0022-538X/91/010155-07$02.00/0
Copyright © 1991, American Society for Microbiology
Expression of
Yeast L-A
Double-Stranded RNA Virus
Proteins
Produces Derepressed Replication: a ski- Phenocopy
REED B.WICKNER,* TATEO ICHO,t TSUTOMU FUJIMURA, ANDWILLIAM R. WIDNER Section on the Genetics ofSimple Eukaryotes, Laboratory ofBiochemicalPharmacology, National Instituteof
Diabetes andDigestive
andKidney
Diseases,
Bethesda,
Maryland
20892Received 15 August 1990/Accepted 14 October 1990
The plus strand of the L-A double-stranded RNA virus ofSaccharomyces cerevisiae has two large open reading frames, ORF1, which encodes the major coat protein, and ORF2, which encodes a single-stranded RNA-binding protein having a sequence diagnostic of viral RNA-dependent RNA polymerases. ORF2 is expressed onlyasaGag-Pol-type fusion protein with ORF1. We have constructed a plasmid which expresses theseproteins from the yeast PGK1 promoter. We show thatthisplasmidcansupportthereplication ofthe killer toxin-encoding M1 satellite virus in the absence of an L-A double-stranded RNA helper virus itself. This requires ORF2 expression, providing a potential in vivo assay for the RNA polymerase and single-stranded RNA-binding activities of thefusion protein determined by ORF2. ORF1 expression, like a host si- mutation, cansuppressthe usual requirement ofM1 for the MAKI], MAK18, and MAK27 genes and allow a defectiveL-A (L-A-E) to support M1 replication. These results suggest that expression of ORF1 from the vector makes the cellaski-phenocopy. Indeed, expression of ORF1 in awild-type killer makes it a superkiller, suggesting that atargetof the SKI antiviral system may be the
major
coatprotein.Saccharomyces cerevisiae carries three double-stranded
RNA(dsRNA) viruses (35, 36), several retroviruses (2), and a single-stranded circular RNA virus (21a). The plus strand
ofthe 4.5-kb L-A dsRNAvirushas two open readingframes (19). The 5' open readingframe ORF1 encodes the 80-kDa
majorcoat protein (5, 18, 19), and acombination of ORF1 and the 3' ORF2 encodes the 180-kDa Gag-Pol fusion protein
(Fig.1) (13, 19). TheC-terminaldomain of the fusion protein has single-strandedRNA(ssRNA)-binding activity (13) and an amino acid sequence pattern diagnostic ofviral
RNA-dependentRNApolymerases (19). Within the 130-bp
over-lap of ORF1 and ORF2 is a "slippery site" at which ribosomal frameshifting occurs (5a, 19) by a mechanism
similar or identical to that described for retroviruses in
forming their Gag-Pol fusion proteins(17, 20,21).
Inaddition toprovidingthese proteins for its own
repli-cation, L-A can support one of several satellite dsRNA viruses, called M1, M2, etc., each of which encodes a secretedprotein toxinandimmunitytothattoxin(reviewed
inreferences 3and36). L-Acanalso support thereplication ofsmallerdeletionmutantsderived eitherfromL-Aitselfor
from M1 (8, 9). While L-A particles contain only one L-A dsRNA molecule per particle, those containing a satellite
genome(Moradeletionmutantof M or L-A)containone to
eightdsRNA molecules perparticle. Ineach case, the plus strand is the species packaged to form new particles and
replication occurs untiltheparticle is full("head-full repli-cation"). Thennew plus strands are all extruded from the
particles (6, 8, 10-12).
The yeast SKI genes are an antiviral system that are essential for hostgrowthathighand low temperaturesonly in the presence ofM dsRNA (7, 23, 24, 26, 27). The SKI
proteins repress replication of M1, M2, L-A, and L-BC
*Correspondingauthor.
tPresent address:TokyoMedicaland DentalUniversity, Tokyo, Japan.
dsRNAs(1,24,27)aswellasthesingle-stranded circular 20S
RNAvirus ofyeast (21a).
Theinteractions ofL-Aandits naturalvariantswiththe M
satellitegenomeshavebeenstudiedgenetically.Avariant of
L-A(L-A-E)unabletosupportM1orM2in awild-typehost has been previously described (24, 25, 33). L-A-E can, however, support M in a ski- host. This defect can be
complementedfromanormal L-A,andthisactivity is called [HOK] (helper of killer) orjust H, as in L-A-H (24, 39). [NEX](orN) is another L-Atrait(15, 33,34).
Replication or maintenance of M dsRNA requires the
products ofatleast 30 host genes, called MAK(maintenance ofkiller) genes (31, 32, 37, 38). Ofthese genes, onlythree, MAK3,
MAKIO,
andPET18,areneeded forL-Areplication
(25, 39). Certain variants of L-A can bypass M1's usual requirement formanyoftheMAKgeneproducts. This trait
is called [B] (bypass), andL-As carrying it are designated
L-A-B,orL-A-HNBif theyhave[HOK] and [NEX] activity
aswell (1, 27, 30). It wasfrom suchan L-A-HNB thatwe
isolated our cDNA clone (19). M1's requirement for most MAK genes is also suppressed inaski- host (29).
In ordertodissect the molecular mechanisms of
expres-sion of L-A-encoded information, L-A replication, and the L-Agenetics,wehaveconstructedanL-Aexpressionvector andstudied itsbiologicalactivities. This workhas ledus to the hypothesisthat onetarget ofthe antiviral SKI genes is the L-Amajor coatprotein.
MATERIALS AND METHODS
Media and strains.Themedia used forgrowthof yeast and assayofthe killerphenomenonwereasdescribedelsewhere (31). The yeast strains used are listed in Table 1.Escherichia coliHB101wasthe host formost
plasmid
manipulations.
E. coli CJ236 (F+ dut ung) and MV1190 (F+dut+
ung+),
obtained from Bio-Rad, were used for
oligonucleotide-di-rected, site-specificmutagenesis
byfollowing
the Bio-RadMutagene kit instructions.
Construction of mutable L-A expression vector.
pTIL05
155
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156 WICKNER ET AL.
2072
1939 /
1
JAA
-1 ribosomal frameshifting site
Encapsidation Signal
4546
IRE
\
r
s4579
site for replication
ORF2
UAA80 kDa_
major coat protein 180 kDa
coat protein
domain
'W
RNA
RNA
binding
polymerase
SG...T...NT..N -- GDD
FIG. 1. Expression of codinginformation fromL-Aplus strands (modified from reference 19). ORF1 encodes the majorcoatprotein. ORF1andORF2 arefused by ribosomalframeshiftingtoencodethe180-kDa minor viralcoatprotein. IRE, Internal replication enhancer.
(19) is the Bluescript vector SK+ with the entire L-A sequence (except for 8 bp at the 3' end thataredownstream of the coding region) inserted in the SmaI site. p375 is a
derivative ofYEpIPT(16)into which amultiple cloning site has beeninsertedintheunique EcoRI site(15a). In order to carryoutlocalized mutagenesis, weinserted the fl ori on a
0.52-kb Sall fragmentfrompDM1 (22)intotheunique Sall sitein the tetgene ofp375, forming
p12.
The L-Asequence on anXhoI-SstlfragmentfrompTIL05
wasinsertedbetweentheXhoIand SstIsites of p375andofp12, forming
pTIL131
(pORF1+2) and pI2L2,respectively. FrompTIL131 aNotI
[image:2.612.159.476.80.247.2]fragmentthat included
nearly
allof ORF2(from
base 2580ofTABLE 1. Strains andplasmids
Genotypeordescription
Strains 2928
2604
2966 (2604H-K-) 4219-2A
4219-2C 2955rho°
2128 1074
1101
1368
2629
3012 2057 1717 1720
Plasmids pTIL05 p375
pTIL131(pORF1+2) pDM1
p12 pI2L2
pTIL141 (pORFl)
M10(pORFloc+2) Mll (pORF1+2oc)
M2(pORFlfus2)
aura3his3trpl GAL'L-A-HN
Ml
atrpl leu2his3pho3pho5L-A-HN
Ml
atrpl Ieu2his3pho3phoSL-A-o M-o atrplIeu2ski2-2L-A-EMl
atrplhis3ski2-2L-A-EMl
a
trpi
his3adelmaklO-I L-A-oM-o[rho°]aura3-52trplcdcl6-1makil-l L-A-HNM-o aleukarl-i L-A-HN
Ml
ahis4-15 karl-I L-A-HN
Ml
ahis4-lS karl-i L-A-HNB
Ml
aleul karl-i L-A-HNBM1a hismetl4trpl ura3makl8-1L-A-HN M-o aura3trpl leu2rnal mak27-1L-A-HN M-o aaro7
1eu2
trpl uralmak3-1 L-o M-o aaro7leu2trpllys2 mak3-1 L-o M-oEntireL-A-HNB cDNA(except8basesat3' end)in theSmaIsite ofBluescript
vectorSK+
Yeastexpression plasmid withPGKI promoter,TRPI,2,umDNAorigin, pBR322,
andamultiple cloningsite
XhoI-SstIfragmentfrompTIL05 carrying entireL-Ainsertligated into
XhoI-SstI-cutp375; L-A'sORF1and ORF2 areintact
pAT153with a0.52-kb fragment carryingthefl oriintheSail site p375withflori on aSaiIfragment frompDM1
XhoI-SstIfragmentfrompTIL05 carrying entireL-Ainsertligatedinto
XhoI-SstI-cut12;L-A'sORF1andORF2areintact
pTIL131 fromwhich the2.0-kbNotIfragment(includingmostof ORF2)has been
removed
Ochremutationataminoacid 20ofORF1;frompI2L2
Ochre mutationin ORF2 atthird codonafter theend ofORF1;derived from pI2L2 ORF1and ORF2placed in frame by addition ofan Aresidueatthesite ofthe
ribosomal frameshift(Sa);derived frompI2L2
AUG
ORF1
Strainorplasmid Reference
Thiswork Thiswork Thiswork This work This work This work
30 30 30 30 30 32 38 37 37
19
16
Thiswork
22
This work This work This work This work Thiswork This work
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[image:2.612.67.564.398.720.2]L-A
Yeast
PGK
ORF1
Promoter
Major
Coat
Prc
RNA polymerase consensus
[image:3.612.64.555.72.194.2]EcoRV EcoRI
FIG. 2. Plasmid forexpressingL-A-encoded proteinsinS. cerevisiae.
the L-A plus strand to the 3' end) was removed. The
remaining plasmid was ligated to form pTIL141 (pORF1). M2, M10, and Mll were constructed by
oligonucleotide-directed localized mutagenesis of pI2L2 (Table 1).
Testing for suppression of mak mutations. Plasmids were
introduced into trpl mak hosts, and then L-A andM1 were
introduced by cytoduction using the karlmutantdefective in nuclear fusion (4). Whenakarl strainmates,binucleatecells
carrying the cytoplasmic elements (viruses, mitochondria, etc.) of bothparentsareformed. The nuclei failtofuse and separate at the next cell division. When a karl strain
carrying L-A, M1, and mitochondrial DNA
([rho'])
is used to mate with a mak M-o[rho']
strain, the net effect is to transfer cytoplasmic elements from the karl parent (called thedonor)tothe makparent(called the recipient). Cytoduc-tions were performed as described previously (39), except thatcytoduction mixtureswereplatedon media thatsimul-taneously selected for retention of the plasmid and against the cytoduction donor.
RESULTS
L-A expression vector. The expression vector, p375, has the PGK1 promoter, amultiple cloning site, the 2,um DNA
replication origin, the yeast TRP1 gene, and pBR322
se-quences. Inordertobe abletodo site-directed mutagenesis and express the mutated L-A sequence in yeast cells, we
insertedthe florigin ofreplication into theyeastexpression vector p375 (Fig. 2). The L-A sequence was inserted
be-tween the PGK1 promoterand the 2,um DNA terminatorto make pORF1+2 (pTIL131).
[HOK] activity requires only ORF1. To test whether the L-A information was being expressed, pORF1+2 was put intoaSKI+ L-o strain, and the transformantswerecrossed
withaski2-2 L-A-E M1 strain (Table 2). The diploids formed wereSKI+ andwould have lost M1 if L-A information had
notbeenexpressed from the clone, since L-A-E is defective in itsabilitytomaintain M1. However, thediploidswere all
stable killersaslongasselection for theexpression plasmid
pORF1+2wasmaintained. Allowing the plasmidtobe lost by growth onnonselective medium showed that loss of the
L-A expression plasmid resulted in the loss ofM1. Thus, pORF1+2 has[HOK] activity, the abilitytoprovideM1with the helper function that is deficient in the natural variant L-A-E. Thesameresultwasobtained whenpORF1wasused instead of pORF1+2, indicating that the ssRNA-binding RNA polymerase domain (ORF2) is not necessary for [HOK]. Whenweintroducedanochre mutationinplaceof
amino acid 20 ofORF1,theresulting plasmid (pORFloc+2) had no [HOK] activity, indicating that the protein encoded
byORF1 and notjustthe RNA wasessential for[HOK]. As
expected, introducinganochre mutation in ORF2 inplace of thethird aminoacidafter the endofORF1(pORF1+2oc)did notinterfere with [HOK]activity.
[B] activity requires onlypORF1.Certain isolates ofL-A,
called L-A-HNB and includingthatfrom which our cDNA clone was isolated, make several MAK genes, such as MAKI
J,
dispensableforM1 replicationor maintenance(30). When pORF1+2, pORF1, and p375 (vector alone) weretransformed intoamakll-l strain andL-A-HN and M1 were then introduced by cytoduction, the same pattern as for [HOK] activity was seen, namely, only ORF1 was needed for[B] activityandORF1 RNA was not sufficient; an ochre
mutation earlyinORF1 prevented expressionof[B]fromthe vector(Table 3).
pORFi
suppresses makl8 and mak27 mutations. While many mak mutations are suppressed by L-A-HNB (1), mutations in makl8and mak27 are among those which are notsuppressed.Wefound, however, thatexpression ofL-Aproteins from pORF1+2 or from pORF1 suppress both
makl8and mak27mutations(Table 4). These results could
suggest that the MAK18andMAK27productsareconcerned
with the expressionofL-Aproteins, particularly the major
coatprotein encodedby ORF1. Alternatively, since makl8 andmak27mutationsaresuppressed by ski mutations,these results could mean thatexpression of pORF1makes thecells
aski- phenocopy.
pORFl makes cells superkillers. Introduction of
pORFi
into the wild-type killer strains 2604 and 2928 made them
TABLE 2. [HOK] requires onlyORF1 proteina
Characteristics ofdiploids formed bycrossing with: Plasmid Insert
4219-2A(ski2-2 4219-2C(ski2-2
L-A-EM1) L-A-EM1)
None None All K- All
K-p375 None All K- All
K-pTIL131 ORF1 + ORF2 AllK+ AllK+
pTIL141 ORF1 All K+ AllK+
M1o ORFloc + ORF2 All K- All
K-M11 ORF1 + ORF2oc All K+ All K+
a Plasmids weretransformedintostrain2966(L-A-oM-o),and transfor-mants werematedwith the ski2-2L-A-EM1 strains.Findingthatalldiploids
werekillers(K+) indicatedthat theplasmidwasexpressing[HOK] activity.
Atleast 10diploidsinglecolonieswereisolatedonplateslacking Trp(-Trp)
to ensureretentionoftheplasmidand testedfor K1. SubcloningonYPAD resultedinfrequentlossoftheexpressionplasmidandconcomitantloss of
M1. Subcloning on -Trp plates ofdiploids carrying pTIL131 or pTIL141
yielded only K+single colonies. K-, Nonkiller.
Yeast
pBR322O0l
fl
Jl11
BgI 11/
1
BamHI
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[image:3.612.317.559.552.655.2]158 WICKNER ET AL.
TABLE 3. [B] requires ORF1only
Donor charac- Recipient
characteristicsb
Characteristics of No. ofcyto-teristics' cytoductants ductants
L-A-HN M1 makll-l L-A-HN All K- >500
L-A-HNBM1 makll-l L-A-HN AllK+ 15
L-A-HN M1 makll-lL-A-HNp375(vector) All K- >30
L-A-HN M1 makll-l L-A-HNpORF1+ORF2 AllK+c 20
L-A-HNBM1 makll-l L-A-HNpORF1+ORF2 AllK+ >15
L-A-HNM1 makll-l L-A-HNpORF1 AllK+c 8
L-A-HNBM1 makll-l L-A-HNpORF1 AllK+ >15
L-A-HNM1 makll-l L-A-HNpORF1+2oc AllK+ >20
L-A-HNM1 makll-lL-A-HNpORFloc+2 All K- >20
aCytoductiondonors werestrains 1101 (L-A-HNM1)and 1368(L-A-HNBM1).
bCytoduction recipients werestrain2128 (makll -I L-A-HN M-o) carrying one of the L-A expression plasmids. Cytoductions were done on SD agar containing His and uracil to keep in the L-Aexpression plasmid and plated on SD agar containing uracil to select against donor cells and retain the plasmid.
cSubcloning these cytoductants on -Trp plates to retain the plasmid produced onlyK+ subclones. Subcloning on YPAD to allow loss of the L-A expression plasmid produced only K- and a few weak killer (KW) subclones.
superkillers, as judged by their zone of killing of a sensitive
strain at 30°C. In contrast, the vector p375 or the vector
pORFloc+2, which has an ochre mutation early in ORF1, had no such effect.
maklO-1, but not mak3-1, is suppressed bypORF1+2. The
MAK3, MAKIO, and PET18 genes are needed for L-A
replication(25),although Toh-e and Sahashi (28) have shown thatPET18 is needed only at temperatures above 30°C. The lossof M1bymaklO-1 strains wassuppressed by pORF1+2, asshown by both meiotic crosses (Table 5) and cytoduction
experiments (Table 6). However, pORFi did not detectably suppress
maklO-J.
Todetermine whether pORF1+2 allowed replication in the maklO-J host of the L-A virus itself, themaklO-J
pORF1+2 M1 strains (Table 6) were mated witheitheroftwotrplmak3 L-A-ostrains(1717 and 1720) and the
diploids were examined to determine whether they were
killers beforeand after mitotic loss of pORF1+2. Since the mak3 and
makJO
mutations complement each other, and since mak3 mutants lack L-A, the diploids, after loss ofpORF1+2, are expected to be killers only if pORF1+2
allows replicationof L-A in the mak/O host. In each case, all
TABLE 4. Suppression of mak27-1 andmakl8-1 by pORF1orpORF1+2
Donor Recipient Cytoductant
charactenisticsReiinCyoutt
(karaet)a
characteristicsb characteristicsL-A-HNM1 mak27-1 All
K-L-A-HNB M1 mak27-1 All
K-L-A-HNM1 mak27-1 pORF1+2 AllK+c
L-A-HNB M1 mak27-1 pORF1+2 AllK+
L-A-HNM1
mak27-1
pORF1 AllK+cL-A-HNB M1 mak27-1 pORF1 AllK+
L-A-HNM1
mak18-1
AllK-L-A-HNM1 makl8-1 All
K-L-A-HNM1 mak18-1 p375 All
K-L-A-HNM1 makl8-1 pORF1 AllK+c
L-A-HNM1 makl8-1 pORF1+2 AllK+c
aCytoductiondonorswere strains 1101 and 1074(L-A-HNM1)and strains 1368and2629(L-A-HNBM1).
bThemak27-1 recipient was strain 2057, and themakl8-1 recipient was
strain 3012.
cFourcytoductants from each of these cytoductions were subcloned on -Trp plates, and all were stably K+. Subcloning the same cytoductants on YPAD plates showed that all Trp- mitotic segregants had become K-,
showing that thesuppression of mak27-1 or ofmakl8-1 depended on the L-A
expressionplasmid.
diploidswerestable killers whilepORF1+2 was present and
nonkillers when pORF1+2 was lost. The same result was obtained by examination of isolated viralparticles for L-A dsRNAby Northern(RNA) blot hybridization (Fig. 3). This shows that pORF1+2 did not allow the replication of L-A-HN in the the makJO background and suggests that MAKIO is needed for thereplication or maintenance of L-A
independent of the supply of L-A-encoded proteins. Even
thoughtheproteinsarebeing supplied in amounts sufficient
for M1,L-Acannotreplicate. This result also indicatesthat M1 does not need the L-A genome itself for its replication but needsonly the products ofL-A.Italsosuggeststhatthe MAKIOproductis not necessary for the translation ofL-A products. These results also show thatpORF1+2 can main-tain M1 in awild-type host. In contrast, crosses carriedout with mak3-1 andpORF1+2showed noevidenceof
suppres-sion(Table 5).
DISCUSSION
Wereport the establishment of an expression system for the information encodedbythe L-A dsRNA virus of yeast. That both open reading frames are in fact expressed from this clone is shown by its maintenance of M1 in a mak/O strain (or ina wild-type strain) in the absence of the L-A dsRNAgenome, an activity dependent onboth ORF1 and ORF2. This activity proves that ourclone ofL-A encodes
biologically active proteins. That our sequences of three
completeindependentcDNAclones ofL-A were, except for asinglenucleotide, incomplete agreement with each other also supportsthisconclusion (19).
Using this
expression
system, we havebeguntoexaminethe molecular basis of several phenomena we have previ-ously described. L-A-E can support M1onlyin a ski- host defective in the antiviral system. [HOK] is the ability of
many L-AisolatestosupplementL-A-EinsupportingM1 in a wild-type host. That [HOK] activity is expressed from pORF1+2 and
requires
only ORF1, which encodes themajorcoatprotein, indicates thatthe defect in L-A-E is in themajorcoatprotein and is eitherqualitative or
quantita-tive. L-A-E must,however, besupplyingthefusionprotein. Since [HOK] is needed only ina
SKI'
host, this suggests that onefunction ofthemajorcoat protein is to protectMl
(and presumablyL-Aaswell)from theSKIantiviralsystem.
[B] is the ability of certain L-A natural variants (called
L-A-HNB)tosupportM1incertainmak-hosts fromwhich
it wouldotherwisebelost. [B] suppresses many mak muta-J. VIROL.
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[image:4.612.64.304.515.651.2]TABLE 5. pORF1+2suppresses maklO-I but not mak3-1
Parents Segregationa No. of tetrads
1717(mak3-1 L-oM-o) x 4224-8B(pORF1+2 L-A-E M1) 2K-:2K+ 10
2956(maklO-IL-oM-o) x 4224-8B(pORF1+2L-A-E M1) 4K+:0 12
2957(maklO-IL-oM-o) x 4224-5A(pORF1+2L-A-EM1) 4K+:0 12
2604(L-A-HN M1) x 2955(pORF1+2) (maklO-I L-opORF1+2) 4K+:0 7
2604 (L-A-HNM1) x 2955(pTIL141) (maklO-I L-opORF1) 2K+:2K- 8
aAll crosses werecarriedout, diploids weresporulated,tetradsweredissected, andsporesweregerminatedin the absence oftryptophanin ordertoinsure thecontinuedpresenceof pTIL131.ThedependenceofM1in thesegregantson pTIL131wasdemonstrated by streaking each spore clone of threetetradsof the cross2957x4224-5Aoneither-Trpplatesor on YPADplates. Allthesingle colonies on-Trpplateswerekillers, whileallthoseonthe YPADplateswere either nonkillers-or weakkillers.
1 2 3 4 5 6 7 8 9 1011 121314 1 <
1 2 3 4 5 6 7 8 9 10111213 14 A
FIG. 3. Maintenance of M1 butnotL-A inamaklO-l strai pORF1+2. Viral particleswerepreparedaspreviously describe
8, 13, 14) fromstationary-phase cells of strain 2955(pORF1+2) whichL-A-HNBand M1 from strain 2629 had been cytoduced. final step in the purification was a CsCl gradient. RNAs
extracted from each fraction of the gradient and analyzed nondenaturingagarosegel. Ethidium bromide staining of the shown intheupperpanel. The theRNAsweredenatured in the blotted onto a nylon sheet, and hybridized with a 32P-lat
L-A-specific probe. An autoradiogram of the sheet is shown it lowerpanel. Lanes 1to14aretheCsCl gradientfractions, with 1beingthe bottomfractionandlane14being thetopfraction. L and L-A show A HindIII markers and purified L-A dsR respectively. The positions of L-A andMldsRNAsareshown andM1, respectively,ontheright. Note that therearetwotyp,
M1viralparticles (6);one,which containstwoM1 dsRNAmole(
perparticle(M1-H), formsabandatfraction4, and theother,w
containsoneM1dsRNA moleculeperparticle(Ml-L),formsa atfraction7. Theexpecteddensity ofmatureL-Aparticlesisth fraction3.The dsRNAwiththesamemobilityasthat of L-A
throughoutthegradientis L-BC.
tions but not makl8-1 or mak27-1. The expression of [B]
fromourcDNAclone requires only ORF1. Furthermore, the
same pORF1 suppresses at least two mak mutations not normally suppressed by [B] (Table 7). That pORF1 sup-pressesmutationsinmanyMAKgenesis consistentwith the notionthat thesegenes areconcerned with theefficiency of
L production ofthe majorcoat protein from L-A transcripts.
Alternatively, since these mak mutations are also those
which are suppressed by ski mutations, they could be
involved in
Ml's
defense against the SKI antiviralsystemorbeoneof the actualtargets of theSKIsystem.
Aunifying hypothesistoexplain the interactions of[HOK], [B], pORF1, MAKgenes, andSKIgenes. The SKIproducts reduce the copy numbers of M1, M2, L-A, and L-BC dsRNAs, and this function is essentialoratleastimportant for cell growth, depending on the temperature and other cytoplasmicelements in the strain(7, 24, 27). However,the precisetargetof SKIgeneaction isnotyetknown.L-A-E's defect in maintaining M1 is suppressed by skimutationsor
by supplying majorcoatprotein fromanormal L-A([HOK]
function)orfrom pORF1.This is consistent with the major
coatprotein beingthetarget of theSKI antiviral systemor
L with the coat
protein protecting
the actualtarget
(genomic
RNA?)from theSKIsystem. L-Ascarrying [B] makemany
MgI
MAKgenesdispensableforM1,and thesameL-A maintainsM1atahigher-than-normalcopynumber(30).Hereweshow thatpORF1 is sufficient for [B] and for the suppression of
twomak mutationsnotsuppressed by [B]. However,all the mak mutationssuppressedbypORF1aresuppressed by ski mutations. If the target of the SKI system were the major
coat protein, then its overproduction by pORFi might
swamp the SKI system, making the cell a skiphenocopy. in by
d(6,
into TABLE 6. L-A expression vectors allow Mlreplication
TheX11%, in amaklO-1 strain were
on a
gelis
gel, beled
nthe
ilane
,anes
tNA,
asL esof
cules vhich
band atof seen
Characteristics Characteristics ofrecipi- Killerphenotypeof
o onor
s(rain
entstrain2955rho°
cytoductantsaL-A-HNB M1 maklO-I All
K-L-A-HNB M1 maklO-Ip375 All
K-L-A-HNB M1 maklO-I pORF1+2 AllK+
L-A-HNB M1 maklO-I pORF1 All
K-L-A-HNB M1 maklO-I pORFlfus2 All
K-L-A-HNB M1 maklO-I pORFloc+2 All
K-L-A-HNB M1 maklO-I pORF1+2oc All
K-aCytoductionmixtureswereplatedonmediaselectingagainstthe
cyto-duction donorand for retentionofthe L-Aexpressionplasmid.Cloneswere
testedforgrowthonminimal medium(toscreenoutdiploids),forgrowthon
glycerolplates (toidentify clonesthat hadreceiveddonorcytoplasm),andfor
killingofasensitive strain. Atleast 10cytoductantsfromeachcytoduction
werescored.
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[image:5.612.55.296.209.537.2] [image:5.612.315.555.577.674.2]160 WICKNER ET AL.
TABLE 7. Comparison ofeffects of pORF1,pORF1+2, and askimutation
Suppression by:
Trait Definition
ski- pORFl pORF1+2
[B] Ability ofanL-A tomakeMAKI]dispensable forM1 replication + + +
[HOK] Ability ofan L-A tocomplementdefectiveL-A-E insupporting + + +
M1 inaSKI' host
makl8, mak27 LoseM1 but not L-A or L-BC + + +
maklO LoseM1and L-A but not L-BC - - +
mak3 LoseM1 andL-AbutnotL-BC
This would havethe effect of suppressing many mak
muta-tions andallowing L-A-E to supportM1 replication. Infact,introduction ofpORF1into awild-type killer strain
makes it a superkiller, as predicted by this model. This
finding is most easily explained ifthe major coatproteinis
the target or the first molecule recognized by the SKI
products.Supplying an excess of this target would titrate out the SKIproducts, allowing unrestrained replication ofM1, and allofthegenetic consequences ofthe cell'sbeing ski-would follow. L-A-B could be interpreted, in accord with
this model, as avariant ofL-A that produces more major
coat protein and as a result has a partial Ski- phenotype.
L-A-B both suppresses some (but not all) of the mak
mutations suppressed by ski mutations and produces a
partial superkiller phenotype (with larger killing zones and
higherM1 copynumber thanwild-type strains). An
alterna-tive model would have the excess of major coat protein providingbetterprotection of M1from the SKI products.
pORFl+2support of M1 in a maklO-1 host provides an in vivo assay for ORF2 functions. pORF1+2 supported M1
replication inamaklO-1 host. Both genetic andbiochemical
testsshowed that L-A was lost from the strain.Subsequently making the strain MAK+ by mating it with a mak3 strain produced wild-type cellsinwhich pORF1+2 was
maintain-ing M1intheabsence ofL-A.Thisindicates thatM1depends on the products ofL-A for its replication but not on the presence ofa replicating L-A genome. Moreover, M1 does not
directly
require the MAKIOproduct forits replication;MAKIO is needed only because L-A products are needed and L-A needs MAKIO. SincepORF1 didnot supportM1 in
a
maklO-J
host, M1 needs both ORF1 and ORF2. An ochremutation early in ORF1 andan ochre mutationinORF2just afterthe endof ORF1 both eliminate theability ofpORF1+2 tomaintain M1. Thisprovides asimpleinvivoassayforthe
activity ofORF2 whichcanbe used tostudy the roleofthe RNApolymeraseconsensus sequence in ORF2 (19) and the
ssRNA-binding activityof ORF2 (13).
That pORF1+2 allowsM1, but not L-A, to replicate in a
maklO host points again to the distinction between the
mechanisms of replicationof the L-A replicon and those of its satellites, M1, M2, and X (a deletion mutant of L-A).
Biochemical studies indicate that both follow the same
replicationcycle, except for the head-full replication mech-anismshown by the smaller replicons, the fact that
M1
and X require many MAK genes not needed by L-A, and the fact that M1 and X must borrow coat proteins synthesized by L-A. pORF1+2also supportsM1
in awild-type host (in theabsence of L-A), showing that this is not a phenomenon
peculiarto maklO strains. Previous studies of L-A
replica-tion in a
maklOfs
host showed that L-A dsRNA-containing particleswereunstable, although L-A ssRNA particles wereapparently unaffected(11). Ifthat difference were due to the L-A dsRNA-containing particles being full while the L-A
ssRNA-containing particles were not, then the present re-sults would notbe unexpected. This is consistent with the MAKIOproduct being essential for the stability of full viral
particles but not for that ofparticles that are only partially
filled.
WhilepORF1+2 supported M1 in a maklO host, it didnot do so in a mak3 host. This suggests that the MAK3product
is more directly needed for M1 replication, not simply
through itsbeingneeded for L-A as in the case ofMAKIO.
The availability of a complete, expressed clone of L-A makes possible the detailed dissection of the role of L-A-encodedproteinsin thereplicationprocessandin interaction with satellite genomes and with the host. Since viral plus
strands are thespeciespackaged to make new viral particles, plus strands made from the vector with the correct ends should replicate. This "launching" of L-A from the vector should allow its use as anRNAvirusexpressionvector.
ACKNOWLEDGMENTS
WethankRonHitzeman and Genentech forgenerouslyproviding thep375vector.
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![TABLE 3. [B] requires ORF1 only](https://thumb-us.123doks.com/thumbv2/123dok_us/1317393.85272/4.612.64.304.515.651/table-b-requires-orf-only.webp)
