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Viruses in fungi Infection of yeast with the K1 and K2 killer viruses

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Viruses in fungi Infection of yeast with the K1 and K2 killer viruses Powered By Docstoc
					   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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