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. The probe for extracted FHV genomic RNA1 and RNA2 and, at intervals, (+) URA3 was complementary to the 3' 598 nt of the URA3 tested for infectious FHV virion production by plaque assay on ORF plus 140 nt downstream. Drosophila cell monolayers. As seen in Fig. 2, plaque forming Transformation and Transfection of Yeast. Plasmid DNAs units (pfu) were recoverable by 12 hr posttransfection (hpt), were introduced into YPH500 (a ura3-52 lys2-801 ade2-101 increased beyond 60 hpt, and plateaued by 72 hpt. These pfu trpl-A63 his3-A200 leu2-AJ) cells as described (28). FHV corresponded to FHV virions, as their infectivity was abolished virion RNA and in vitro transcribed RNAs were transfected into YPH500 spheroplasts using PEG and CaCl2 (2, 29). Yeast 4 were grown at 26°C in media selective for plasmid and RNA replicon maintenance. Virion, RNA, and DNA Analysis. Yeast spheroplasts were pelleted at 5000 rpm for 5 min, resuspended in FHV buffer (50 0 2 1. a) mM NaHEPES, pH 7.0/0.1% 2-mercaptoethanol/0.1% bo- D) m vine serum albumin) and stored at -80°C. The lysate was IL thawed and cleared of cell debris by centrifuging at 9000 rpm 0- at 4°C for 30 min. Total virion content was determined by 4) assaying the cleared culture and spheroplast lysate for plaque 0 formation on Drosophila cell monolayers. ay) Total yeast RNA was extracted with phenol/chloroform as -2 described (30) except that glass beads were omitted for sphero- 0 plasts. Total Drosophila RNA was isolated with phenol/ 20 hr after transfection. URA3 sequences were chloroform (26) 84 96 amplified from 1.25 ,ug total yeast DNA or 0.25 ng YEp24 as 0 12 24 36 48 60 72 described (2) except that Taq Extender (Stratagene) was used. Hours posttransfection For Northern blot analysis, RNAs were denatured with FIG. 2. Single step FHV growth curve in FHV virion RNA- glyoxal (27) and electrophoresed on a 0.8% agarose/10 mM transfected yeast spheroplasts. Yeast spheroplasts (5 x 108) were phosphate gel. For Southern blot analysis, PCR products were transfected with 5 ,ug FHV virion RNA, resuspended in 10 ml media, electrophoresed on a 1% agarose gel. Nucleic acids were and incubated 96 hr. At 12-hr intervals, aliquots were assayed for pfu transferred to Zetaprobe (Bio-Rad) membranes that were on Drosophila cell monolayers. The total number of pfu per ml divided prehybridized for 4 hr, hybridized overnight in 50% formamide by the number of infected spheroplasts per ml (as determined by at 600C with 5 x 106 cpm/ml per probe, and washed at 60°C endpoint dilution assay; see Results) is plotted on a log scale versus hpt. Downloaded by guest on September 28, 2021 Biochemistry: Price et al. Proc. Natl. Acad. Sci. USA 93 (1996) 9467 by incubation (1 hr at 37°C) with a 1:10,000 dilution of FHV-neutralizing antiserum, while 0.1 mg/ml ribonuclease A had no effect. Consistent with the expected intracellular origin, Hours posttransfection 1v,t -90% of these pfu were recoverable only after deliberate lysis of the yeast by freeze thawing. The exponential increase in A 0 12 24 36 48 60 72 84 96 4S Z 4' FHV pfu between 0 and 48 hpt (see log scale in Fig. 2) suggested that these virions resulted from encapsidating the products of significant FHV RNA replication in yeast, rather than simply translating and encapsidating the input inoculum r~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~...... RNA. Conclusive evidence of this, including temporal and quantitative correlation between newly synthesized RNA and pfu accumulation, is given below. In parallel controls, spheroplasts were mock transfected or transfected with intact FHV virions. Pfu were never detected 1 2 3 4 6 7 8 9 10 11 12 in mock-transfected spheroplasts. Under the conditions used, .w. there was no evidence of significant viral replication in sphero- plasts transfected with FHV virions, rather than FHV RNA. z *uw,w Pfu were recovered in the media and the number of pfu cr 42 remained constant from 0 to 4 days posttransfection. Thus, +0- under the conditions of the experiment, extracellular virions ,. s.....: were stable but unable to infect new spheroplasts. is~,- 43 Transfection Efficiency and Yield of Pfu. The pfu yields 11 suggested that both transfection efficiency and pfu yield per C infected spheroplast were high. To determine both parame- ,
RNA1: I m + +. I - +* + faWe P N - -w DU: I- - +1 - _ + -I .. ..',.... A B ...-f::. .- RNAI
RNA3 > 1 2 2 34.56 7 D.
DU
FIG. 5. Replication of in vivo transcribed FHV RNA1 and transfected DU RNA in yeast cells. Yeast cells containing the indicated RNAs were grown in liquid media to late log phase. Total RNA was extracted and processed as in Fig. 3, using the probes described below. (A) Northern analysis of (+) strand FHV RNA. Lanes: 1, P contains 1 ng each of in vitro transcribed (+) strand FHV RNA1 and DU (see Fig. 1); 2, N contains 0.01 ng each of in vitro transcripts of (-) strand RNA1 and a truncated DU (-) strand lacking the 3' 264 nt; 3-5, total RNA extracted from Drosophila cells 20 hr after transfection with ddH20, with FHV RNA1 alone, or with RNA1 and DU, respectively. One microgram of total yeast RNA was used as a carrier in each of lanes 1-5. Lanes 6-8 contain 2.5 j.Lg total RNA extracted from yeast cells containing YEplacll2 alone, plR alone, plR and DU, or pl(fs)R alone (see Fig. 1), respectively. The blot was hybridized to a probe complementary to (+) strand RNA3. The positions of RNA1 and subgenomic RNA3 are indicated at left. The positions of two RNAs, one comigrating with RNA1 in lanes 7 and 8 and one migrating just above RNA1 in lanes 7-9, are indicated with open arrows (see Results). (B) The same as A except that 4-times more RNA was loaded per lane, the blot was hybridized to a probe complementary to (-) strand FHV RNA3, and the image was printed at 10-times the intensity level. (C) The same as A except that 70-times more Drosophila RNA was loaded in lanes 3-5 and the blot was hybridized to a probe complementary to (+) strand URA3. (D) The same as C except that 4-times more RNA was loaded per lane, the blot was hybridized to a probe complementary to (-) strand URA3, and the image was printed at 10-times the intensity level.
upper open arrow in Fig. 5A, lanes 7-9). This species may and B). The dramatic increase in (+) RNA1 and RNA2 represent a transcript derived from plR and pl(fs)R by between 0 and 84 hpt, as well as the appearance of (-) strand upstream initiation by a cellular DNA-dependent RNA poly- RNA1 and RNA2 replication intermediates and subgenomic merase. RNA3, which were not present in the inoculum, all demon- As seen in Fig. 5 C and D, (+) and (-) strand-specific URA3 strated de novo RNA synthesis. As expected for RNA- probes revealed no bands in total RNA from any YPH500 dependent viral replication, accumulation of (-) strand RNA (ura3-52) yeast cells not transfected with DU RNA (lanes 6, 7, peaked first, followed in turn by (+) strand RNA, and then and 9) or in total RNA from mock- or RNA1-transfected FHV pfu (Fig. 3C). The sensitivity of these pfu to FHV- Drosophila cells (lanes 3 and 4). However, total RNA extracted neutralizing antibodies, but not to ribonuclease, showed that from Drosophila cells transfected with RNA1 and DU RNA or they represented virions. Moreover, these virions resulted from DU-transfected Ura+ yeast contained (+) and (-) from packaging RNA synthesized in yeast, rather than from DU-sized RNA bands (lanes 5 and 8). Therefore, DU RNA- translation and repackaging ofthe inoculum: pfu accumulation transfected Ura+ yeast prototrophs, at least 30 generations increased exponentially between 12 and 48 hpt, (+) genomic after transfection, contain RNAs that hybridize to (+) and (-) RNA accumulation foreshadowed, temporally and quantita- URA3 probes and have mobilities indistinguishable from DU tively, virion accumulation, and cell-associated inoculum at 0 RNA replicated in Drosophila cells. hpt was insufficient to account for the number of FHV virions. Robustness of FIV RNA Replication and Virion Synthesis DISCUSSION in Yeast. FHV replication in yeast mirrored that in Drosophila cells in multiple ways. (i) The molar ratios of all FHV RNAs The results reported here establish that S. cerevisiae can in yeast were similar to those in Drosophila cells. (ii) The support robust FHV genomic replication and infectious virion calculated yield of 2000 pfu per infected spheroplast was synthesis. They also show that an FHV-derived RNA replicon comparable to the yield of 2400 pfu per infectedDrosophila cell expressing a selected marker gene can be maintained persis- (31). This was initially surprising because yeast cells are tently in yeast, conferring heritable phenotypic changes that normally much smaller than Drosophila cells. However, yeast should be useful in further genetic analysis of viral replication spheroplasts are unable to regenerate their cell wall in liquid and gene expression. media (34), and our microscopy showed that spheroplasts De Novo Synthesis of FHV RNA and Infectious Virions in increased in size from 70 ,m3 (35) at 0 hpt to about the size Yeast. Following transfection of yeast spheroplasts with (+) of a Drosophila cell, 360 ,um3, by 24 hpt, independently of strand FHV RNA1 and RNA2, all expected RNA products of infection. (iii) The ratio of (+) strand RNA to pfu showed that FHV RNA replication and transcription appeared (Fig. 3 A the specific infectivity of yeast-derived FHV virions was at Downloaded by guest on September 28, 2021 9470 Biochemistry: Price et aL Proc. Natl. Acad. Sci. USA 93 (1996) least as high as that of virions from Drosophila cells. Overall, 4. Miller, L. K. (1996) in Fields Virology, eds. Fields, B. N., Knipe, the results imply surprisingly high compatibility between FHV D. M. & Howely, P. M. (Lippincott-Raven, Philadelphia), Vol. 1, and yeast factors involved in replication. pp. 549-551. Previously, when (-) strand RNA from FHV replication in 5. Selling, B. H., Allison, R, F. & Kaesberg, P. (1990) Proc. Natl. BHK (baby hamster kidney) cells was analyzed by electro- Acad. Sci. USA 87, 434-438. phoresis, (-) strand RNA2 6. Ball, L. A., Amann, J. M. & Garrett, B. K. (1992) J. Virol. 66, sequences were found to migrate 2326-2334. in at least two bands: a less prevalent band of monomeric 7. Selling, B. H. & Rueckert, R. R. (1984) J. Virol. 51, 251-253. RNA2, and a predominant band with mobility equivalent to 8. Gallagher, T. M. (1987) Ph.D. thesis (Univ. of Wisconsin, Mad- dimeric RNA2 or double-stranded RNA2 (10). Although both ison), pp. 84-111. bands were observed (Fig. 3A), monomeric (-) RNA2 pre- 9. Dasmahapatra, B., Dasgupta, R., Saunders, K., Selling, B., dominated in both insect and yeast cells under the conditions Gallagher, T. & Kaesberg, P. (1986) Proc. Natl. Acad. Sci. USA described here. 83, 63-66. Expression and Maintenance of Heterologous Markers in 10. Ball, L. A. (1994) Proc. Natl. Acad. Sci. USA 91, 12443-12447. Yeast by FHV RNA Replication. Viral components required 11. Zhong, W., Dasgupta, R. & Rueckert, R. (1992) Proc. Natl. Acad. for FHV RNA replication were provided from FHV RNA1 Sci. USA 89, 11146-11150. cDNA plasmid p1R. RNA species comigrating with (+) and 12. Ball, L. A. & Li, Y. (1993) J. Virol. 67, 3544-3551. (-) RNA1 and RNA3 were present in total RNA extracted 13. Zhong, W. (1993) Ph.D. thesis (Univ. of Wisconsin, Madison), from cells containing p1R, but not from cells transfected with pp. 119-138. 14. Wu, S.-X. & Kaesberg, P. (1991) Virology 183, 392-396. a derivative with a frameshift in the FHV replication gene A. 15. Wu, S.-X., Ahlquist, P. & Kaesberg, P. (1992) Proc. Natl. Acad. When Ura- cells containing plR were transfected with in Sci. USA 89, 11136-11140. vitro transcripts of DU, an FHV RNA2-derivative containing 16. Scotti, P. D., Dearing, S. & Mossop, D. W. (1983)Arch. Virol. 75, the URA3 gene, Ural colonies were obtained. Three results 181-190. indicated that the Ural phenotype was due to URA3 expres- 17. Dasmahapatra, B., Dasgupta, R., Ghosh, A. & Kaesberg, P. sion from DU, which was replicated as a free RNA and (1985) J. Mol. Biol. 182, 183-189. inherited by daughter cells. (i) Colony formation depended on 18. Gallagher, T. M., Friesen, P. D. & Rueckert, R. R. (1983)J. Virol. FHV components in trans and in cis. (ii) PCR showed that the 46, 481-489. Ura+ phenotype was not a result of reverse transcription of the 19. Friesen, P. D. & Rueckert, R. R. (1981) J. Virol. 37, 876-886. RNA to DNA. (iii) DU RNA and its (-) strand replication 20. Ball, L. A. (1995) J. Virol. 69, 720-727. intermediate were found in Ural prototrophs derived from 21. Guarino, L. A., Ghosh, A., Dasmahapatra, B., Dasgupta, R. & DU-transfected spheroplasts many generations after transfec- Kaesberg, P. (1984) Virology 139, 199-203. 22. Friesen, P. D. & Rueckert, R. R. (1982) J. Virol. 42, 986-995. tion. 23. Dasgupta, R. & Sgro, J.-Y. (1989) Nucleic Acids Res. 17, 7525- The ability of S. cerevisiae to support full FHV RNA 7526. replication and encapsidation, plus the ability of FHV to direct 24. Gietz, R. D. & Sugino, A. (1988) Gene 74, 527-534. yeast phenotypes, means that the extensive resources of yeast 25. Botstein, D., Falco, S. C., Stewart, S. E., Brennan, M., Scherer, S., can be used to analyze FHV. Among other opportunities, this Stinchcomb, D. T., Struhl, K. & Davis, R. W. (1979) Gene 8, provides powerful host genetics for identification and charac- 17-24. terization of host factors required for FHV genomic replica- 26. Zhong, W. & Rueckert, R. R. (1993)J. Virol. 67, 2716-2722. tion, gene expression, and assembly. 27. Kroner, P. & Ahlquist, P. (1992) in Molecular Plant Pathology, eds. Gurr, S. J., McPherson, M. J. & Bowles, D. J. (IRL, Oxford), We thank L. A. Ball for pFHV1[1,0], M. Culbertson for the S288C Vol. 1, pp. 23-34. (URA3) yeast strain, and W. Sugden, M. Salvato, and P. Kaesberg for 28. Ito, H., Fukuda, Y., Murata, K. & Kimura, A. (1983)J. Bacteriol. helpful comments on the manuscript. This research was supported by 153, 163-168. the National Institutes of Health under Public Health Service Grants 29. Russell, P. J., Hambidge, S. J. & Kirkegaard, K. (1991) Nucleic GM51301 and GM35072 to P.A. and AI22813 to R.R.R. Acids Res. 19, 4949-4953. 30. Carlson, M. & Botstein, D. (1982) Cell 28, 145-154. 1. Wickner, R. B. (1991) in The Molecularand CellularBiology ofthe 31. Schneemann, A., Zhong, W., Gallagher, T. M. & Rueckert, R. R. Yeast Saccharomyces, eds. Broach, J. R., Pringle, J. & Jones, (1992) J. Virol. 66, 6728-6734. E. W. (Cold Spring Harbor Lab. Press, Plainview, NY), pp. 32. Benton, B. M., Eng, W. -K., Dunn, J. J., Studier, F. W., 263-296. Sternglanz, R. & Fisher, P. A. (1990) Mol. Cell. Biol. 10,353-360. 2. Janda, M. & Ahlquist, P. (1993) Cell 72, 961-970. 33. Rose, M. & Winston, F. (1984) Mol. Gen. Genet. 193, 557-560. 3. Quadt, R., Ishikawa, M., Janda, M. & Ahlquist, P. (1995) Proc. 34. Ne6as, 0. (1961) Nature (London) 192, 580-581. Natl. Acad. Sci. USA 92, 4892-4896. 35. Sherman, F. (1991) Methods Enzymol. 194, 17. Downloaded by guest on September 28, 2021