identifying such factors remains difficult. Plasmids. pB12 (laboratory designation pB12VG1) expresses Brome mosaic virus (BMV), a member of the alphavirus-like BMV 1a and 2a from the GAL1 and GAL10 promoters, respec- superfamily of human-, animal-, and plant-infecting (ϩ)RNA viruses, has been studied as a model for viral RNA replication, Abbreviations: BMV, brome mosaic virus; (ϩ)RNA, positive-strand RNA; (Ϫ)RNA, negative- encapsidation, recombination, and other processes (3). BMV has strand RNA; ER, endoplasmic reticulum; sg, subgenomic; Rluc, Renilla luciferase; GUS, three genomic RNAs. RNAs 1 and 2 encode the interacting, ␤-glucuronidase; RLU, relative light unit. multifunctional 1a helicase-like and 2a polymerase RNA repli- †Present address: Department of Biology, Dickinson College, Carlisle, PA 17013. cation factors (4, 5), which form endoplasmic reticulum (ER) §Present address: Center for the Study of Hepatitis C, The Rockefeller University, New York, membrane-associated RNA replication complexes with func- NY 10021. tional similarities to the replicative cores of retrovirus and ¶Present address: Department of Bacteriology, University of Wisconsin, Madison, WI 53726. double-strand (ds)RNA virus virions (6). RNA3 encodes protein ʈTo whom correspondence should be addressed at: Institute for Molecular Virology, Uni- 3a that enables infection spread between cells in natural hosts. versity of Wisconsin, 1525 Linden Drive, Madison, WI 53706. E-mail: [email protected]. The negative-strand [(Ϫ) RNA]3 replication intermediate also © 2003 by The National Academy of Sciences of the USA 15764–15769 ͉ PNAS ͉ December 23, 2003 ͉ vol. 100 ͉ no. 26 www.pnas.org͞cgi͞doi͞10.1073͞pnas.2536857100 Downloaded by guest on September 25, 2021 Fig. 2. (A) Rluc activity assayed on intact WT BY4743 yeast transformed with Fig. 1. (A) Viral cDNAs (bold lines) and flanking expression elements in pB12, the indicated combinations of pB12, pB3Rluc, and͞or pB3, a plasmid express- expressing 1a and 2a, and pB3, expressing RNA3. X, the BMV coat protein gene ing WT RNA3 (20). (B) Northern blot analysis of BMV RNA replication products, or any gene replacing it, such as Rluc. (Right) pB3 transcription produces an RNA3Rluc and sg mRNA RNA4Rluc, in WT YPH500 or isogenic ded1i yeast cells initial (ϩ)RNA3 inoculum, used by 1a and 2a to direct synthesis of (Ϫ)RNA3, transformed with pB12 and pB3Rluc. (C) Rluc activity assayed on intact WT which then is used as a template to greatly amplify (ϩ)RNA3 and produce YPH500 or isogenic ded1i yeast transformed with pB12 and pB3Rluc. sgRNA4. An, poly(A) signal; GAL1͞GAL10, yeast promoters; Rz, self-cleaving ribozyme. (B) pB12 supports WT RNA3 replication at a level similar to that of independent plasmids expressing 1a and 2a. 2a and RNA3 in yeast (20). To facilitate high-throughput transformation, we developed a two-plasmid BMV system. pB12 tively (Fig. 1A). To construct pB12, the PacI–BamHI 1a- (Fig. 1A) inducibly expresses 1a and 2a from the bidirectional, containing fragment of pB1YT3H (4) replaced the PacI–BamHI galactose-induced GAL1-GAL10 promoters (24). Yeast trans- 2a-containing fragment of pB2YT5 (4) to make pB1VG17, then formed with pB12 and a plasmid expressing WT RNA3 sup- the 2a ORF and ADH1 poly(A) site (T4 DNA polymerase- ported RNA3 replication and sgRNA4 synthesis to levels similar blunted PstI–SphI fragment from pB2YT5) were inserted into to those when 1a and 2a were expressed from individual plasmids EcoRI-linearized͞blunted pB1VG17. pB3Rluc, based on (Fig. 1B). pB3RQ39 (20), uses a truncated GAL1 promoter [GALL (21)] As sgRNA4 is templated by the (Ϫ)RNA3 replication inter- to express RNA3 with the coat protein ORF replaced by the Rluc mediate (Fig. 1A), its synthesis depends on, and can serve as, a ORF (from pRL-null; Promega). pB3VG1 is pB3RQ39 with the reporter for BMV RNA replication (20). To assay sgRNA4 more GAL1 promoter replaced by the CUP1 promoter, which was simply and rapidly than by Northern blotting, we used a whole- PCR-amplified from pSal1 (22) and contained a 5Ј EcoRI site cell reporter gene assay based on pB3Rluc, a plasmid expressing used for subcloning into EcoRI- and SnaBI-opened pB3RQ39. RNA3 with Rluc replacing the capsid gene. In the presence of To make pCUP1B3 (laboratory designation pB3VG1ura), the 1a and 2a, such RNA3 derivatives are replicated and express the EcoRI–SalI fragment from pB3VG1, containing the CUP1 substituted foreign gene (Fig. 1A and refs. 7 and 25). To avoid ␦ promoter, WT RNA3, and a hepatitis ribozyme, was subcloned the need to lyse yeast to measure BMV-directed Rluc activity, we into EcoRI- and SalI-opened pRS316 (23). pB3GUSura was used a whole-cell assay (based on refs. 26 and 27). In this constructed by subcloning the EcoRI–SalI fragment from whole-cell assay, the Rluc signal from galactose-induced yeast pB3MI20 (M.