Proc. Natl. Acad. Sci. USA Vol. 93, pp. 9465-9470, September 1996 Biochemistry Complete replication of an animal virus and maintenance of expression vectors derived from it in Saccharomyces cerevisiae (flock house virus/host factors/Nodavirus) B. DUANE PRICE*t, ROLAND R. RUECKERT*t, AND PAUL AHLQUIST*t§ *Institute for Molecular Virology and Departments of tPlant Pathology and *Biochemistry, University of Wisconsin, 1525 Linden Drive, Madison, WI 53706-1596 Contributed by Paul Ahlquist, June 12 1996 ABSTRACT Here we describe the first instances to our ref. 4). FHV has many key features as a model for analyzing knowledge of animal virus genome replication, and of de novo (+) strand RNA viruses. Its 4.5-kb genome is one of the synthesis of infectious virions by a nonendogenous virus, in smallest among animal viruses. Although FHV virions are the yeast Saccharomyces cerevisiae, whose versatile genetics infectious to insect cells, naked FHV genomic RNA is infec- offers significant advantages for studying viral replication tious to insect, plant (5), and mammalian (6) cells, suggesting and virus-host interactions. Flock house virus (FHV) is the that host factors necessary for its replication are highly con- most extensively studied member of the Nodaviridae family of served. An FHV plaque assay (7) and neutralizing antibodies (+) strand RNA animal viruses. Transfection of yeast with (8) allow sensitive detection of FHV virions. Infectious tran- FHV genomic RNA induced viral RNA replication, transcrip- scripts from full-length cDNAs can be produced in vitro (9) and tion, and assembly of infectious virions. Genome replication in vivo (10). The cis elements needed for genome replication and virus synthesis were robust: all replicating FHV RNA have been characterized (11, 12) and FHV RNAs have been species were readily detected in yeast by Northern blot anal- used to carry and express heterologous sequences (13). In ysis and yields of virions per cell were similar to those from addition, a replicase capable of full in vitro replication of added Drosophila cells. We also describe in vivo expression and FHV RNA has been isolated from infected cells (14, 15). maintenance of a selectable yeast marker gene from an The FHV genome is bipartite (Fig. IA) (16), with both engineered FHV RNA derivative dependent on FHV-directed RNAs packaged in the same particle (7, 8). RNA1 (3.1 kb) (17) RNA replication. Use of these approaches with FHV and their encodes all viral contributions to the FHV RNA replicase and possible extension to other viruses should facilitate identifi- can replicate autonomously (18). RNA1 serves as mRNA for cation and characterization of host factors required for protein A (112 kDa) (19), which contains the GDD amino acid genomic replication, gene expression, and virion assembly. sequence motif characteristic of RNA polymerases and is essential for FHV RNA replication (20). An RNA1-encoded The understanding of virus-host interactions, including the subgenomic RNA, RNA3 (0.4 kb) (21), serves as mRNA for identity of the host factors with which viruses interact to protein B (10 kDa) (22). Protein B is not required for mediate their replication and cytopathic effects, remains a production of (+) or (-) strand RNA1 or RNA3 (20). RNA2 challenging frontier in virology. For a number of viruses, (1.4 kb) encodes the virion coat protein precursor, a (43 kDa) specific host factors have been implicated in some important (19, 23). Here we demonstrate that S. cerevisiae spheroplasts infection processes such as cell attachment, DNA transcription transfected with FHV RNA support full replication of FHV, and replication, and others. However, for many important including assembly of infectious virions, and we describe a virus functions, such as genome replication by (+) strand RNA system for in vivo expression and maintenance of heterologous viruses, the nature of the host factors involved remains almost marker genes dependent on FHV RNA replication. completely unknown. For most viruses, identification of rele- vant host factors would be greatly assisted by ready genetic dissection of the host. Particularly effective eukaryotic genetics MATERIALS AND METHODS exists for the yeast Saccharomyces cerevisiae, as illustrated by Plasmids. To construct plR and pl(fs)R (see Results), the prior identification of host genes required for the maintenance 5.3-kb ScaI-NarI fragment of pFHV1[1,0] (10), containing an of its endogenous L-A double-stranded RNA virus (1). FHV RNA1 cDNA flanked 1 nt from its 5' end by a T7 To utilize this genetic potential for (+) strand RNA viruses, promoter and 0 nt from its 3' end by a hepatitis delta virus systems were recently developed that demonstrate the RNA- ribozyme and a T7 terminator, was transferred to the 3.0-kb dependent replication, transcription, and persistence in yeast NarI-ScaI fragment of the yeast shuttle plasmid YEplac12 of derivatives of brome mosaic virus (BMV), a plant-infecting (24). For pl(fs)R, the EagI site at position 373 of the FHV member of the alphavirus-like superfamily (2, 3). Yeast ex- RNA1 cDNAwas digested, filled in, and religated. The 0.17-kb pressing the viral components of the BMV RNA replicase Sacl fragment containing the T7 terminator was deleted from support replication and gene expression by BMV RNA deriv- plR and pl(fs)R. atives. Such RNAs are transmitted through mitosis, so that To construct the plasmid pDU (see Results), a 0.9-kb Sacl Ura+ strains can be obtained by transfecting Ura- yeast with fragment containing a modified URA3 gene from YEp24 (25) a BMV RNA replicon expressing the URA3 gene. This system was transferred to the Sacl site of pBS-DI634 (11, 26). The 5' provides a basis for selecting mutations in host genes required end of the modified gene was a 238-bp SacI-NcoI fragment of for BMV RNA replication. a product amplified by polymerase chain reaction (PCR) with Given the feasibility of this approach, it was attractive to the primers 5'-GCGGAGCTCTGTCGAAAGCTACATAT- extend it to other viruses. Flock house virus (FHV) belongs to AAGGAA-3' and 5'-AGCGGCTTAACTGTGCCCTCC-3', the Nodaviridae family of (+) strand RNA viruses, whose and the 3' end was a 649-bp NcoI-SacI fragment with a Sacl members infect invertebrates and vertebrates (for review, see Abbreviations: BMV, brome mosaic virus; DI, defective interfering; The publication costs of this article were defrayed in part by page charge FHV, flock house virus; hpt, hours posttransfection; pfu, plaque payment. This article must therefore be hereby marked "advertisement" in forming units. accordance with 18 U.S.C. §1734 solely to indicate this fact. §To whom reprint requests should be addressed. 9465 Downloaded by guest on September 28, 2021 9466 Biochemistry: Price et al. Proc. Natl. Acad. Sci. USA 93 (1996) A A (Polymerase) RNA1 (3.1 kb) |Capsid RNA2 (1.4 kb) Subgenomic RNA2 D1634 RNA3 (0.4 kb) (0.6 kb) B (- MCS -- ----) YEplac112 C A (Polymerase) p plR 71MM ~~DU (1.5 kb) LWl ---------------------7-l(fs)R V DUAT (1.4 kb) TRPf 21 _ FIG. 1. Structure of the FHV genome and derivatives employed in this study. (A) Schematic drawing of FHV genomic RNA1 and RNA2. Subgenomic RNA3 is shown below RNA1. A derivative of RNA2, defective interfering RNA 634 (DI634), is shown below RNA2. Open boxes represent ORFs and lines represent nontranslated regions. The bent arrow below RNA1 represents the 5'-most nucleotide of RNA3. ORF A (polymerase) and ORF B overlap in different frames. The open triangles above DI634 represent two deleted RNA2 sequences. (B) Schematic drawing (not to scale) of plasmids YEplacll2, p1R, and pl(fs)R. MCS indicates the multiple cloning site of YEplacll2. The full-length FHV RNA1 cDNA is depicted as a heavy line. A hepatitis delta virus ribozyme cDNA is denoted with a shaded box. The filled triangle in pl(fs)R represents a 4-nt insertion following nt 377. The relative position of the TRPI gene and yeast 2,u origin of replication are indicated. (C) Schematic drawing of DU and DUA3' RNAs. Sequences derived from FHV D1634 are represented as heavy lines. The URA3 ORF, indicated by the filled box, is fused in-frame to the first 12 codons of the FHV capsid protein, indicated by the open box. The last 100 nt of DU are deleted in DUA3', as indicated by the brackets. linker inserted at the SmaI site in the 3' noncoding region. To following the manufacturer's protocols. Quantitative results construct pDUA3', the 106-bp MscI-BamHI fragment of pDU were obtained with a Molecular Dynamics PhosphorImager containing the 3' 100 bp of the D1634 cDNA was deleted. digital radioactivity imaging system. In Vitro Transcripts. Unlabeled in vitro transcripts were Drosophila Cells. The WR strain (19) of Drosophila cells was generated using an Ampliscribe kit (Epicentre Technologies, propagated (26), infectious virions were assayed on Drosophila Madison, WI), treated with DNase I, and purified by phenol- cell monolayers (31), and FHV virions were treated with chloroform extraction and sephadex G50 (Pharmacia) spin neutralizing FHV antibody (8) as described. Drosophila (2 x column chromatography. (+) DU and (+) DUA3' contained 106) cells were lipofectin-transfected (26) with 40 ng gel- 4 nonviral nucleotides at their 3' ends. purified viral RNA1 or 40 ng gel-purified viral RNA1 plus 100 Strand-specific 32P-labeled in vitro transcripts were gener- ng in vitro-transcribed DU RNA. ated as described (27). The probes for (-) and (+) RNAs 1 and 3 corresponded to or were complementary to nt 2718-3064 of RESULTS FHV RNA1. The probe for (-) RNA2 corresponded to nt 425-1080 of FHV RNA2. The probe for (+) RNA2 was Infectious FHV Virions from FHV RNA-Transfected Yeast. complementary to nt 62-1400 of FHV RNA2. The probe for As a sensitive assay for biological activity of FHV RNA in (-) URA3 corresponded to the entire 804-nt URA3 open yeast, yeast spheroplasts were transfected with phenol- reading frame (ORF) plus 140 nt downstream.