Systematic, Genome-Wide Identification of Host Genes Affecting Replication of a Positive-Strand RNA Virus

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. Ishikawa and P.A., unpublished results), contain- with pB12 and pB3Rluc was 50-fold higher than the background ing the GAL1 promoter and RNA3 with the ␤-glucuronidase in yeast with pB3Rluc alone or with pB12 and a plasmid GENETICS (GUS) gene replacing the coat protein sequence, into pRS316. expressing WT RNA3 (Fig. 2A). We also compared WT and isogenic ded1i yeast, in which BMV 2a polymerase translation RNA and Protein Analyses. Transformed yeast were inoculated into and thus BMV replication are selectively inhibited by mutation liquid medium containing galactose, were grown overnight, of an RNA helicase encoded by DED1, a yeast gene essential for subcultured, and grown overnight to mid-exponential phase cell viability (11). Northern blots showed that WT RNA3 and (approximate OD ϭ 0.6). Yeast containing pCUP1B3 were 600 RNA3-Rluc replication in ded1i yeast were 4% (11) and 10% grown without added copper to use basal CUP1 promoter (Fig. 2B) of that in WT yeast. In the whole-cell assay, Rluc activity. For Rluc assays, yeast were pelleted and washed twice ͞ activity for ded1i was 15% of WT, confirming the ability to detect with assay buffer (10 mM NaPi,pH7 20 mM EDTA, pH 8). For single-tube assays, cell lysates or a fixed number of cells based on defects in BMV replication (Fig. 2C). ␮ OD600 reading were assayed in assay buffer plus 1 M coelenterazine in 0.1% methanol by using a Pharmingen Monolight Identification of Genes Facilitating BMV-Directed Replication and 3010 luminometer. For 96-well assays, fixed volumes of cells were Expression of Rluc. The BY4743 homozygous diploid yeast dele- similarly assayed by automated coelenterazine injection with a tion strain array is composed of 4,792 yeast strains, each with both alleles of a specific, nonessential gene deleted, arrayed in MicroLumat Plus (Perkin–Elmer). The OD600 of a second fixed volume of cells was measured to determine relative light units 96-well plates (16). The diploid nature of the strains protects (RLU) per cell. For each pass through the Ϸ4,500 deletion against phenotypic effects from any spontaneous mutations in strains, BMV-directed Rluc expression in each strain was cal- untargeted loci. Two hundred thirty five strains annotated as culated as the ratio of the strain RLU per 5 ϫ 105 cells to the pass ‘‘slow͞petite’’ or ‘‘quality control failures’’ were not assayed. average RLU per 5 ϫ 105 cells, and converted to a percentage. Another group of 66 strains with similar notated growth anom- Total yeast RNA isolation, protein extractions, Northern and alies (plate 0372) transformed inefficiently and was only tested Western blots, and GUS assays were as described (11). once. The remaining 4,491 strains were tested twice in a wholly independent manner. In each of these passes, pB12 and pB3Rluc Results were cotransformed into the deletion strains, and the resulting Initiating and Assaying BMV RNA Replication. We previously used strains were grown and assayed for Rluc activity as described in three plasmids to express BMV RNA replication factors 1a and Materials and Methods. Complete RLU data for both passes

Kushner et al. PNAS ͉ December 23, 2003 ͉ vol. 100 ͉ no. 26 ͉ 15765 Downloaded by guest on September 25, 2021 through the deletion strain array plus notes on occasional Table 1. Yeast deletion strains in which BMV-directed Rluc strain-specific variations in transformation efficiency or post- expression was impaired at least 3-fold transformation growth rate are in Table 3, which is published as Average supporting information on the PNAS web site. Strains with an percent average change in BMV-directed Rluc expression of at least Rluc 3-fold, and at least a 2.5-fold change in each pass, are listed in Yeast ORF Gene expression* Function͞phenotype Tables 1 and 2. Identity of these strains was verified by strain- specific PCR (16). YGR063c SPT4 1 Ϯ 0 Transcription elongation Table 1 lists genes whose deletion inhibited BMV-directed YBR173c UMP1 3 Ϯ 0 Proteasome activator Rluc expression. The 58 ORFs in Table 1 include 48 for which YDL160c DHH1 4 Ϯ 1 mRNA decapping, translation at least suggested functions were previously annotated in the YDR156w RPA14 5 Ϯ 1 RNA polymerase I subunit Saccharomyces Genome Database (www.yeastgenome.org). A YDR069c DOA4 8 Ϯ 5 Ubiquitin-speciﬁc protease number of these genes are implicated in regulating varied aspects YGL025c PGD1 8 Ϯ 1 RNA polymerase II regulation of RNA function or turnover (CBC2, STO1, DHH1, LSM1, YPL178w CBC2 8 Ϯ 1 RNA cap-binding protein LSM6, and PAT1). Prior work from our group showed that LSM1 YGR037c ACB1 9 Ϯ 2 Fatty acid transport, metabolism is required for efficient template selection for BMV RNA YJL101c† GSH1 10 Ϯ 2 Glutathione synthesis replication (10). Other genes (ACB1 and SCS2) are involved in YGR188c BUB1 11 Ϯ 4 Protein tyrosine kinase activity membrane lipid synthesis or metabolism, and may modulate YJL101c† GSH1 11 Ϯ 3 Glutathione synthesis activity of the membrane-associated viral RNA replication com- YDR363w-a SEM1 11 Ϯ 3 Exocytosis͞cell cycle regulation plex by affecting membrane lipid composition, as previously YHR167w THP2 13 Ϯ 1 Transcription elongation found for fatty acid desaturase OLE1 (12), which is essential for YNL229c URE2 13 Ϯ 4 Nitrogen utilization cell viability and therefore not tested in the deletion array. For YKL213c DOA1 14 Ϯ 1 Ubiquitin metabolism some Table 1 genes, the link(s) to BMV RNA replication were YML062c MFT1 15 Ϯ 1 Transcription elongation less obvious. Examples include genes involved in protein turn- YJL148w RPA34 16 Ϯ 9 RNA polymerase I subunit over (e.g., DOA1, PRE9, and QRI8), glutathione synthesis YMR125w STO1 16 Ϯ 4 RNA cap-binding protein (GSH1), nitrogen utilization (URE2), etc. Table 1 also includes YPL084w BRO1 19 Ϯ 3 Protein ubiquitination 10 hypothetical ORFs of Ͼ100 amino acids for which functions YHL011c PRS3 19 Ϯ 1 Ribose phosphate diphosphokinase have not yet been determined (www.yeastgenome.org). The YGR184c UBR1 19 Ϯ 1 Ubiquitin ligase varied possible contributions to BMV replication by the Table 1 YGR135w PRE9 19 Ϯ 2 20S proteasome core ␣ subunit genes are considered further in Discussion. YNL032w SIW14 20 Ϯ 5 Protein tyrosine phosphatase BMV-directed Rluc-expression was inhibited to varying de- YGL125w MET13 20 Ϯ 4 Methionine biosynthesis grees by deleting a number of genes that facilitate transcription YNL056w 20 Ϯ 4 Hypothetical ORF͞unknown function Ϯ ͞ elongation, including SPT4, THP2, MFT1, and DST1 (Table 1). YPL055c LGE1 20 2 Protein ubiquitination cell size control Ϯ Several of these are involved in modifying chromatin structure YML013w SEL1 21 10 Secretion regulation Ϯ ͞ and have effects on chromosome segregation. Deleting these or YGL042c 21 0 Hypothetical ORF unknown function Ϯ ͞ other genes with effects on transcription (PGD1, SNT1, and YHL029c 22 5 Hypothetical ORF unknown function Ϯ SNF1) might affect DNA-directed transcription of BMV 1a or 2a YFR010w UBP6 22 6 Protein deubiquitination Ϯ mRNAs, BMV RNA3, or yeast genes required for BMV repli- YDL020c RPN4 23 2 Proteasome and cell cycle regulation Ϯ cation. THP2 and DST1 deletion decreased 2a and 1a plus 2a YPL226w NEW1 23 7 ATP-binding cassette transporter Ϯ protein levels, respectively (see below). The possibility that such YOR246c 23 1 Oxidoreductase activity Ϯ deletions also might act by modulating expression of other yeast YGL194c HOS2 23 4 Histone deacetylase Ϯ ͞ genes was emphasized by our finding that deleting ELP1, -2, -3, YER119c-a 24 1 Hypothetical ORF unknown function Ϯ -4,or-6, the five nonessential components of another transcrip- YCR077c PAT1 24 9 mRNA catabolism, translation Ϯ tion elongation complex, increased BMV-directed Rluc expres- YDR241w BUD26 24 9 Bud site selection Ϯ ͞ sion (see Table 2 below) and that deleting the transcription- YML010c-b 24 9 Hypothetical ORF unknown function Ϯ suppressing histone deacetylase HOS2 or its cofactor SNT1, YKL160w ELF1 24 5 Unknown function Ϯ inhibited Rluc expression (Table 1). YGL043w DST1 25 6 Transcription elongation Ϯ ͞ Eighteen deletion strains from Table 1 were selected for more YDR067c 26 5 Hypothetical ORF unknown function Ϯ ͞ detailed analysis (Fig. 3). These strains were selected from those YJL124c LSM1 26 13 mRNA catabolism RNA cap-binding YGR001c 27 Ϯ 2 Hypothetical ORF͞unknown function for which BMV-directed Rluc expression was decreased by at Ϯ ͞ least 3-fold on each of the two independent passes through the YCR095c 28 7 Hypothetical ORF unknown function YCR033w SNT1 28 Ϯ 6 Histone deacetylase deletion strain array. In addition, the genes deleted were chosen Ϯ to represent a range of different functions and pathways, and YGL127c SOH1 28 8 DNA repair YPR046w MCM16 28 Ϯ 7 Chromosome segregation varied levels of virus inhibition to test the linearity of the Rluc Ϯ results. WT BY4743 and each selected deletion strain were YER120w SCS2 28 1 Myoinositol metabolism YML010w-a 29 Ϯ 4 Hypothetical ORF͞unknown function transformed with pB12 and pCUP1B3, a plasmid expressing WT Ϯ ͞ RNA3 from the CUP1 promoter. WT RNA3 crosschecks for YDR378c LSM6 29 0 mRNA catabolism splicing YLR055c SPT8 30 Ϯ 1 Histone acetylation possible defects specific to the Rluc reporter and the CUP1 Ϯ promoter crosschecks for possible defects specific to the GAL1- YBL027w RPL19B 30 1 Ribosome component YDR477w SNF1 30 Ϯ 9 Protein kinase promoted B3Rluc transcription used in the Rluc assays. More- YHR179w OYE2 31 Ϯ 4 NADPH dehydrogenase over, for these and the prior Rluc assays, yeast were grown on YNL099c OCA1 31 Ϯ 5 Protein tyrosine phosphatase activity galactose, requiring galactose induction of chromosomal GAL1 YKL010c UFD4 31 Ϯ 1 Protein ubiquitination and GAL10, whose promoters were used to express BMV 1a and YMR022w QRI8 31 Ϯ 6 Ubiquitin conjugating enzyme 2a (Fig. 1A), and 1a and 2a expression were verified directly (Fig. YIL107c PFK26 31 Ϯ 4 Phosphofructokinase 3). The yeast were grown to midexponential phase, and total YBR103w SIF2 32 Ϯ 5 Histone acetylase RNA and proteins extracted for Northern and Western analyses. Whereas yeast ACT1 and ADH1 mRNA levels in the deletion *Average Rluc expression of both passes. strains were similar to WT, RNA4 levels generally were reduced †A few deletions, such as YJL101c, are present twice in the library.

15766 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.2536857100 Kushner et al. Downloaded by guest on September 25, 2021 Table 2. Yeast deletion strains in which BMV-directed Rluc expression was enhanced at least 3-fold Average percent Rluc Yeast ORF Gene expression* Function͞phenotype

YCR063w BUD31 1377 Ϯ 385 Bud site selection YLR373c VID22 856 Ϯ 168 Vacuolar protein catabolism YGL214w† 769 Ϯ 46 Hypothetical ORF͞unknown function YPR189w SKI3 606 Ϯ 35 mRNA catabolism͞antiviral protein YEL003w GIM4 529 Ϯ 98 Tubulin binding͞folding YCR094w CDC50 522 Ϯ 124 Transcription regulation YKL149c DBR1 493 Ϯ 10 RNA catabolism Ϯ ͞ YOR076c SKI7 454 34 mRNA catabolism antiviral protein Fig. 3. Northern and Western analyses of BMV RNAs and proteins from WT YGR200c ELP2 444 Ϯ 112 Transcription elongation BY4743 yeast and selected deletion strains with reduced BMV-directed Rluc YGL213c SKI8 440 Ϯ 81 mRNA catabolism͞antiviral protein expression. Each strain was transformed with pB12 to express 1a and 2a and YNL119w 428 Ϯ 15 Hypothetical ORF͞unknown function with pCUP1B3 to express WT RNA3. Percent of WT sgRNA4 accumulation and YJR074w MOG1 422 Ϯ 27 Nuclear protein import Rluc expression are indicated. Northern blots of yeast ACT1 and ADH1 mRNAs YJL183w MNN11 414 Ϯ 144 Mannosyltransferase͞glycosylation are shown for comparison. Although not listed in Table 1, SCP160 was included YPL086c ELP3 409 Ϯ 16 Transcription elongation due to its membrane association. YHR004c NEM1 407 Ϯ 18 Nuclear membrane͞ER morphology YOL064w MET22 399 Ϯ 65 Methionine biosynthesis 1a and 2a protein levels were seen on deleting DHH1, which we YAL026c DRS2 395 Ϯ 21 Phospholipid translocating ATPase recently found is required for efficient translation of BMV YPL102c 381 Ϯ 28 Hypothetical ORF͞unknown function genomic RNAs and their mRNA derivatives (ref. 13 and A.O.N. YHR111w UBA4 374 Ϯ 53 Ubiquitin-activating enzyme YJL204c RCY1 373 Ϯ 2 Endocytosis, membrane recycling and P.A., unpublished results), or on deleting DST1 (see above). YHR207c SET5 367 Ϯ 93 Unknown function Reduced 2a protein levels were seen on deleting ELF1, QRI8, YKL110c KTI12 367 Ϯ 47 Carbon utilization͞toxin resistance THP2,orYGL042 (which overlaps DST1). YBL079w NUP170 366 Ϯ 51 Nucleocytoplasmic transport YLR398c SKI2 359 Ϯ 61 mRNA catabolism͞antiviral protein Yeast Genes Inhibiting BMV-Directed Replication and Expression of YLR357w RSC2 355 Ϯ 2 Chromatin modeling Rluc. Table 2 lists genes whose deletion stimulated BMV-directed YOR051c 354 Ϯ 2 Hypothetical ORF͞unknown function Rluc expression. The 39 ORFs in Table 2 include 32 ORFs for YPL101w ELP4 348 Ϯ 52 Transcription elongation which proposed functions were previously annotated, and 7 YCR098c GIT1 346 Ϯ 90 Glycerophosphoinositol uptake hypothetical ORFs. They include four SKI genes (SKI2, -3, -7, YPR061c 343 Ϯ 22 Hypothetical ORF͞unknown function and -8) that are involved in degrading nonpolyadenylated RNAs YGL214w† 342 Ϯ 35 Hypothetical ORF͞unknown function and inhibit the yeast L-A dsRNA replicon (28). Other genes YNL148c ALF1 340 Ϯ 28 Tubulin folding listed are implicated in lipid or membrane synthesis, transport or YLR384c ELP1 337 Ϯ 39 Transcription elongation remodeling (NEM1, DRS2, RCY1, GIT1, and SPO7), tubulin YOR014w RTS1 337 Ϯ 47 Protein phosphatase folding (GIM4 and ALF1), and other processes. YGL211w 336 Ϯ 69 Hypothetical ORF͞unknown function Thirteen deletion strains with increased BMV-directed Rluc YML017w PSP2 333 Ϯ 42 Unknown function expression were selected for more detailed analysis. In these YNL120c 333 Ϯ 21 Hypothetical ORF͞unknown function strains (Fig. 4), yeast ACT1 and ADH1 mRNAs accumulated to GENETICS YJL128c PBS2 328 Ϯ 61 Osmoregulatory MAP kinase WT levels but, in keeping with the increased Rluc expression, YMR312w ELP6 324 Ϯ 53 Pol II transcription elongation BMV RNA3 and sgRNA4 accumulated to increased levels, YAL009w SPO7 323 Ϯ 71 Nuclear membrane͞ER morphology except in yeast lacking ELP3. Intriguingly, 1a and 2a protein Ϯ YLR320w MMS22 306 36 Ubiquitination, DNA repair levels generally were slightly reduced from WT. *Average Rluc expression of both passes. †A few deletions, such as YGL214w, are present twice in the library.

to a level comparable to the reduction in Rluc expression. Thus, reduced Rluc activity in these strains was not due to defects in RNA4 translation, but in RNA4 synthesis and͞or accumulation. Yeast lacking ACB1 or CBC2, e.g., had 8–9% of WT Rluc activity and Ϸ1–4% of WT RNA4 levels. Other deletions with similar behavior included YNL056w, YOR246c, STO1, DHH1, ELF1, PRS3, and PRE9. Yeast lacking DOA1, QRI8, THP2,or SCS2 had lesser defects in WT RNA3 replication and WT RNA4 production than in BMV-directed Rluc expression, suggesting defects exacerbated by B3Rluc. Consistent with inhibition of viral RNA replication, levels of (Ϫ)RNA3, the replication intermediate that serves as a template Fig. 4. Northern and Western analyses of BMV RNAs and proteins from WT for sgRNA4, generally were reduced in parallel with RNA4, as Ϫ BY4743 yeast and selected deletion strains with enhanced BMV-directed Rluc were the levels of the other ( )RNA3-templated product, expression. As in Fig. 3, WT RNA3 from pCUP1B3 was used as the replication (ϩ)RNA3. For most strains in Fig. 3, 1a and 2a proteins template. Percent of WT sgRNA4 accumulation and Rluc and GUS expression accumulated to normal or somewhat increased levels. Reduced are indicated. NT, not tested.

Kushner et al. PNAS ͉ December 23, 2003 ͉ vol. 100 ͉ no. 26 ͉ 15767 Downloaded by guest on September 25, 2021 Many tested deletions, including RNA catabolism genes SKI2, DHH1 are defective in supporting RNA replication steps besides -3, -7, -8, and DBR1, left (Ϫ)RNA3 levels close to WT (Fig. 4), 1a and 2a accumulation. suggesting that the increased (ϩ)RNA3 and sgRNA4 levels Further links to translation and ribosome biogenesis were might primarily be due to effects on RNA stability rather than found for RPA14 and RPA34, whose deletion decreased Rluc synthesis. By contrast, (Ϫ)RNA3 levels were increased by de- expression 20- and 6-fold (Table 1). RPA14͞34 are RNA poly- leting DRS2, PBS2,orRCY1, encoding factors related to mem- merase I subunits that are dispensable for cell growth individ- brane composition, trafficking and function (Table 2 and ually, but not simultaneously, suggesting related functions. Discussion). Rpa14p forms an ssRNA-binding heterodimer with Rpa43p, an The increases in WT sgRNA4 levels for the SKI2, -3, -7, -8, RNA polymerase I subunit that binds the polymerase I tran- YGL214w, DBR1, ELP3, and YNL119w deletion strains relative scription factor encoded by RRN3. RRN3 is essential for cell to WT yeast were Ն2-fold lower than the increase in Rluc activity viability, but we recently identified rrn3 mutations that preserve (Fig. 4). This result may be because WT RNA3 replicates to yeast growth while inhibiting BMV RNA replication (A.O.N. Ϸ6-fold higher levels than B3Rluc even in WT yeast, leaving less and P.A., unpublished results). Moreover, BMV-directed Rluc potential to increase RNA yield after removing the antagonistic expression was inhibited Ͼ3-fold by deleting RPL19B, one of two influence of these host genes. To test this hypothesis, we assayed genes encoding identical ribosomal protein L19 sequences. the same strains for GUS expression from B3GUS, an RNA3 Mutations in other 60S ribosomal proteins or other mutations derivative that has the coat gene replaced by GUS and replicates reducing free 60S ribosome levels inhibit replication of the M1 to levels similar to B3Rluc (9). As predicted, GUS activity in the satellite RNA of yeast dsRNA replicon L-A, apparently by SKI2, -3, -7, -8, YGL214w, and YNL119w deletion strains in- preferentially inhibiting translation of nonpolyadenylated creased relative to WT yeast in parallel with Rluc (Fig. 4). In mRNAs such as L-A mRNAs (28) and BMV RNA replication yeast lacking DBR1 and ELP3, GUS activity was higher than in products, (ϩ)RNA3 and sgRNA4. WT yeast but lower than corresponding Rluc levels, suggesting BMV-directed Rluc or GUS expression was increased 4- to some Rluc-specific effects in these strains. 6-fold by deleting SKI2, -3, -7,or-8, which encode factors for exosome-mediated 3Ј to 5Ј mRNA decay and inhibit accumula- Discussion tion of nonpolyadenylated mRNAs like those of L-A (28, 36, 37). The continuing emergence of pathogenic viruses like the SARS Consistent with a similar effect on BMV RNA stability, all four coronavirus, another (ϩ)RNA virus, underscores the impor- SKI deletions increased WT (ϩ)RNA3 and sgRNA4 accumu- tance of advancing understanding of virus–host interactions. lation with little change in (Ϫ)RNA3 (Fig. 4). Here, we used a yeast gene deletion library (16) and the unusual ability of BMV to replicate in yeast to conduct a systematic Regulated Levels and Interaction of Viral Replication Factors. Dele- search for host genes affecting viral replication. The assay was tion of PRE9, the only nonessential 20S proteasome core com- validated by finding several genes previously implicated in BMV ponent, reduced BMV-directed Rluc expression and WT RNA3 replication. Overall, we identified nearly 100 yeast genes that, replication by 5- to 6-fold (Table 1 and Fig. 3). PRE9-containing when absent, altered viral-directed Rluc expression by 3-fold to proteasomes localize primarily to the nuclear envelope–ER over 25-fold. The assays likely underestimated the contribution network (38), the site of BMV RNA replication (6, 39), and of many of these functions to viral replication because many BMV 2a polymerase accumulation increased significantly in nonessential yeast gene deletions are partially compensated by yeast lacking PRE9 (Fig. 3). Like retroviruses, many (ϩ)RNA other genes (29). Moreover, just as high multiplicity of infection viruses down-regulate expression of their polymerases, and this overcomes antiviral resistance in some cell lines (e.g., ref. 30), down-regulation is linked to higher replicative fitness (ref. 6 and the high level, continuous expression of BMV replication pro- references therein). The 2a-homologous nsP4 polymerase of teins and RNA replication templates used in this initial screen Sindbis virus, another member of the alphavirus superfamily, is likely reduced virus dependence on some host functions, relative regulated by the ubiquitin͞proteasome-dependent N-end rule to natural infections initiated by a single copy of viral RNA. degradation pathway (40). Absence of several other yeast genes Below, we discuss selected genes and pathways and their poten- involved in ubiquitination or protein turnover also dramatically tially diverse roles in virus replication. reduced BMV-directed Rluc expression (Table 1). However, some of these genes (e.g., DOA1, UBP6, UBR1, and UMP1) had Regulation of Viral RNA Translation, Replication, and Degradation. little or no effect on accumulation of WT BMV sgRNA4 (Fig. Among the genes identified, we previously found three, LSM1, 3 and data not shown). Whether the more selective effects of LSM6, and PAT1, to be involved in translating BMV RNAs and these genes were due to the higher replicative fitness of WT recruiting them from translation to RNA replication, and to link RNA3 compared to the RNA3-Rluc derivative (see Results)or these steps to a cellular mRNA turnover pathway (10, 13). Here, to other effects specific to Rluc requires further study. BMV 2a we also identified additional host factors involved in RNA also is regulated at the translational level, in part through specific regulation, including CBC2, STO1, and DHH1. CBC2 and STO1 dependence on host RNA helicase DED1 (11). encode a cap-binding complex (31), which was implicated in Deletion of protein tyrosine phosphatases SIW14 or OCA1, initial rounds of translation (32). CBC2 or STO1 deletion did not inhibited BMV-directed Rluc expression by 5- and 3-fold (Table impair translation of the capped and polyadenylated 1a and 2a 1). These phosphatases might contribute to BMV replication by mRNAs (Fig. 3), but may have affected entry of the capped but jointly counteracting N-terminal phosphorylation of 2a polymer- nonpolyadenylated WT RNA3 templates into translation, from ase, which, for a closely related bromovirus, has been shown to which they must be recruited for replication (33). The role of occur in vivo and to inhibit 2a interaction with 1a to promote DHH1 in BMV RNA replication may be interrelated, because replication complex assembly (41). Thus, replication complex the encoded Dhh1p promotes mRNA decapping (34) and com- assembly may be regulated by cell phosphatases as well as plexes with Lsm1p (35). On DHH1 deletion, 1a and 2a protein kinases. levels were slightly reduced (Fig. 3), which was consistent with our recent finding of a role for DHH1 in translating some viral Host Membrane Involvement. All (ϩ)RNA viruses replicate their mRNAs and their derivatives (ref. 13 and A.O.N. and P.A., RNA genomes on host cell membranes. BMV RNA replication unpublished results). However, the modest reduction in 1a and occurs in virus-induced invaginations of the outer perinuclear 2a appears insufficient to explain the 12- to 20-fold reduction in ER membrane (6) with locally altered lipid composition (42). Rluc expression and RNA4 levels, implying that cells lacking Mutations in the essential ⌬9 fatty acid desaturase OLE1 cause

15768 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.2536857100 Kushner et al. Downloaded by guest on September 25, 2021 a reduction in unsaturated fatty acids that blocks BMV RNA YML010c-b, YNL119w, and ELP4, respectively) whose deletion synthesis after viral RNA template recruitment (12). Here, we had parallel effects on BMV. Thus, the former deletions might found that replication of WT BMV RNA3 was inhibited 25-fold affect BMV because they inactivate the latter ORFs on the (Fig. 3) by deleting ACB1, encoding an acyl-CoA-binding factor opposite strand. Consistent with a possible lack of biological involved in membrane sphingolipid biosynthesis (43). This find- function, the former ORFs are not conserved in closely related ing underscores the importance of membrane properties to yeasts (45). Conversely, we found 11 uncharacterized ORFs BMV RNA replication and implies that multiple classes of (YCR095c, YDR067c, YGL211w, YGR001c, YHL029c, membrane lipids are required for a functional RNA replication YML010c-b, YNL056w, YNL119w, YOR051c, YOR246c, and complex. YPR061c) that do not overlap other ORFs and are conserved in Deleting SEL1, encoding a transmembrane protein that in- other yeasts (45). The effects of deleting these ORFs on BMV hibits secretion, suppressed BMV-directed Rluc expression further supports their biological relevance and should shed light 5-fold (Table 1). Lesser defects in Rluc expression and WT on their functions. RNA3 replication were caused by deleting SCS2 (Table 1 and The high-throughput, functional genomics approach described Fig. 3), encoding an ER protein involved in ER targeting of other here identified many host factors that affect BMV replication proteins and in inositol metabolism, with localized homology to and implicated previously unconsidered pathways in the virus a human VAMP-associated protein that interacts with hepatitis life cycle. Further studies will determine more directly how C virus RNA replication factor, NS5A (44). Conversely, Rluc Ϫ implicated host factors affect the virus and how such effects expression, WT BMV RNA replication, and ( )RNA3 levels illuminate cellular functions and pathways. Additional screens were increased by deleting DRS2, RCY1,orPBS2, encoding a more closely duplicating natural infection likely will reveal phospholipid-translocating ATPase, a membrane recycling fac- further relevant host factors. Finally, additional studies should tor, and a MAP kinase required for resistance to osmotic stress, further illuminate the roles of host genes essential for cell respectively, any of which might affect replication complex- growth, which necessarily were not present in the deletion associated membranes (Table 2 and Fig. 4). BMV-directed Rluc library, but which we have already found to make major con- expression similarly increased on deleting NEM1 or SPO7, tributions to BMV replication (refs. 11 and 12 and unpublished encoding interacting factors controlling nuclear membrane ex- pansion and morphology, or GIT1, encoding a factor involved in results). glycerophosphoinositol uptake (Table 2). As in prior virus–host interaction studies, further studies on We thank Chris Bradfield and Guang Yao for valuable advice on the yeast deletion strain array and data analysis, April Wicks and Kimberly the genes discussed above may provide important insights to host Luke for technical assistance, other members of our laboratory for cell function. Moreover, Tables 1 and 2 list 17 hypothetical ORFs valuable discussions, and Paul Lambert for comments on the manuscript. of unknown function whose deletion affected BMV-directed This work was supported by National Institutes of Health Grant Rluc expression. Six of these ORFs (YER119c-a, YGL042c, GM35072. P.A. is an Investigator of the Howard Hughes Medical YGL214w, YML010w-a, YNL120c, and YPL102c) reside on the Institute. D.B.K. was partially supported by National Institutes of Health opposite strand from another ORF (SCS2, DST1, SKI8, Training Grant T32 CA09075.

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