From the Laboratory of Biochemistry and Genetics, NIDDK, National Institutes of Health, Bethesda, Maryland 20892-0830
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ABSTRACT |
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The L-A double-stranded RNA virus of yeast
encodes its major coat protein, Gag, and a Gag-Pol fusion protein made
by a 1 ribosomal frameshift, a coding strategy used by many
retroviruses. We find that cells expressing only Gag from one plasmid
and only Gag-Pol (in frame) from a separate plasmid can support the
propagation of M1 double-stranded RNA, encoding the
killer toxin. We use this system to separately investigate the
functions of Gag and the Gag part of Gag-Pol. L-A contains two fusion
protein molecules per particle, and although N-terminal acetylation of
Gag is essential for viral assembly, it is completely dispensable for
function of Gag-Pol. In general, the requirements on Gag for viral
assembly and propagation are more stringent than on the Gag part of
Gag-Pol. Finally, we directly show that it is Gag that instructs the
incorporation of Gag-Pol into the viral particles.
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INTRODUCTION |
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The L-A dsRNA1 virus of
Saccharomyces cerevisiae closely resembles dsRNA viruses of
animals and plants both structurally and in its replication cycle
(reviewed in Refs. 1, 2). L-A is an icosahedral T = 1 virus with
an assymetric unit consisting of a dimer of the major coat protein, Gag
(3, 4), a structure similar to the cores of other dsRNA viruses (5-8).
L-A has a 4.6-kilobase single segment genome encoding its major coat
protein, Gag, and an RNA-dependent RNA polymerase, Pol,
synthesized as a Gag-Pol fusion protein formed by a 1 ribosomal
frameshift (9, 10). This structure has proven typical of a large group
of viruses of fungi, parasitic microorganisms and plants, the
Totiviridae. A satellite dsRNA, M1, encodes a
polypeptide killer toxin lethal to strains not carrying M1
and is useful as a phenotype for following L-A functions
genetically.
The coding strategy of L-A suggested both a parallel with retroviruses, and a mechanism of virus assembly and packaging (9, 11). It was proposed that the fusion protein was incorporated into the viral particles by the association of the Gag part of the Gag-Pol fusion protein, with free Gag molecules. Because the Pol domain of the fusion protein bound single-stranded RNA, it was suggested that this association led also to packaging of the viral (+) strands. It was shown that most of Pol was dispensable for incorporation of Gag-Pol into viral particles but that the N terminus of Pol contains the domain necessary for packaging viral RNA (12, 13). However, it was impossible to test whether part or all of Gag of the Gag-Pol fusion protein was necessary for incorporation of Gag-Pol into viral particles without changing both Gag itself and the Gag part of the fusion protein.
The ribosomal frameshift site that forms the Gag-Pol fusion is not at the C terminus of Gag but lies 35 amino acids upstream from that point, so that the Gag part of Gag-Pol lacks these C-terminal 35 residues present in Gag. For the same reason, the most N-terminal part of the Pol ORF is encoded by the same sequence as is the C-terminal 35 residues of Gag. Its function could also not be tested without simultaneous alterations of Gag. The Gag protein is sufficient to form viral particles (12), but what parts of Gag are essential has not been defined. The N termini of Gag and Gag-Pol are acetylated by Mak3p, a modification necessary for viral assembly (14-16), but whether both Gag and Gag-Pol need to be acetylated or whether myristoylation can substitute for acetylation was not known.
In this work, we address the functions of Gag, the Gag part of the fusion protein, and the part of Pol that overlaps Gag by expressing Gag and Gag-Pol (and their mutants) separately and using this combination to support the M1 satellite dsRNA encoding the killer toxin.
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MATERIALS AND METHODS |
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Strains--
S. cerevisiae strains JR3 (MATa
ura3 his3 trp1 L-A-o L-BC), JR5 pI2L2 K+ (MAT
kar1 ura2 leu2 trp1 L-A-o pI2L2 M1), JR8
(JR3
o), JR13 (MATa trp1 ura3 leu2 his3
pep4::HIS3 nuc1::LEU2 L-A-o L-BC-o), and 5x47
(MATa/MAT
his1/+ trp1/+ ura3/+ M-o) were used. Escherichia coli strains MV1190 and CJ236 (Bio-Rad) and
DH5
F'IQ (Life Technologies, Inc.) were used.
Plasmids and DNA Techniques-- The L-A cDNA expression plasmids pI2L2 (TRP1 selection, PGK1 promoter (17)) and pJR63 (HindIII-BamHI cDNA fragment from pI2L2 ligated into the S. cerevisiae expression plasmid pVT101U (URA3 selection, ADH1 promoter (18)) were used. pM2 is pI2L2 with an A inserted at the frameshift site of L-A to express only the fusion protein Gag-Pol (17). pJR99 is the HindIII-BamHI L-A cDNA fragment from pM2 inserted into pVT101U cut with the same enzymes. pJR13 is pI2L2 with the change in the L-A slippery site 1958-GGGTTT-1963 to 1958-AGGTTT-1963 that essentially abolishes frameshifting, makes no change in the amino acid sequence of Gag, and produces no detectable Gag-Pol (10). pJR133 is pJR13 cut with NotI in the L-A region and with BamHI, blunt-ended and religated, avoiding any residual expression of full-length Gag-Pol. pJR96 is the HindIII-SnaBI L-A fragment (deleting Pol) from pJR13 inserted into pVT101U cut with HindIII and PvuII to express only Gag.
pTF143 is pI2L2 cut with SalI and BamHI and is ligated to make an intact Gag and a truncated Gag-Pol. pJR111 is pJR99 cut with SalI and BamHI and ligated to make a truncated Gag-Pol. pJR139 contains the full sequence of Gag and the full sequence of Pol in frame (the normal Gag-Pol sequence lacks the last 35 amino acids of Gag). It was made by inserting a XhoI site in pJR99 before the Pol sequence to make pJR134 and inserting a XhoI site in pJR96 after the end of Gag to make pJR135. pJR136 contained the large HindIII-XhoI fragment of pJR134 (including vector and Pol sequences) and the HindIII-XhoI Gag fragment of pJR135. XhoI was removed, and Gag and Pol were put in frame by site-directed mutagenesis (SDM) of pJR136 to make pJR139. pJR138 contained the entire Gag sequence and the Pol sequence lacking its first 35 amino acids and was made by SDM of pJR13. pJR146 has the ADH1 promoter-X cDNA sequence-ADH1 terminator fragment from pJR58 (13) inserted in pLitmus38 (New England Biolabs) cut with SphI. pJR147 has the ADH1 promoter-X cDNA sequence-ADH1 terminator on a SalI-EagI fragment from pJR146 inserted into pRS423 (HIS3 selection (19)) cut with SalI and EagI. pJR98 is pJR63 cut with PvuII and BamHI and religated to produce a C-terminal deletion of 27 amino acids in Gag. pJR143, pJR148, pJR149, pJR177, pJR176, pJR145, pJR152, and pJR153 were made by SDM introducing termination codons into the Gag sequence of pJR96 to make C-terminal deletions of Gag of 5, 10, 15, 33, 35, 37, 41, and 45 amino acids, respectively. pJR113 is pJR63 cut with XhoI (inserted at amino acid 627 of Gag, pJR105) and BamHI and religated producing a C-terminal deletion of 52 amino acids in Gag. pJR150 and pJR151 are pJR96 with N-terminal deletions created by SDM of amino acids 9-13 and 9-18 of the Gag sequence, respectively. pJR156, pJR157, pJR158, pJR169, and pJR175 are pJR99 with C-terminal deletions created by SDM removing residues 644-645, 641-645, 636-645, 536-645, and 436-645, respectively, of the Gag part of Gag-Pol. pJR165 and pJR166 contain the Gag-Pol sequence with N-terminal deletion of amino acids 9-13 and 9-18, respectively, by cutting pJR150 and pJR151 with ClaI and BamHI and introducing the corresponding ClaI-BamHI fragment from pJR99. pJR174 is pJR99 with the N-terminal deletion of Gag-Pol created by SDM of amino acids 9-118. pJR161 and pJR162 are pJR96 with the N-terminal substitutions MLRFPreparation of Particles Made in Yeast from an L-A cDNA
Clone--
Viral particles from strains JR3 or JR13, harboring
different L-A constructs, were prepared from CsCl gradients by a
modification of our method (13). Stationary phase cells from a 1-liter
culture were suspended in 1.6 ml/g (wet weight) of 100 mM
Tris-HCl, pH 7.6, 20 mM -mercaptoethanol, 1.4 M sorbitol, and 6 mg/ml zymolyase 20T, incubated for 55-60
min at 37 °C, and collected by centrifugation at 1500 × g for 20 min. Cells were suspended in 30 ml of buffer A (50 mM Tris-HCl, pH 7.6, 150 mM NaCl, 10 mM EDTA, 10 mM
-mercaptoethanol) and lysed
by passage through a French pressure cell. Cell debris was removed by
centrifugation at 13,000 × g for 20 min, and a sample
was taken as "cell extract" (see figures). The supernatant was
centrifuged at 100,000 × g for 90 min. The pellet was
resuspended in buffer A, clarified, and made 1.32 g/ml with CsCl (for
empty particles from strain JR13) or 1.35 (for M1 dsRNA
containing particles from strain JR3) in a total volume of 13 ml. 18 fractions of 0.5-0.6 ml were collected.
Electrophoresis and Western Blot Analysis of Viral Proteins-- Fractions were diluted four times in SDS loading buffer, boiled for 4 min, and 4 µl (empty particles) or 20 µl (M1 particles) were analyzed by SDS-7.5% polyacrylamide gel electrophoresis and Western blotting. The upper part of the membrane was incubated with rabbit polyclonal anti-Pol and the lower part with mouse monoclonal anti-Gag antiserum as described (13). Detection was by an alkaline phosphatase-conjugated second antibody (Promega).
In Vivo Killer Assay of Altered L-A Expression Vectors-- Cytoduction (transfer of cytoplasm from donor to recipient strain without changing the nuclear genotype (24)) and the killer assay were done as described (13, 25). M1 dsRNA was introduced from the donor strain JR5 pI2L2 K+ to the recipient strain JR8, expressing Gag and Gag-Pol (or their mutant forms).
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RESULTS |
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Gag and Gag-Pol, Expressed Independently, Make Functional
Virus--
When Gag and Gag-Pol are translated from the mRNA of
L-A, the fusion protein is expressed at about 2% the efficiency of Gag (10). However, expressing Gag and Gag-Pol from two independent clones
(pJR96 + pM2, Fig. 1A,
sample 4), produced particles with roughly the same ratio of Gag
to Gag-Pol as was produced from the intact L-A clone (pI2L2, Fig.
1A, sample 1) or is found in the L-A virus (data
not shown). As previously shown (12), Gag alone was able to make
particles (Fig. 1A, sample 2), but Gag-Pol did
not (Fig. 1A, sample 3). For unknown reasons, the
level of Gag-Pol (and its mutants) was only 3-4-fold higher when
expressed from these clones than when expressed by the 1
frameshifting mechanism (see cell extract of Fig. 1A,
samples 3 and 4, and below). The separately
expressed Gag and Gag-Pol made virus particles containing the fusion
protein, and these particles could stably maintain the killer
toxin-encoding M1 satellite dsRNA when M1 was
introduced by cytoduction (Fig. 1C, sample 4) and
showed the same profile of M1-containing particles in CsCl
gradients as that from an L-A clone (Fig. 1B). A higher
Gag-Pol ratio achieved by expressing Gag-Pol alone from one plasmid and
both Gag and Gag-Pol (by frameshifting) from the normal L-A clone also
stably maintained M1 dsRNA (data not shown). A very low
Gag-Pol:Gag ratio supplied by pJR13, mutated in the frameshift
sequence, could propagate M1 dsRNA at very low copy number,
with just detectable killer activity (data not shown). In strain JR3,
this very weak activity was stably propagated, but it was unstable in
host strain JR13. The fusion protein was undetectable on CsCl gradients
of particles (data not shown). In the experiments described below, Gag
(and its mutant derivatives) was provided from pJR13 derivatives that had been deleted for Pol sequences.
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The L-A Virus Contains at Least Two Fusion Proteins per Particle-- When a fusion protein, truncated at residue 414 of the Pol domain, is expressed, even though in 3-4-fold excess, together with the full-length L-A, the former does not interfere with the ability of the latter to propagate M1 stably. The truncated Gag-Pol is nonfunctional, lacking the RNA-dependent RNA polymerase consensus regions known to be essential for propagation of M1 (25). We examined M1 dsRNA containing viral particles in two strains: one with a full-length L-A clone and a truncated Gag-Pol clone (Fig. 2A) and the other with a truncated L-A clone (that makes Gag and a truncated Gag-Pol) and a full-length Gag-Pol clone (Fig. 2B). Because of its smaller size, the truncated Gag-Pol can be distinguished on Western blots from the full-length Gag-Pol. CsCl gradients of M1-containing particles from both strains showed peaks of empty particles, light particles (one M1 dsRNA molecule per particle), and heavy particles (two M1 dsRNA molecules per particle). In both strains, the truncated Gag-Pol was incorporated into the fractions of M1 dsRNA-containing particles. Each particle with the truncated Gag-Pol that has M1 dsRNA must also have a second (functional) Gag-Pol. The only qualification of this experiment is that the deleted part of Gag-Pol could be the part that identifies the molecule as Gag-Pol to the assembling virus. However, if this were the case, higher amounts of the truncated fusion protein should be found in virions as it would be incorporated as a Gag, rather than as a Gag-Pol. In fact, the particles formed in cells with excess truncated fusion protein have no more truncated than full-length fusion protein (Fig. 2A), whereas the cells making excess full-length fusion protein incorporate it more efficiently into particles than the truncated protein (Fig. 2B). X dsRNA, a deletion mutant of L-A dsRNA, is propagated by L-A virus-encoded proteins and is found in particles containing from one to eight X molecules per particle (26). When X is supported by a full-length L-A clone and a truncated Gag-Pol, the truncated protein is found in all fractions containing virus particles but always in amounts equal to or less than the full-length fusion protein, supporting the results above (data not shown).
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Why Does Gag-Pol Lack the Last 35 Amino Acids of Gag?-- A Gag-Pol with the last 35 amino acids of Gag added between the Gag and Pol sequence was incorporated into the particles (Fig. 3, A, sample 5, and C, sample 5), and these particles were able to maintain the killer dsRNA (Fig. 3C, sample 5). Like the native Gag-Pol, this construct cannot make particles by itself (data not shown), so the failure of native Gag-Pol to form viral particles or to be incorporated in higher amount into particles is not simply due to the absence of these 35 residues present in its Gag part. This construct, like the normal Gag-Pol, was only 3-4-fold overproduced in total cell extracts (Fig. 3A, compare samples 1 and 5).
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Gag-Pol Has Different N-terminal Requirements from Gag-- L-A proteins are acetylated at their N terminus, and the absence of this modification leads to failure of particle formation (14-16). When the Gag N terminus was modified by changing MLRF to MLAF or MLEF, changes previously shown to prevent acetylation (16), no viral proteins were detected in cell extracts or a CsCl gradient, as expected. However, when these changes were introduced into Gag-Pol and expressed with normal Gag, viral proteins including the fusion protein were made and assembled into particles detected on CsCl or sucrose gradients; these particles were able to stably maintain M1 dsRNA (data not shown).
Other viruses, such as hepatitis B virus (27) or HIV (28), myristoylate their N terminus rather than acetylating it. When L-A Gag's N terminus was modified to that of cAMP-dependent protein kinase, or of HIV Gag, each of which are recognized and modified by the yeast N-myristoyl-transferase (20), no viral proteins could be detected in cell extracts or CsCl or sucrose gradients. With the same Gag-Pol modifications, leaving Gag normal, the proteins were detected in cell extracts and particles were made, but Gag-Pol was barely detectable in sucrose gradients, and these particles were unable to propagate the M1 dsRNA (data not shown). This means that the Gag modifications have different effects on Gag and Gag-Pol. Failure to acetylate Gag results in absence of accumulation of viral proteins, probably due to failure of assembly and consequent instability of viral proteins, but acetylation of Gag-Pol is dispensable. In contrast, changing either Gag or Gag-Pol to a substrate for myristoylation disrupts their function. However, even in the case of myristoylation, Gag-Pol is less affected than Gag, as the former is not degraded and is partially incorporated into particles. To examine the importance of the N-proximal part of Gag for function, deletions were made, leaving the 8 N-terminal residues unchanged to maintain acetylation (see Ref. 16). Deletion in Gag of amino acids 9-13 did not affect its ability to form particles or to propagate M1 (Fig. 4C, sample 1), although its amount in total cell extracts decreased slightly (Fig. 4A, sample 1). Further deletion of residues 9-18 abolished the ability to make particles or to propagate the killer (Fig. 4A, sample 2), and the protein was barely detectable in total cell extracts, suggesting that its failure to form particles made it unstable (Fig. 4A, sample 2).
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Gag C-terminal Modifications Differentially Affect Gag and Gag-Pol-- Gag-Pol lacks the last 35 residues of Gag without affecting its ability to be incorporated into particles. To determine if these residues play a specific role for Gag in assembly and viral activity, a series of deletions was made supplying Gag-Pol from a separate plasmid. The last 10 amino acids were dispensable for Gag (Fig. 5C, samples 1-3), but deletion of 15 residues made the protein unable to support M1 propagation (Fig. 5C, sample 4), although viral particles were still made. Gag with 33 residues deleted remained able to make particles incorporating the fusion protein (Fig. 5A, sample 6), but deletion of two more amino acids from Gag (to residue 645) resulted in failure to make particles (Fig. 5A, sample 7), although this is the normal composition of the Gag part of Gag-Pol, which is incorporated into the particles. This and further deletions appeared spread over the light half of the CsCl gradient with amount of protein decreasing with increasing deletion. This decrease of Gag was also detected in total cell extracts, suggesting that failure of assembly leads to destabilization of Gag and degradation (Fig. 5A).
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DISCUSSION |
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Like the Totiviruses, of which L-A is the type species, many retroviruses synthesize their reverse transcriptase (Pol) as a Gag-Pol fusion protein, often using a ribosomal frameshift to fuse the reading frames in a minority of the protein molecules. Expressing Gag and Gag-Pol from separate plasmids produces functional viral particles and allows independent control of their structure and the amounts produced.
Although we were able to produce three to four times more than the normal proportion of fusion protein relative to Gag, we never observed more than the normal proportion of fusion protein in the particles. This amount has been roughly estimated based on the normal efficiency of ribosomal frameshifting (1.8%, Ref. 10) and on Coomassie Blue staining to be about 2 molecules per particle. Because of evidence that L-A could only tolerate a 2-fold change in the Gag:Gag-Pol ratio (29, 30), we were surprised that this apparent overproduction of Gag-Pol relative to Gag did not adversely affect viral propagation. The earlier studies involved altering the ratio of proteins produced from a single mRNA (viral or plasmid), whereas our experiments involved proteins made from two different mRNAs.
We show that there is no cis-assembly, that is, no preference for the Gag and Gag-Pol molecule to originate from the same mRNA. Previous work has suggested that there is cis-packaging, the preferential binding (packaging) by Gag-Pol of the mRNA from which it was translated (22, 26, 31), although direct evidence on this point is not yet available.
We show here that there are at least two fusion protein molecules per particle. What limits the amount of Gag-Pol to about 2 molecules per particle? What tells the assembling virus not to regard a particular molecule as a Gag monomer but to treat it as a Gag-Pol fusion protein? There is no unique part of Pol whose deletion results in increased incorporation of fusion protein (deletions of residues 9-204, 205-413, 415-860 of Pol behave the same as intact Pol).2 The Gag part of Gag-Pol lacks 35 residues present in Gag, but we show here that this is not what limits the amount of Gag-Pol in viral particles. Perhaps it is simply the presence of any sizable sequence attached to the Gag C terminus that limits incorporation. Alternatively, it is possible that there is a specific association of Pol domains, that Gag-Pol dimerization is rate-limiting, and dimers of Gag-Pol prime particle formation. This Gag-Pol dimer primed capsid assembly may thus kinetically exclude more fusion protein molecules. Once a Gag-Pol dimer is formed, it rapidly becomes a complete particle. Because Gag is sufficient to make viral particles, Gag-Pol is not necessary, but it may speed the initiation of particle formation. It will be of interest to determine whether the two fusion protein molecules in each particle are in fact part of the same dimer in the structure of the shell (4), as is the case in retroviruses (32).
The deletions of the Gag part of Gag-Pol confirm the hypothesis (11) that the Gag region is directing its incorporation into particles. However, the Gag requirements of Gag-Pol to interact with Gag proteins are less stringent than those of Gag itself. The integrity of the particle structure is determined by Gag, whereas the Gag part of Gag-Pol need only associate with other Gag molecules to be incorporated. Similar results have been obtained for incorporation of HIV Gag-Pol into particles composed mainly of HIV Gag (33).
N-Acetylation of Gag, but not of Gag-Pol, is necessary for particle assembly, a result in perfect parallel with that found for HIV (34, 35). Rous sarcoma virus cores assemble without myristoylation of Gag but do not properly localize to the membrane. Myristoylation does not substitute for acetylation in L-A assembly. Even Gag-Pol, whose acetylation is dispensable, is nonfunctional if myristoylated. Understanding the precise role of acetylation will require more detailed knowledge of L-A structure.
We expect that a detailed molecular dissection of L-A viral proteins, combined with structural studies, will provide a deeper understanding of the mechanisms of its assembly and replication.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Bldg. 8, Rm. 225, National Institutes of Health, 8 Center Drive MSC 0830, Bethesda, MD
20892-0830. Tel.: 301-496-3452; Fax: 301-402-0240; E-mail: wickner{at}helix.nih.gov.
1 The abbreviations used are: dsRNA, double-stranded RNA; SDM, site-directed mutagenesis; HIV, human immunodeficiency virus.
2 J. Carlos Ribas and R. B. Wickner, unpublished results.
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REFERENCES |
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