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Proc. Nati. Acad. Sci. USA Vol. 84, pp. 4293-4297, June 1987 Microbiology Viruses in fungi: Infection of yeast with the K1 and K2 killer viruses (double-stranded RNA/Saccharomyces cerevisiae/Reoviridae/mycovirus) MOHAMED EL-SHERBEINI AND KEITH A. BOSTIAN Section of Biochemistry, Division of Biology and Medicine, Brown University, Providence, RI 02912 Communicated by Martin Gibbs, February 9, 1987 (received for review July 5, 1986) ABSTRACT We demonstrate here that yeast killer virus- Transmission of yeast viruses has been thought to occur es, previously thought to be transmitted only by cytoplasmic only by cytoplasmic mixing of cells during budding, mating, mixing during division, mating, or other induced forms of cell cytoduction, or protoplast fusion. Numerous attempts by fusion, are capable of extracellular transmission. Viral parti- many workers have failed to demonstrate de novo infection cles from standard K1 and K2 killer strains were used to of yeast cells or yeast spheroplasts by the S. cerevisiae inoculate sensitive cells of Saccharomyces cerevisiae, rendered viruses (ScV) (6). In the present study we demonstrate that competent by spheroplasting, lithium acetate treatment, or by the type 1 and type 2 killer viruses are capable of extracellular natural mating. Extracellular transmission of the killer viruses transmission during natural mating of cells or under circum- was judged by the following criteria and controls. (i) Filter- stances such as removal of the cell wall or stimulation of sterilized virus inocula were shown to be free of viable yeast uptake by lithium acetate treatment. The newly infected cells, and host cells treated in the absence of added virus did not clones possess the genotype of the sensitive host parent and yield killer progeny. (ii) Infected clones originating from the killer phenotype (K1 or K2) of the infecting killer virus. spheroplasts or lithium acetate-treated cells were shown to In their new genetic background the infecting viruses repli- possess the genotype of the host strain and the killer phenotype cate to a level similar to that present in the original parental of the infecting virus. (iii) Infected clones derived from killer strain. It is concluded that the yeast dsRNA viruses complementary mating pairs were found to be wild-type possess all of the attributes required for a segmented virus diploids, whose meiotic segregants exhibited 2:2 segregation for except the ability to permeate the yeast cell wall. unlinked nutritional markers and 4:0 segregation for the killer phenotype. This technique is generally applicable to the study MATERIALS AND METHODS of interactions between yeast viruses and different hosts and suggests that extracellular transmission may be a natural route Yeast Strains and Media. Strain GG100-14D (a his3 ura3-50 for the inheritance and dissemination of mycoviruses. trpl phoS pho3) contains L1A and L1Bc and was derived from the mating of two nonkiller strains, DB4 (a ura3-50 pho3 Viruses with double-stranded RNA (dsRNA) genomes are phoS) and DB13-1A (a his3-532 trpl gal2 suc2). The tester widely distributed in nature, found in a wide variety of strains K7.S1 (a arg9) and S6 (a/a) lack M1 dsRNA and are hosts-namely, animals (1), plants (2), insects (3), fungi (4), sensitive to yeast killer toxin. The virus donor strains K7 (a and bacteria (5). They are associated with the production of arg9)s and Y110 (a/a) contain L1A M1 and L2A L2BC M2, toxins in fungi, with hypovirulence of plant pathogens, respectively, and are standard K1 and K2 killers. Strain including the chestnut blight fungus, which has virtually K23.A (a ade2) is a killer strain containing L1A L1BC and M1, eliminated the American chestnut, and with rotaviral infec- used here and in previous work as a control for dsRNA and tions, the major cause of childhood diarrhea. The killer virus protein determinations. YEPD, methylene blue, and phenomenon in Saccharomyces cerevisiae is unique among minimal media are described elsewhere (9, 10). YEPD pH 6.5 eukaryotic dsRNA viral systems in the detail with which medium is YEPD medium adjusted to a final pH of 6.5. interactions between viral and host components have been Buffered YEPD pH 6.5 medium is YEPD medium buffered explored and in the actual number ofhost components known with 0.2 M Na2HPO4/citrate to pH 6.5. to be involved (6). Preparation of Viral Particles. Virus preparations were Type I killer strains (K1) of S. cerevisiae contain two species made from cells of strains K7 and Y110 grown in YEPD of dsRNA, L1 [4.7 kilobase pairs (kb)] and M1 (1.9 kb), medium by Zymolyase/glass bead cell disruption, clarifica- encapsidated in viruses called ScV-Ll and ScV-Ml, respective- tion by centrifugation, and purification of virus on 10-14% ly. The ScV-Ll viruses are helper particles that provide the (wt/vol) sucrose gradients as described in detail elsewhere (7) capsid protein for both viruses (7). The ScV-Ml viruses are with the following exceptions. Cells were not treated with defective particles having a positive requirement for the pres- 2-mercaptoethanol but rather were harvested, washed in ence of ScV-Ll (8). The M1 genome encodes the killer toxin and SEKS buffer (1 M sorbitol/0.1 M EDTA/0.1 M Na2SO3/0.8 the resistance function that renders killer cells immune to their M KCl), resuspended in the same buffer, and treated with own toxin (9). Several additional killer systems exist in yeast, Zymolyase-20T (Seikagaku Kogyo) at 0.25 mg/g of wet cells. each distinguishable on the basis of the killer toxin produced Virus used for inoculating spheroplasts and LiOAc-treated (10). The type 2 killer strains (K2), first isolated as brewery cells was suspended in STC buffer (10 mM Tris-HCl, pH contaminants, are readily distinguishable from K1 killers not 7.5/1.2 M sorbitol/10 mM CaCl2). Virus preparations used only physiologically, on the basis of distinct toxins, but also by for inoculating mating cells were suspended in PKE buffer (30 their constituent M genomes. The M2 genome of K2 killers is of mM NaPO4, pH 7.6/150 mM KCI/10 mM EDTA). In both lower molecular weight and shows little sequence homology to cases virus preparations were used within 1 day and were M1 by RNA blot hybridization. kept at 4°C. Virus preparations stored refrigerated for 3 weeks became noninfectious. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviations: dsRNA, double-stranded RNA; ScV, Saccha- in accordance with 18 U.S.C. §1734 solely to indicate this fact. romyces cerevisiae virus. 4293 4294 Microbiology: El-Sherbeini and Bostian Proc. Natl. Acad. Sci. USA 84 (1987) Preparation and Inoculation of Spheroplasts. Spheroplasts RESULTS used in infection studies were prepared by the method of Hinnen et al. (11) as modified below. Cells of a sensitive Infection of Spheroplasts with the K1 and K2 Killer Viruses. recipient strain were grown to early logarithmic phase in 200 Spheroplasts were prepared from the sensitive host strain ml of YEPD medium, harvested, and washed twice with 1 M GG100-14D and inoculated with filter-sterilized virus pre- sorbitol by low-speed centrifugation. Cells were suspended in pared from K1 and K2 killer strains. Since the killer viruses 20 ml of 1 M sorbitol containing 168 pl of 2-mercaptoethanol do not carry a directly selectable marker, spheroplasts were and incubated at 30'C for 15 min. Cells were washed twice as infected in the presence of the plasmid YEp24, conferring above, suspended in 20 ml of 1 M sorbitol containing 1% upon transformed cells a readily selectable uracil prototroph- (vol/vol) glusulase, and incubated at 30'C for 1 hr. The ic phenotype. Prototrophs obtained originate from regener- treated cells were again washed as above, suspended in 1 M ated spheroplasts that have taken up at least one molecule of plasmid. These were screened for viral uptake by their killer sorbitol, and kept on ice until inoculated with virus in the character. GG100-14D carries the stable ura3-50 mutation presence of the plasmid YEp24. Approximately 8 x 107 and contains only L dsRNA (L1A and L1BC). YEp24 contains treated cells were gently centrifuged and suspended in 100 A.l the URA3 gene in addition to the 2-,um origin of replication of STC buffer containing 1.5 ,g of YEp24 DNA, 10 pug of calf and transforms ura3 auxotrophs to uracil prototrophy at high thymus DNA, and 30 ,ug of the killer virus and incubated on frequency (15). Prior to infection, virus inocula were plated ice for 10 min followed by incubation at 280C for 20 min. Virus directly onto regeneration medium and no colonies were concentrations were calculated using the known biophysical obtained. In parallel, YEp24 transformants generated in the properties and composition of the virus (12), based on capsid absence of virus inocula were obtained and several hundred protein determinations. Virus preparations from gradient analyzed were shown to be sensitive nonkillers. fractions of strain K7 were -60% ScV-L, with the remainder One hundred single uracil prototrophic colonies obtained ScV-M (see Fig. 2A, lane b). Based on the calculated from regenerated spheroplasts that had been exposed to the composition of the individual species (12), the composite K1 viruses (from strain K7) were "toothpicked" onto master protein content is 82%. Using a similarly derived composite plates, allowed to grow to full colonies, and then tested for molecular weight of 12.8 x 106, the approximate number of the killer phenotype on methylene blue plates seeded with a virus particles added to spheroplasts was 1 x 1012. Ten sensitive background lawn (strain S6). Of these, 67 showed volumes of PEG reagent (20% polyethylene glycol 4000/10 the killer phenotype, due to viral uptake. All showed the mM CaCl2/10 mM Tris HCl, pH 7.5) was then added to the genotype of the host strain, with the exception of ura3. The cell suspensions, which were incubated an additional 10 min infected clones were replicated three successive times onto at 28°C. Spheroplasts were collected, resuspended in 150 ,u YEPD plates and tested for the killer phenotype at each of 1 M sorbitol/33% YEPD medium/6.5 mM CaCl2/6 ,g of stage. All replicates showed the killer phenotype of the infecting virus, suggesting a stable inheritance of the killer uracil per ml, and then added to 10 ml of regeneration viruses in infected host cells. Decreasing the virus concen- agar (minimal medium containing 1.2 M sorbitol, histidine, tration in the inoculum as well as lowering the temperature tryptophan, and 3% agar), and selection was made for during infection lead to a decrease in the percentage of URA3+ transformants by plating onto uracil-deficient medi- infected clones obtained, with little effect on transformation um. frequency (data not shown). As a further test that the newly Preparation and Inoculation of LiOAc-Competent Cells. infected clones contained the type 1 killer virus, the three The LiOAc procedure of Ito et al. (13) was used as modified infected clones, designated inkl.1, inkl.2, and inkl.3, were below to render sensitive host cells competent for transfor- tested for their ability to kill a standard K2 killer strain, Y110. mation. Cells were grown to late logarithmic phase in 50 ml The inkl clones killed the K2 killer strain in a fashion of YEPD medium, harvested, and washed once in 10 ml of 10 identical to the donor K1 killer (Fig. 1). mM Tris.HCl/1 mM EDTA, pH 7.5, by low-speed centrifu- Virus stability was determined in greater detail for four of gation. Cells were suspended in 20 ml of LiOAc/TE buffer the infected clones grown successively for 40 generations in (10 mM Tris HCl, pH 7.5/0.1 M LiOAc/1 mM EDTA) and liquid YEPD medium, as described in the legend to Table 1. mixed gently by shaking at 30°C for 1 hr. Approximately 8 x Cultures were buffered at pH 6.5 to avoid possible selection 107 treated cells were harvested, suspended in 100 ,u1 of 0.1 for cells carrying the killer virus. At pH 6.5 the M-encoded M LiOAc/TE buffer containing 1.5 ,ug of YEp24 DNA, 50 pug toxin is unstable and inactive. Data are shown for inkl.1, of calf thymus DNA, and 18 ,ug of virus, and incubated at inkl.2, and inkl.3. All of the progeny cells tested, except one, 28°C for 30 min. Seven volumes of filter-sterilized PEG exhibited the killer phenotype. Moreover, the size of the reagent (40% polyethylene glycol 4000/0.1 M LiOAc/10 mM killing zone produced by each of the infected clones was Tris-HCl/1 mM EDTA, pH 7.5) was added, mixed, and comparable to that of the virus donor strain K7. Propagation incubated at 28°C for 1 hr and then incubated at 42°C for 5-10 of the K1 killer virus in the infected clones analyzed thus amounted essentially to 100% after 40 generations of growth. min. Finally, cells were collected, resuspended in water, and In contrast, with the 67% infection of URA3+ transform- plated onto uracil-deficient selective medium. ants with K1 killer virus, only 3.5% of the 200 tested L1A L1BC Inoculation of Mating Cells. Genetic crosses undertaken in URA3+ transformants inoculated with the K2 virus exhibited the presence of killer virus were done using sensitive, the killer phenotype (Table 2). All seven of the K2-infected complementing auxotrophs devoid of ScV-M. Parental cells clones, designated ink2.1-ink2.7, were shown to be the were pregrown in separate YEPD cultures to midlogarithmic appropriate haploid killers, possessing the genotype of the phase, harvested by centrifugation, washed once, and resus- host strain and the killer phenotype of the infecting virus, pended in the same medium. Cells were mixed at a ratio of ScV-M2 (Fig. 1). Stability of the K2 virus in the ink2 clones 1:1, to give a total of 107 cells in 200 ,l, and 60 ,ug (100 pul) of was examined in the same fashion as was done for the inkl virus was added. The mixture was incubated at 28°C for 2 hr strains. Data for four ink2 clones are shown in Table 1. In with occasional gentle shaking. Aliquots were then serially each case, the killer phenotype was lost in =20% of the diluted in minimal medium and resulting diploids were earliest subcloned progeny assayed. This ratio remained selected by plating onto minimal medium agar. Sporulation of essentially constant for an additional 40 generations of diploids and tetrad analysis were performed by standard growth. In contrast, the killer phenotype of the virus donor genetic techniques (14). strain Y11O was lost at a lower rate (by a factor of 100) (Table Microbiology: El-Sherbeini and Bostian Proc. Natl. Acad. Sci. USA 84 (1987) 4295 A B c D FIG. 1. Killer:sensitive assay of clones infected with LAWN: K7 Yvl 0 inK2-1 inKl14 K1 and K2 killer viruses. Clones from host cells (GG100- ad"ub. K7 Y110 K7 14D) infected with K1 (designated inkl.1) or K2 (desig- nated ink2.1) killer viruses were tested for their killer _- inKK-1 inK1-1 __M GG100-14D OM phenotype on methylene blue agar plates seeded with Y110 -w inK2-1 standard killer strains of either K1 (strain K7) or K2 3GI00-14D *O-* nK 241 _ inKl-1 (strain Y110) killer types (A and B, respectively). The %mm GG100-14D same newly infected clones (ink2.1 and inkl.1) were used inK241 qo:a~- GG100-14D as background lawns (pH 4.2 and 4.7, respectively) for -- Y110 KK7 Y110 . 11- inK2-1 testing the killer types of each other (C and D, respec- ..... ..eS tively). As controls, standard K1 (K7) and K2 (Y110) killer strains as well as the sensitive host GG100-14D were also tested. 1), similar to the loss of the killer phenotype in Kl-infected used to produce spheroplasts were treated with LiOAc (13), clones. Three of the K+ URA3' subclones from the initial and the cells were inoculated with K1 killer virus in the growth of ink2.5 (ink2.5a, ink2.5b, ink2.5c) were further presence of the plasmid YEp24. Selection was then made for subcultured and assayed for the killer phenotype. All sub- URA3 transformants. Infection with the K1 virus occurred in clones tested were found to be killers, indicating that in these 3% of the uracil prototrophs tested. This is lower by a factor progenies a stable M2 virus was maintained. of -20 than observed for infection of spheroplasts (Table 2), The killer progenies from initial growth were unusual in their although the virus-to-cell ratio with LiOAc-treated cells was ability to take up methylene blue from the killer assay plates, only half that used for spheroplasts. Again, the infected suggesting that only partial expression of immunity occurred. clones showed the genotype of the host strain and the killer This could be due to low copy number of the virus or slow phenotype of the infecting killer virus. changes in the cell wall or plasma membrane necessary to Infection of Mating Yeast Cells with the K1 Killer Virus. We express immunity. The ability to take up stain was lost after an also tested the ability of yeast cells to take up killer virus additional 40 generations of growth. Stain uptake was not under more natural circumstances. This was done by expos- observed with Y11 orinkl clones. Combined, the observations ing mating cells to virus preparations. Two appropriate on the ink.2 clones suggest that M2 is subject to exclusion in haploid yeast stains lacking M dsRNA (Table 3) were early generations after infection due to poor replication or subjected to mating in liquid complete medium in the pres- segregation of virus at initially low copy number. ence of the killer virus. Of 50 randomly isolated diploids Infection of LiOAc-Treated Cells with K1 Killer Virus. tested as described in Fig. 1, 3 showed the killer phenotype. Yeast cells were also tested for their ability to incorporate the All three killer clones sporulated when plated onto sporula- killer virus without removal of the cell wall, by stimulating tion medium, unlike the parental haploids. Tetrad analysis of uptake with LiOAc. Cells from the same sensitive host strain the three killer diploids upon microdissection revealed a 4:0 Table 1. Stability of K1 and K2 killer phenotypes K-/K+, % Initial +10 +20 +40 Strain growth generations generations generations K7 1 1(00)0 (009) 10 0 210 1009 1049 (0.09) Y110 3 (0.3) 2(0.38) 1(0.19) 12(0.19) inkl. inkl.1 I 0 10500 0 210 0~ ~ ~ 210° 1 20.19) TO5(0 inkl.2 0 0 0 0() 525 210 210 )525 inkl.3 0 0 1 0 525 (-) ~~(0.47) ~~~~~~ ~ ~ (- ink2.1 26247 (21.3) (22.3) 46 (21.9) (21.6) ink2.5 412 (19.5) 204 (19.4) 43 (20.4) 201(19.1) 846 (19.4) 414- ink2.6 2 (22.6) 50 (23.8) 49 (23.3) 239(22.7) ink2.7 2419 (23.7) 52 (24.8) 48 (22.9) 802 (23.6) ink2.5a 1048 (0.19) 0 1 (0347) (0.28) ink2.5b 523523 (0.38) 0 TOO~(- 1 47) 2 (0.47)52-3 (0.38) ink2.5c 1(0.19) 0 0 2 0.38) Colonies were toothpicked from transformation plates onto a master plate, which was incubated overnight at 30'C, and the resulting cells were suspended in buffered YEPD pH 6.5 medium and plated for single colony formation. The single colonies were then assayed for the killer phenotype (initial growth) as in Fig. 1. The suspended cells were also used as inoculum for a culture grown serially four times for 10 generations each. Single colonies were obtained from each and assayed for the killer phenotype (+10, +20, and +40 generations). 4296 Microbiology: El-Sherbeini and Bostian Proc. Natl. Acad. Sci. USA 84 (1987) Table 2. Extracellular transmission of ScV virus into sensitive host strains Genotype and phenotype of % of Genotype and phenotype of donor parents (virus inocula Genotype and phenotype transformants host parent prepared from K7 and Y110) of infected clones* infected Infection of spheroplasts with K1 and K2 killer viruses a ura3 his3 trpi (LIBL1A -) S a arg9 (L1AMi) K1 his3 trpl (L1BL1AMi) K1 67 a ura3 his3 trpl (LiBLLA -) S a/a wt (L2AM2) K2 his3 trpl (LlBLlAM2) K2 4 Infection of LiOAc-treated cells with K1 killer viruses a ura3 his3 trpl (LIBLlA -) S a arg9 (- L1AMi) K1 his3 trpl (LiBLUAM,) K1 2 S denotes the sensitive character; K1 and K2 refer to the K1 and K2 phenotypes, respectively. *Infected clones were tested and shown to be haploids. segregation for the killer:sensitive phenotype and a 2:2 virus thus provides a powerful basis for studying the prop- segregation for the four unlinked nutritional markers present erties of mycoviruses and the nucleo-cytoplasmic interac- in the parental strains, confirming that the killer diploids were tions within and among mycoviral systems, including exam- derived from mating of two sensitive parents (Table 3). The ination of host gene functions involved in virus maintenance exact stage in the mating process at which cells are capable and replication, viral gene expression, dsRNA genome in- of taking up exogenous virus awaits determination. teractions, exclusion phenomena, and other issues of host Molecular Characterization of Virus-Infected Cells. Cells incompatibility. Previous studies along these lines of inves- infected with K1 or K2 virus were analyzed at the molecular tigation (6) have been limited to techniques of cell mating or level for their viral constituents. dsRNAs were extracted cell fusion between strains that together contain one or more from the virus donor strains K7 and Y11O and from the K1- viral species at a steady-state concentration. The infection and K2-infected clones inkl.1 and ink2.1 and analyzed by protocol provides an efficient alternative approach, allowing agarose gel electrophoresis (Fig. 2A). K7 and the clone inkl.1 the capability of following the kinetic properties of viral possess comparable amounts of M1 dsRNA (Fig. 2A, lanes c propagation, particularly with regard to amplification, copy and e, respectively). Similarly, Y110 and the clone ink2.1 number control, interference, and exclusion. possess comparable amounts of M2 (Fig. 2A, lanes b and d, Since conditions have not been fully optimized, the esti- respectively). These data suggest that the M genomes (M1 and mates of infection percentages given here could be consid- M2) replicate in the newly infected clones until they reach copy ered as a minimum and improvements may be anticipated. numbers similar to those present in the standard killer strains. For example, it is unclear from existing experiments whether Viral particles prepared from the same strains were ana- plasmid DNA plays any role in the infection process other lyzed by NaDodSO4/polyacrylamide gel electrophoresis (7) than as an aid in selection. Its presence at high concentrations (Fig. 2B). Strain K7 possesses Mr 88,000 capsid protein may act indirectly to stimulate membrane uptake, and ex- (VLlA-Pl) derived from LlA, whereas Y110 possesses a periments to test this possibility are necessary. Clearly, major capsid protein of Mr 84,000 (VL2A-P1) and a minor plasmid DNA is not essential for the viral uptake process by capsid of Mr 83,000 (VL2B-P1) not indicated on the gel (Fig. mating cells, since it was absent from the reaction mixture. 28, lanes a and b, respectively). The recipient strain pos- Moreover, direct selection methods for isolating infected sesses VLlA-P1 (Mr 88,000) derived from LlA and a capsid colonies are conceivable, and these could be developed to species VLlBC-Pl (Mr 82,000) derived from L1BC (Fig. 2B, further streamline the procedure. Other refinements may lane c). Initial transformants infected with the K1 (Fig. 2B, ensure conditions for single virus uptake, readily allowing for lanes d-f) or K2 virus (Fig. 2B, lanes g-n) and their successive the isolation of viral mutants, and variant viral species. subclones all showed the same profile of capsid polypeptides In a different context, the experiments reported here tacitly as the recipient host. Interestingly, the K2 killer virus- imply that extracellular transmission is a natural, productive infected cells shown to possess M2 dsRNA (Fig. 2A) lack the route for viral inheritance, provided that the conditions used VL2A-P1 capsid protein, indicating that L1 (most likely, L1A) mimic similar situations occurring in nature-that is, cell lysis is capable of fulfilling the capsid requirement for maintenance or liberation of viral particles in the same habitat stimulating of M2. This is in agreement with and confirms the data of cell surface transport. Several natural cell-surface alter- Hannig et al. (16). ations, in addition to those responsible for infection of mating cells, such as the budding process occurring during cyto- DISCUSSION kinesis, mechanical damage, or conditions of nutritional The data presented here demonstrate that limited numbers of stress stimulating membrane transport, may provide addi- viral particles are sufficient for the extracellular transmission tional targets for viral penetration. Virus particles are found of viral genomes into compatible yeast strains, following the in vivo at levels as high as several hundred thousand per cell, procedures' outlined above, at frequencies similar to those providing a feasible pool for infection. Although the predom- observed for transformation of yeast with autonomously inant route for inheritance of virus within a species is most replicating plasmids (15). Infections were performed at virus- probably vertical inheritance, horizontal exchange may be an to-cell ratios of about 1000:1, yielding -102 infected trans- important feature of viral exchange among different, non- formants per /ig of virus by the spheroplast method. This mating fungal organisms. Using the procedure described here technique of infecting yeast spheroplasts and yeast cells with for infecting yeast spheroplasts with yeast killer virus we Table 3. Extracellular transmission of ScV virus into sensitive mating cells % of No. of tetrads Genotype and phenotype of diploids Genotype and phenotype analyzed from Cross (parents) parents infected of meiotic segregants* each diploid GG100-14D a ura3 his3 trpl (LIBLIA -) S 6 4 (LBLlAM) K1:0 S 3 K7.S1 a arg9 (L1A -) S LBlM)K: S denotes the sensitive character; K1 refers to the K1 phenotype. *Unlinked nutritional markers of the meiotic products segregated 2:2 in the tetrads analyzed. Microbiology: El-Sherbeini and Bostian Proc. Natl. Acad. Sci. USA 84 (1987) 4297 A B mkt (16). The properties of GG100-14D with respect to these a bcde f ab c d e fg h jk mn genetic elements are presently unknown. -23 In both situations, a low copy number for ScV-M2 could be -9.4 135- 6.5 caused by inefficient encapsidation of the M2 dsRNA by the -4.3 K1 capsid, resulting in low levels of M2 dsRNA. Whereas the -2.3 -2.1 92- concentrations of M2 in cells from the original ink2.1 clone PJ AML - and stable subclones derived from ink2.1 and ink2.2 were VLlA M2 Pl- comparable to that of the original ScV-M2 host, Y11O, VL2A.P1- measured by RNA blot hybridization (7) of total yeast RNA VLeC-P1 (data not shown), cells from the original ink2.2 clone showed much lower M2 levels. No differences were observed be- 53- tween the capsid polypeptide profiles of the host and the initially infected clones or their killer or sensitive derivatives FIG. 2. Viral components of K1- and K2-infected clones. (A) obtained by subculturing (see Fig. 2B). Agarose gel electrophoretic analysis of dsRNAs from S. cerevisiae It is also possible that successful infection by M2 occurs strains prepared by LiCl precipitation and CF11 chromatography (7). Lanes b and c, dsRNA from clones infected with K2 or K1 killer virus only when accompanied by coinfection with L2, which is (ink2.1 and inkl.1, respectively); lanes d and e, dsRNA from the donor subsequently excluded by LiA. Its presence could be tran- K2 and K1 killer strains (Y110 and K7, respectively). As a control, sient, however, since L2 gene products are absent from virus dsRNA from strain K23.A (lane a) was also analyzed. The Ml content preparations made from the progeny of the M2-infected cells of this strain is unusually low. X DNA-HindIH digest is shown in lane (Fig. 2). Exclusion among different L species is well docu- f. (B) Capsid protein profile of sucrose gradient-fractionated viruses. mented. L2 could bolster M2 copy number in early cell Viral particles were prepared, purified by 10-40%o sucrose gradient generations, providing partial escape from virus loss, or it centrifugation, and analyzed as described (7). Lanes: a, K7 (LIA, Ml); could exclude other interfering species of L present in the b, Y110 (L2A, L2B, M2); c, GG100-14D (LlA, L1BC); d-f, infected clones host, or both. A requirement for coinfection with L2 would inkl.1, inkl.2, and inkl.3, respectively; g-j, a stably infected clone also explain the much lower percentage of transformants ink2.1, a sensitive subclone (ink2.1.1s), and two killer subclones (ink2.1.lk and ink2.1.7k) derived from ink2.1, respectively; k-n, an infected with ScV-M2 compared to that for ScV-Ml. Further unstably infected clone ink2.2, a sensitive subclone (ink2.2.1s), and two genetic analysis of the L genome of GG100-14D with respect killer subclones (ink2.2.1k and ink2.2.7k) derived from ink2.2, respec- to NEX and EXL functions may therefore prove useful in tively. Positions of molecular weight markers (shown as Mr x 10-s) are interpreting this data. as indicated. Finally, as pointed out by Herskowitz (17), yeast shares with animal cells many of the cellular processes and compo- have, in fact, been able to infect S. cerevisiae with virus nent structures that are objects of intensive study, such as the particles derived from the dimorphic yeast Yarrowia ability to internalize medium constituents by endocytosis. lipolytica (unpublished data). S. cerevisiae clones were found Endocytosis is believed to be the infectious route for many to stably maintain the Y. lipolytica viral particles, either alone human and animal viruses, and preliminary results (data not or along with ScV-L1 and ScV-Ml. shown) suggest that virus uptake in yeast follows an endo- The initial loss of ScV-M2 from K2-infected cells is cytotic pathway. Whether the process is receptor mediated, dramatically different from the stable incorporation exhibited follows fluid-phase endocytosis, or involves SEC gene prod- for ScV-M1 infections. Apparently ScV-Ml-infected cells ucts (18) remains to be determined. The killer system there- replicate the K1 virus at a sufficiently high enough rate during fore provides a facile approach to studying these processes in outgrowth of the initial transformed cell to allow for parti- yeast and may prove to be an eminent paradigm, by exploit- tioning of virus to all progeny. Based on this stable acquisi- ing the powerful traditional and modem genetic techniques tion of ScV-M, by the ScV-L-containing host, the stability of available in this system. ScV-M2 in its normal host, and the eventual stability of ScV-M2 in GG100-14D, we conclude that it is the process of We thank Stephen Sturley and Stephen Parent for useful com- ments and Naasa Ramadan for technical assistance. The work was de novo acquisition of the virus and the dsRNA genome supported in part by Public Health Service Grants GM32496 and and/or genetic composition of the recipient strain that are GM36441. responsible for the differences in inheritance. 1. Joklik, W. K. (1974) in Comprehensive Virology, eds. Frankel-Conrat, The pattern of ScV-M2 maintenance observed suggests H. & Wagner, R. R. (Plenum, New York), Vol. 2, pp. 231-334. that its concentration remains low enough during replication 2. Grill, L. K. & Garger, S. J. (1981) Proc. Nati. Acad. Sci. USA 78, 7043-7046. of the infected host cell and its close progeny to cause loss of 3. Scott, M. P., Fostel, J. M. & Pardue, M. L. (1980) Cell 22, 929-941. virus to varying extents in individual cells in the resulting cell 4. Buck, K. W. (1980) in The Eucaryotic Microbiol Cell, eds. Gooday, G. W., lines. This could occur by a passive mechanism, in which Lloyd, D. & Trinci, A. P. G. (Cambridge Univ., England), pp. 329-375. 5. Van Etten, J., Lane, L., Gonzales, C., Partridge, J. & Vivader, A. (1976) initial low copy populations of virus are unequally segregated J. Virol. 18, 652-658. during replication and division of the host cell. As the copy 6. Tipper, D. J. & Bostian, K. A. (1984) Microbiol. Rev. 48, 125-156. number of the virus increases, it segregates normally within 7. El-Sherbeini, M., Tipper, D. J., Mitchell, D. J. & Bostian, K. A. (1984) Mol. Cell. Biol. 4, 2818-2827. cells retaining the virus, resulting in mixed populations of 8. El-Sherbeini, M., Bevan, E. A. & Mitchell, D. J. (1983) Curr. Genet. 7, cells that have either maintained or lost the virus. Alterna- 63-68. 9. Hanes, S., Burn, V., Sturley, S., Tipper, D. J. & Bostian, K. A. (1986) tively, a host process or L helper virus property may interfere Proc. Natl. Acad. Sci. USA 83, 1675-1679. directly with replication, packaging of M2, or some other 10. Young, T. W. & Yagiu, M. (1978) Antonie van Leeuwenhoek 44, 59-77. essential function, thereby attenuating virus copy number or 11. Hinnen, A. J., Hicks, J. B. & Fink, G. R. (1978) Proc. Natl. Acad. Sci. USA 75, 1929-1933. completely excluding the virus. Interference could be subject 12. Bruenn, J. A. (1980) Annu. Rev. Microbiol. 34, 49-68. to negative control, so that eventually stable virus popula- 13. Ito, H., Fukuda, Y., Murata, K. & Kimura, A. (1983) J. Bacteriol. 153, tions are attained. It is known that the M2 genome is 163-168. 14. Mortimer, K. & Hawthorne, D. (1974) in The Yeasts, eds. Rose, A. H. excludable by certain species of L dsRNA. For example, L & Harrison, J. S. (Academic, New York), Vol. 1, pp. 386-460. dsRNA carrying EXL (16) excludes M2 in the absence of 15. Parent, S. A., Fenimore, C. & Bostian, K. A. (1985) Yeast 1, 83-138. 16. Hannig, E., Leibowitz, M. J. & Wickner, R. B. (1985) Yeast 1, 57-65. NEX. Also, NEX (carried on certain L species) can exclude 17. Herskowitz, I. (1985) Nature (London) 316, 678-679. M2 in the presence of certain chromosomal mutations such as 18. Riezman, H. (1985) Cell 40, 1001-1009.
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