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J. Biol. Chem., Vol. 275, Issue 47, 37118-37126, November 24, 2000
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andFrom the Instituto de Microbiología Bioquímica/Departamento de Microbiología y Genética, Consejo Superior de Investigaciones Científicas/Universidad de Salamanca, Salamanca 37007, Spain
Received for publication, June 16, 2000, and in revised form, July 27, 2000
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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The encapsidation signal of the yeast L-A virus
contains a 24-nucleotide stem-loop structure with a 5-nucleotide loop
and an A bulged at the 5' side of the stem. The Pol part of the Gag-Pol fusion protein is responsible for encapsidation of viral RNA. Opened
empty viral particles containing Gag-Pol specifically bind to this
encapsidation signal in vitro. We found that binding to empty particles protected the bulged A and the flanking-two nucleotides from cleavage by Fe(II)-EDTA-generated hydroxyl radicals. The five
nucleotides of the loop sequence
(4190GAUCC4194) were not protected. However, T1
RNase protection and in vitro mutagenesis experiments
indicated that G4190 is essential for binding. Although the
sequence of the other four nucleotides of the loop is not essential,
data from RNase protection and chemical modification experiments
suggested that C4194 was also directly involved in binding
to empty particles rather than indirectly through its potential base
pairing with G4190. These results suggest that the Pol
domain of Gag-Pol contacts the encapsidation signal at two sites: one,
the bulged A, and the other, G and C bases at the opening of the loop.
These two sites are conserved in the encapsidation signal of M1, a
satellite RNA of the L-A virus.
Double-stranded RNA
(dsRNA)1 viruses encapsidate
their plus single-stranded genomic RNAs into viral particles or inner
cores and subsequently convert them into double-stranded form by the particle-associated RNA-dependent RNA polymerase. Since all
of RNA polymerization reactions, transcription and replication, take place inside the particles, the dsRNA viruses must have efficient and
selective mechanisms to encapsidate not only their genomic RNAs but
also their RNA polymerase machinery into particles.
The yeast L-A dsRNA virus contains a single molecule of linear uncapped
4.6-kilobase dsRNA with two open reading frames (Fig. 1A). The 5' open reading frame
is gag and encodes a 76-kDa major coat protein. The 3' open
reading frame is pol and encodes RNA-dependent RNA polymerase, which is expressed only as Gag-Pol fusion protein by a
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 ribosomal frame-shifting mechanism (1-3). The Pol region of the
fusion protein has a single-stranded RNA (ssRNA) binding activity (1).
L-A virions have an icosahedral symmetry (4). The capsids consist of 60 asymmetric Gag dimers, and one molecule or two of Gag is replaced by
Gag-Pol fusion protein.
View larger version (19K):
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Fig. 1.
Schematic diagrams of L-A gene organization
(A), truncated L-A sequence in pLM1 plasmid
(B), L-A encapsidation signal (C),
and the encapsidation model for the L-A virus
(D). A, L-A plus strands have two
genes, gag and pol. gag encodes the major coat
protein (76 kDa). pol is expressed as a Gag-Pol fusion
protein (170 kDa). The RNA encapsidation activity resides in the
N-terminal quarter of Pol, and the consensus sequences for
RNA-dependent RNA polymerases reside in the second half of
Pol. B, pLM1 contains the L-A sequence (solid
line) with a large deletion (from nt 12 to nt 4090) downstream the
T7 promoter (T7). The dashed line indicates a
60-nucleotide sequence from the vector between the promoter and L-A
sequence. Restriction sites used for T7 run-off transcription and their
locations on the L-A plus strand are shown. The encapsidation signal
located near the 3' end of the L-A plus strand is indicated by a
box. C, the L-A encapsidation signal consists of
a 24-nucleotide stem-loop structure with an A bulge. Nucleotides
protected by empty particles from hydroxyl radicals and T1 ribonuclease
are indicated by arrows and a filled circle,
respectively. C4194 (open circle) is suggested
to have direct contacts with empty particles as shown in this work.
D, the encapsidation domain in Pol of the Gag-Pol fusion
protein specifically recognizes and binds to the encapsidation signal
of the L-A plus strand. Then the N-terminal Gag domain primes capsid
assembly by homologous interaction with Gag (1)
When isolated L-A virions are exposed to low ionic strength conditions, they release L-A dsRNA (5). The resulting opened empty particles retain the Gag-Pol fusion protein and thus can perform transcription and replication reactions in vitro with specificity, if appropriate template RNAs are added (5, 6). Empty particles also bind specifically to the plus strand of viral RNA (7). Using a gel shift assay, the cis signal for binding has been identified as a 24-nucleotide stem-loop with a bulged A at the 5' side of the stem (Fig. 1C) (8). It is located near the 3' end of the L-A plus strand. Subsequently, the binding signal was identified by an in vivo assay as the encapsidation signal for the L-A virus (9). The encapsidation activity resides in the N-terminal quarter of Pol (10). According to the proposed L-A virus encapsidation model (Fig. 1D) (1, 10), the virus has developed a simple, ingenious mechanism to secure the packaging of both the viral plus strand and the RNA polymerase; the encapsidation domain (the N-terminal quarter of Pol) of the Gag-Pol specifically recognizes and binds to the cis encapsidation signal present on L-A plus strands, forming a Gag-Pol·L-A plus ssRNA complex. Then the Gag domain of the fusion protein triggers the capsid assembly by homologous interactions with free Gag proteins (or Gag dimers). The selective packaging of the viral RNA is thus governed by the interaction between Gag-Pol and the viral plus ssRNA.
Since empty particles specifically bind to the L-A encapsidation
signal, this reaction provides valuable information about the nature of
the protein-RNA interactions involved in the selective packaging of the
viral RNA. In this paper we have analyzed the in vitro
binding reaction in detail using chemical and enzymatic probes and also
by in vitro mutagenesis. The results indicate that empty
particles interact with the encapsidation signal at two sites: one, the
bulged A and the surrounding two nucleotides at the 5' side of the
stem, and the other, the G and C bases at the opening of the loop.
Since the natures of the interactions at these two sites are quite
different, it suggests that the fusion protein recognizes these RNA
sites with different contact elements.
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EXPERIMENTAL PROCEDURES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Preparation of Empty Particles-- Intact L-A virions were prepared from strain TF229 (MATa his (3,4), leu2, ski2-2, LA-HN) as described (11). Empty particles were obtained from isolated L-A virions by low ionic treatment and then purified through a CsCl gradient as described (11)
Gel-shift Assay-- Gel-shift assay was carried out as described (12). In some experiments (Fig. 2) tRNA was omitted from binding reaction mixtures.
In Vitro Mutagenesis-- Oligonucleotide site-directed mutagenesis was carried out as described (8). All of the desired changes were confirmed by DNA sequencing.
Plasmids-- pLM1 contains most of the cDNA sequence of X, a deletion mutant of L-A, and has been described (11). It contains the L-A plus strand sequence with a large internal deletion (from nt 12 to nt 4090) (Fig. 1B). Between the T7 promoter and the L-A sequence there are 60 nucleotides derived from the vector.
In Vitro T7 Transcription--
Uniformly labeled RNA for
gel-shift assay was made from EcoRI-digested pLM1 or its
derivatives using T7 RNA polymerase and [
-32P]UTP as
described (7). RNA for enzymatic cleavage or chemical modifications was
made in quantity using a MEGA script T7 kit from Ambion Inc., Austin,
Texas, from pLM1 or its derivatives predigested with XmnI
unless otherwise stated (Fig. 1B).
pCp Labeling-- RNA (2 µg) was 3' end-labeled with 32P-pCp and T4 RNA ligase (Life Technologies, Inc.). The conditions were as suggested by the manufacturer. The labeled RNA was purified through a 5% polyacrylamide gel as described (13), except that the electrophoresis was carried out for 90 min at 8 V/cm. The RNA was cut out from the gel and extracted with 0.6 M ammonium acetate, 1 mM EDTA, and 0.1% SDS overnight at room temperature. The extracted RNA was phenol-treated, ethanol-precipitated, and used for ribonuclease protection experiments. For hydroxyl radical protection experiments, the RNA was further purified using an RNaid w/Spin kit from BIO 101 Inc. CA. As revealed in Figs. 4 and 5, the labeled RNAs contained a small amount (about 20%) of the same RNA species but one nucleotide shorter at the 3' end. This, however, did not affect our conclusions.
Hydroxyl Radical Cleavage-- We basically followed the cleavage procedure as described (14). Since glycerol quenches hydroxyl radicals (15) a stock solution of empty particles was passed through a G-50 mini-spin column (Worthington Biochemical Corp.) pre-equilibrated with 10 mM Tris/HCl, pH 7.5, 50 mM NaCl and then used for protection experiments. RNA labeled at the 3' end (100,000~200,000 cpm) was incubated for 10 min on ice in a buffer (25 µl) containing 10 mM Tris/HCl, pH 7.5, 50 mM NaCl, and 0~10 µl of empty particles (~5 µg of protein/µl). One µl of each of the following four solutions was added to the top of a 1.5-ml Eppendorf tube containing the RNA sample. The solutions were made freshly for each experiment and were 1) 100 mM Na-EDTA, pH 7.5, 2) 50 mM Fe(NH4)2(SO4)2·6H2O (Aldrich), 3) 250 mM sodium ascorbate, and 4) 2.5% H2O2. All solutions were mixed simultaneously by a brief centrifugation at 4 °C, and the tube was kept another 10 min on ice. The cleavage reaction was stopped by the addition of 10 µl of 0.1 M thiourea, and 70 µl of 0.4 M sodium acetate, 0.4% SDS, and 1 µg of tRNA. The RNA was extracted with phenol, phenol/chloroform, and chloroform and precipitated with ethanol. After being washed with 70% ethanol, the RNA was separated on an 8% polyacrylamide sequencing gel. To generate a sequencing ladder, alkaline hydrolysis was carried out at 95 °C for 90 s in a buffer containing 50 mM sodium bicarbonate/carbonate, pH 9.2, 5 µg of tRNA, and the labeled RNA (200,000~400,000 cpm). A T1 ladder was created by incubating the labeled RNA (150,000 ~300,000 cpm) in a buffer containing 25 mM sodium citrate, pH 5.0, 3 µg of tRNA, 7 M urea, and 3 units of T1 nuclease at 55 °C for 15 min.
Ribonuclease Protection Experiments-- 3' end-labeled RNA (100,000 ~200,000 cpm) was incubated on ice for 10 min in a mixture (19 µl) containing 20 mM Tris/HCl, pH 7.5, 50 mM NaCl, 5 µg of tRNA, and various amounts of empty particles (~5 µg of protein/µl). One µl of ribonuclease T1 or T2 (0.02~0.2 units/µl) was added to the mixture, and the cleavage was carried out at 30 °C for 10 min. After the reaction, the RNA was extracted with phenol and phenol/chloroform and analyzed on an 8% acrylamide sequencing gel. To ensure that each RNA molecule would receive no more than a single cleavage, the amounts of T1 and T2 used in Fig. 5 were 0.1 units. In these conditions more than 70% of the labeled RNAs remained undigested.
Dimethyl Sulfate (DMS) Treatment--
Non-labeled RNA (0.5 pmol)
transcribed from EcoRI- or FspI- digested pLM1 by
T7 RNA polymerase was incubated with 0-, 2-, or 5-µl empty particles
(~10 µg of protein/µl) in a buffer (total volume 20 µl)
containing 50 mM sodium cacodylate, pH 7.5 and 50 mM NaCl. Empty particles had been dialyzed twice against
the same buffer for total 2 h at 4 °C. After a 10-min
incubation on ice, the binding reaction mixture received 0.5 µl of
33% DMS (Aldrich) in ethanol and was incubated at 20 °C for another
10 min. The methylation reaction was terminated by the addition of 80 µl of 250 mM Tris/Cl, pH 7.5, 250 mM
-mercaptoethanol, 400 mM sodium acetate, and 2 µg of
tRNA. The RNA (and protein) was precipitated with 300 µl of ethanol,
and the pellet was dissolved in 200 µl of 0.3 M sodium
acetate and 0.3% SDS. Then the RNA was extracted with phenol and
phenol/chloroform and precipitated with ethanol. After an 80% ethanol
wash, the RNA was dried and then served for reverse transcription
reactions. The methylation of RNA by DMS was detected by reverse
transcriptase using 5' end-labeled primers. We used two primers, one
complementary to the L-A plus strand sequence from nt 4277 to nt 4296 and the other from nt 4246 to nt 4265. The primers were labeled at the
5' ends with [
-32P]ATP and T4 polynucleotide kinase
(MBI Fermentas) according to the method recommended by the enzyme
supplier. To the methylated RNA (0.2 pmol), dissolved in 3 µl of
water, were added 7 µl of 4.2 M NaCl, 50 mM
Tris/HCl, pH 7.5, and 7 mM Na-EDTA, 4 µl of the 5'
end-labeled primer (~400,000 cpm), and 30 µl of formamide. Annealing was carried out at 45 °C overnight. The sample was diluted 6-fold with 0.3 M sodium acetate, and the RNA annealed with
the primer was precipitated with ethanol, washed once with 80%
ethanol, and dried. The pellet was dissolved in water, and DNA
synthesis was carried out at 45 °C for 1 h in a volume of 30 µl using 100 units of Superscript RNase H
reverse
transcriptase (Life Technologies, Inc.) in the conditions recommended
by the supplier. The reaction was stopped by the addition of 15 mM EDTA, and the template RNA was digested at 37 °C for 20 min with 5 ng of RNase A. DNA was extracted with phenol/chloroform and chloroform, ethanol-precipitated, and analyzed on an 8%
polyacrylamide sequencing gel. The sequence ladders were created by T7
DNA polymerase with a T7 sequencing kit (Amersham Pharmacia Biotech)
using pLM1 as template, the same but non-labeled primer as mentioned
above, and [-33P]dATP.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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High Binding Affinity--
Empty particles have a high affinity to
bind to ssRNA with the L-A encapsidation signal. We used clones of X
dsRNA, a deletion mutant of L-A, to make ssRNAs in vitro
with the wild type or modified encapsidation signal (Fig.
1B). When empty particles were incubated with a ssRNA
fragment having the wild type sequence, the gel-shift assay revealed a
single species of binding complexes over a wide range of RNA
concentrations (Fig. 2). The
Lineweaver-Burk plot of binding became a straight line (not shown).
These results indicate that empty particles can bind a single molecule
of RNA in the reaction. From the plot, we obtained the maximum number
of binding sites as 11 pmol/mg of protein. Using ~9.3 × 103 kDa as the molecular mass of an empty particle
(118-119 subunits of Gag with a molecular mass of 76 kDa and one or
two Gag-Pol fusion proteins with 170-kDa molecular mass), it is
estimated that 10% of the particle population in this preparation were
active in binding to the encapsidation signal. The dissociation
constant (Kd) was calculated as 0.45 nM.
A similar value has been reported for binding to the encapsidation
signal of M1, a satellite RNA of L-A virus (16). This
Kd gives a 
G°, Gibbs free energy, of
12.8 kcal/mol.
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L-A Encapsidation Signal--
The cis encapsidation
signal of L-A virus consists of a 24-nucleotide stem-loop with an A
bulge at the 5' side of the stem (Fig. 1C). Empty particles
specifically bind to this signal. The importance of the bulged A in the
reaction has been demonstrated by the inability in binding of modified
RNAs whose bulged A were deleted or substituted with any of the other
three nucleotides (9). When the loop sequence
4190GAUCC4194 was modified conservatively, the
modified RNA with 4190AGCUU4194 failed to bind,
indicating the importance of the loop sequence. On the contrary, the
stem sequences themselves do not appear to be critical in binding,
since the substitution of the nucleotide sequence at each side of the
lower stem with the other destroyed the binding activity, whereas the
restoration of the lower stem structure by simultaneously exchanging
those sequences recovered the binding activity (9) (Fig.
3A). When similar exchange
experiments were done to the upper stem sequences, the same results
were obtained (Fig. 3B). Destruction of the upper stem
structure by substituting each of its stem sequences with the other
resulted in the failure in binding. Again, swapping the upper sequences
simultaneously, thus restoring the stem structure, recovered the
binding activity, although slightly less compared with the original
structure. Therefore, these results suggest that the stem
sequences themselves are not important, but that the stem
structure is important to present the bulged A and the loop sequence at
a certain distance or spatial configuration.
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Hydroxyl Radical Footprinting-- We used hydroxyl radical footprinting to analyze the physical interactions between the encapsidation signal and empty particles. Hydroxyl radicals generated by a Fenton reaction from Fe(II)-EDTA and H2O2 are quite ideal as a probe to examine protein-RNA interactions (14, 17). First, the radicals cleave the sugar backbone, and the cleavage is not affected by the bases of the sequence. Second, the size of the probe is small; thus it is suitable for a fine mapping. More importantly, the cleavage is not affected by the secondary structure of RNA as far as the sugar backbone is accessible to the aqueous phase (18, 19).
A small L-A fragment with the encapsidation signal was made in
vitro and labeled at the 3' end with 32P-pCp by T4 RNA
ligase. Then the labeled RNA was treated with hydroxyl radicals in the
presence or absence of empty particles. As shown in Fig.
4, the bulged A and its surrounding two
nucleotides were protected from cleavage upon binding to particles.
Interestingly, the 3' side stem sequence and the loop sequence were not
protected. As a control, the same RNA fragment but with a modified loop
sequence (4190AGCUU4194 ) was used. Since this
RNA has a much reduced binding affinity, almost no protection by empty
particles was observed (Fig. 4). Therefore these results indicate that
empty particles physically contact with the bulged A and the
surrounding few nucleotides but that the loop sequence is not protected
from hydroxyl radical cleavage by binding.
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The Loop Sequence Is Also Important--
Although the loop
sequence of the encapsidation signal is important for binding (9),
hydroxyl radical footprinting failed to detect interactions between the
loop sequence and empty particles. Since the radicals cleave the sugar
backbone, this may suggest that the particles contact with the
nucleotide bases of the loop. In fact, when similar protection
experiments were done using T1 ribonuclease, we could detect such
interactions. As shown in Fig. 5A, the first G (base 4190) of
the loop is accessible to T1 nuclease. The extent of the cleavage,
however, was less compared with other unpaired G residues, thus
suggesting its partially double-stranded nature. In the presence of
empty particles, this G was completely protected from T1 cleavage. This
protection is related to the binding events, since the same G in the
bulged A-deleted RNA was not protected (Fig. 5C).
Ribonuclease T2 cleaved the RNA well at the A and U of the loop
sequence and, at a lesser extent, at the C of base 4193. These T2
cleavages, however, were not inhibited by binding to the particles
(Fig. 5B). Therefore, these results suggest that the first G
(base 4190) of the loop is important for binding. There are two
plausible explanations for this protection. Empty particles directly
interact with this G and protect it from T1 cleavage. Alternatively,
the binding of empty particles may stabilize the upper stem structure
and induce base pairing of this G with C4194, thus forming
a loop with three bases instead of five. Since T1 does not cleave
double-stranded regions, this alternative structure would result in
apparent protection of the G from T1 cleavage.
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To confirm the importance of the first G of the loop in binding, we
modified the loop sequence by in vitro mutagenesis and tested their binding activity. A mutation in the loop
(4191GCU4193) did not significantly affect
binding (Fig. 6). This result is consistent with T2 cleavage experiments (Fig. 5B): that is,
empty particles did not protect these three nucleotides. Since a mutant RNA with 4190AGCUU4194 failed to bind to empty
particles, these results suggest that the remaining two bases of the
loop (G at 4190 and/or C at 4194) are important for binding. In fact,
when these two nucleotides were simultaneously substituted with C
(4190) and G (4194) or A (4190) and U (4194), none of these mutant RNAs
showed binding activity (Fig. 6). To evaluate their importance in
binding, we mutated these two nucleotides individually. When G at 4190 was substituted by any of the other three nucleotides, all of them failed to bind to empty particles (Fig.
7A), confirming that G at 4190 is essential for binding. On the other hand, when C at 4194 was
modified, all of them retained the binding activity substantially. Especially, the RNA with the G substitution (GG mutant) showed a
binding activity only slightly less compared with the wild type RNA.
These in vitro mutagenesis experiments therefore indicate (i) that the G at 4190 is essential for binding, whereas (ii) that C at
4194 is exchangeable with other nucleotides in binding.
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Binding to Empty Particles Does Not Induce GC Base Pairing in the Loop-- As shown above C at 4194 can be replaced by other nucleotides in binding. This does not necessarily mean that this nucleotide does not contact with the empty particles. We rather think that it directly interacts with them, based on the following evidence. When the GG mutant RNA with G at 4194 was treated with T1 nuclease, the two G residues (at 4190 and 4194) in the loop were protected from T1 by binding to empty particles (Fig. 5D). The extent of the protection of each G was the same at a given concentration of empty particles (Fig. 5E). This result thus indicates that the particles directly interact with the first G (at 4190) of the loop and protect it from T1 cleavage rather than inducing the GC base pair at the opening of the loop. The result also indicates that empty particles directly interact with G4194 in the GG mutant RNA (and perhaps with C4194 in the wild type RNA).
To confirm this we did DMS modification experiments. DMS methylates A
(N1) and C (N3) at the Watson-Crick base-pairing positions in the
single-stranded regions, and these modifications can be detected by
reverse transcriptase using 5' end-labeled primers (17, 20). As shown
in Fig. 8, the methylation of two C
residues at positions 4194 and 4206 was greatly stimulated upon binding to empty particles. We used two primers. One of them was complementary to the L-A plus strand sequence from nt 4277 to nt 4296 (Fig. 8) and
the other, from 4246 to 4265 (not shown). Both primers gave the same
results. We also used two RNA templates for DMS methylation. One
contained the intact L-A 3' end region (from nt 4091 to nt 4579),
including the encapsidation signal and the correct L-A 3' end without
extra sequences (Fig. 8). The other was the same RNA but truncated at
nt 4353 (not shown). Again, in both cases we observed the same results.
From these we can mention several points. First, the N3 atom of C at
base 4194 was more methylated by binding to empty particles, thus
indicating that the binding does not induce the Watson-Crick GC base
pairing at the opening of the loop. Therefore it confirms our previous conclusion that empty particles directly interact with G at 4190 and
protect it from T1 cleavage. Second, since G4190 and
C4194 are partially double-stranded in the absence of empty
particles, according to the T1 protection experiment (Fig.
5A), this stimulation in methylation rather indicates that
the binding of empty particles brakes the base pairing between these
two nucleotides and increases the accessibility of the N3 of
C4194 toward DMS. Finally, the modification of C at 4206 (and A4207) by DMS was also stimulated by empty particles.
Since the particles did not protect this C and the surrounding
nucleotides from hydroxyl radical or T2 cleavage (Figs. 4 and
5B), this stimulation is likely to be a secondary effect
caused by the binding. Perhaps the tension induced by binding is
released in this single-stranded region directly attached to the 3'
lower stem. This may be correlated with our previous observation that
the addition of the 10-nucleotide upstream and downstream sequences to
the 24-nucleotide encapsidation signal increased the binding affinity
to empty particles (9).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Interactions at Two RNA Sites--
In this paper we have analyzed
physical interactions between the cis encapsidation signal
of L-A virus and empty L-A viral particles. It is reasonable to assume
that most of the interactions (if not all) exerted by empty particles
can be ascribed to the Pol part of the fusion protein, since the
packaging activity of RNA with the encapsidation signal resides in the
N-terminal quarter of Pol (10) and neither Gag nor the Gag part of the
fusion protein appears to be involved in the recognition of the
encapsidation signal (21). Empty particles specifically bind to the
encapsidation signal with high affinity (Kd ~ 5 × 1010 M) and they
physically contact it at two sites. One is the bulged A4186
and the surrounding few nucleotides (U4185 and
G4187) as demonstrated by protection experiments from
hydroxyl radicals. Since this A cannot be substituted with any of the
other three nucleotides without losing its binding activity, these data
suggest that empty particles extensively contact with the adenine base and its sugar moiety as well. Although U4185 and
G4187 were also protected from hydroxyl radical cleavage,
their contacts with empty particles appear to be limited mainly at
their sugar backbone. These nucleotides can be exchanged by their 3'
side base pair partners without losing binding activity as far as the stem structure is intact (Fig. 3). We have not observed the protection of the 3' side stem by empty particles from hydroxyl radical cleavage. Therefore, these results suggest that empty particles have very localized contact with the 5' side of the stem, namely, at the bulged A
and the sugar moieties of its flanking nucleotides.
The second site is two nucleotides, G4190 and C4194, at the opening of the loop. The other three nucleotides of the loop (4191AUC4193) do not have direct contact with empty particles. G4190 is essential for binding since it cannot be substituted with any of the other three nucleotides. Furthermore, empty particles protected G4190 from T1 cleavage, thus indicating their physical interaction. We have ruled out the possibility that this protection was caused by base pairing of G4190 with C4194, since empty particles protected G4190 well from T1 cleavage even in a modified GG mutant RNA whose C4194 was substituted with G. In addition, methylation at N3 of C4194 by DMS was greatly increased by binding to empty particles. If Watson-Crick base pairing between G4190 and C4194 were involved in the protection, we should observe the opposite, a decrease of methylation at C4194 upon binding to empty particles.
We have several lines of evidence that suggests direct interactions between C4194 and empty particles. In the GG mutant RNA, T1 cleavage at G4194 was inhibited by empty particles, and the pattern of its inhibition was identical to the one observed at G4190, thus implying that the same binding event elicited the protection at both G residues simultaneously. This result therefore indicates that empty particles directly contact with G at position 4194 in the GG mutant. And it is perhaps the same with the wild type sequence, since the binding affinities decrease in the order of C, G, U, and A at position 4194. Bruenn and co-workers (22) have also observed that not only the bulged A and G4190 but also C4194 are conserved among RNA isolates with binding activity in in vitro selection. M1, a satellite RNA of the L-A virus, has these three nucleotides exactly at the same positions as L-A in its encapsidation site (9).
A Possible Mechanism for the Interaction between C4194
and Empty Particles--
The contribution of a single hydrogen bonding
in EcoRI endonuclese/DNA substrate binding is estimated to
be 1.5 kcal/mol on the average (23). The substitution of
C4194 with A decreased its binding affinity about 10-fold.
It corresponds to a decrease in the binding energy of less than 1.4 kcal/mol. Therefore, it suggests that the interactions between empty
particles and C4194 are not extensive. If we assume that
empty particles recognize O2 of C4194, some of the data
presented here could be explained. First, binding of empty particles to
G4190 and O2 of C4194 would break a
Watson-Crick base pairing between them, and it may increase the
accessibility of N3 of C4194 to DMS methylation. Second,
the binding affinities of RNAs substituted at 4194 decreased in the
order of C, G, U, and A. Since adenine has no keto oxygens, the A
substitution has the lowest affinity. Uracil has the keto oxygen (O2)
similar to cytosine, but the hydrogen atom at N3 (or O4) may create
somewhat unfavorable conditions for binding. Then, how is the G
substitution? If two G residues at 4190 and 4194 in the GG mutant form
a non-canonical GG N7-imino base pair, the relative distance and
spatial configuration of O6 of G4194 toward
G4190 may be comparable with those of O2 of C toward G in a
normal Watson-Crick GC base pairing (Fig.
9). Although G4190 and
C4194 of the wild type RNA do not form a Watson-Crick base
pairing in the complex, O6 of G4194 of the GG mutant may
occupy a position similar to that of O2 of C4194 in the
complex and interact with empty particles as a hydrogen bond acceptor.
This also explains why binding to empty particles protected
G4194 in the GG mutant from T1 cleavage (Fig.
5D). Since T1 nuclease interacts with O6 in addition to N1,
N2, and N7 of guanine base (24), such an interaction would surely
inhibit T1 cleavage at G4194 because of steric
hindrance.
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The minor groove of an A-type RNA helix is wide and shallow but provide poor information on hydrogen bond donors and acceptors to interact with proteins. The major groove, on the contrary, has rich information but is very deep and narrow (25). At the end of the helix the major groove becomes wider and accessible to reagents such as diethyl pyrocarbonate, which is comparable in size with protein side chains such as arginine (26). The second binding site G4190 and C4194 is placed at the end of the upper stem or at the opening of the five-nucleotide loop. Although the binding does not require a Watson-Crick base pairing between these nucleotides, T1 cleavage of the wild type sequence suggests their partially double-stranded nature in the absence of empty particles. Perhaps it is important for these bases to be held at a certain spatial configuration or in a dynamic state at the boundary between the upper helix and the loop in order to interact with empty particles. The upper stem between these two sites consists of three base pairs. Since the rotation of an A-type helix is 33° per residue, the sugar backbones of the bulged A and the G4190 may form an angle of about 115°. If a single Pol peptide in the empty particles is responsible for the interactions at these two sites, the contact of empty particles with the GC (4190 and 4194) site may take place through its major groove side.
Comparison of L-A Encapsidation Signal with MS2 Coat Protein
Binding Site--
The L-A encapsidation signal is quite similar to the
RNA hairpin structure found in the MS2 bacteriophage replicase operator to which the MS2 coat protein dimer binds. Extensive structural analyses have been done on the latter system. The MS2 hairpin structure
contains a four-nucleotide loop and a bulged A at the 5' side of the
stem separated from the loop by a two-base pair upper stem.
Interestingly, this bulged A, when unligated, stacks in the
double-stranded helix (27). On the contrary, when it forms a complex
with the coat protein dimer, the bulged A becomes extrahelical and
interacts with the dimer through extensive hydrogen bonding (28, 29).
The encapsidation signal of the L-A virus, therefore, may undergo a
similar structural change at the bulged A upon binding to empty
particles. In the absence of empty particles, the bulged A would be
intercalated between the neighboring base pairs. Its resistance to T2
digestion and non-susceptibility to DMS methylation may be indicative
of its stacking nature. In the complex the bulged A may become
extrahelical so that empty particles can contact extensively with its
adenine base to discriminate it from other bases. The second MS2
binding site is the last adenine of the loop. The MS2 coat protein
dimer contacts with the hairpin structure mainly at these two A
residues, quasi-symmetrically, that is, the same region made of a
-sheet in both subunits contacts with each A similarly. The MS2 coat
protein is small (129 amino acids) but must perform at least two
functions, capsid assembly and binding to the hairpin structure. This
may partly explain why dimer formation is necessary for binding to the
hairpin and why the two A residues are quasi-2-fold-related in
structure in the complex. In the case of the L-A encapsidation signal,
the contact sites with empty particles are quite different: one, the bulged A, but the other, G4190 (and C4194), at
the opening of the loop. As shown in the hydroxyl radical protection
experiments, empty particles interact extensively with the bulged A
including its sugar backbone; on the other hand, their interactions
with the second site appear to be confined to the nucleotide bases. The
Gag part of the fusion protein directs its assembly into capsids,
whereas the region necessary and sufficient for in vivo RNA
packaging has been narrowed down to residues 67-213 of the Pol part
(30) (Fig. 1A), still larger than the MS2 coat protein.
Therefore, these data suggest that the encapsidation domain of Pol has
two different sites to contact with the encapsidation signal.
Does a Gag-Pol Homodimer Exist in the Virion?--
As discussed
above, available data suggest that the encapsidation domain of the
fusion protein has two different regions or sites to contact with the
L-A RNA encapsidation signal. It has been proposed that the fusion
protein forms a homodimer that is responsible for encapsidation (31,
32). If it is the case, one of these different regions in each subunit
may contact with one of the two RNA sites. Alternatively, there is
still the possibility that, like the MS2 coat protein, a single region
in each subunit of the fusion protein dimer can contact with either the
protruding A or GC site. However, we think that neither of them is the
case, as discussed below. Two types of data can be mentioned as the supporting evidence for dimer formation. First, the efficiency of the
1 ribosomal frame-shifting, measured in an RNA fragment encompassing
the frame-shifting site using a reporter gene, was 1.9%. Since L-A
particles contain 120 copies of Gag, it roughly corresponds to two
Gag-Pol molecules per particle. Furthermore, this frame-shifting
efficiency was claimed to be critical for viral reproduction since
either increasing or decreasing the efficiency more than 2-fold
disrupted M1 satellite RNA propagation (31). When the frame-shifting
was measured in a different RNA fragment or even in the same fragment
but in a different cell background, however, much higher efficiencies
(10-20%) were observed (33, 34). One may also wonder if the
frame-shifting efficiency should be the same as the ratio of the coat
proteins assembled. Several host mutants that increased the
frame-shifting efficiency have been isolated. Most of them could
actually maintain M1 at 20 °C. More importantly, none of them had
any effects on L-A virus, even on its copy number (35).
The other line of evidence might be the incorporation of a truncated Gag-Pol protein into active (M1 dsRNA-synthesizing) particles. Since the truncated protein lacks the C-terminal half of Pol including the RNA polymerase domain, thus defective in RNA polymerization, those active particles containing the truncated protein should also contain the intact fusion protein (36). From these results, however, one cannot generalize that L-A virions contain two intact fusion protein molecules per particle. Because the truncated protein can be incorporated into capsids without viral RNA or the intact fusion protein by virtue of its intact Gag domain, the results can also be interpreted as a random distribution of the truncated protein into viral capsids. Furthermore, if the formation of a fusion protein dimer is responsible for the incorporation of the truncated protein into active M1 virions, then the results strongly suggest that RNA polymerase does not require dimerization for its activity. Pol contains a leucine zipper-like sequence (residues 216 to 244) that could be involved in dimerization (2). RNA encapsidation, however, does not require the C-terminal three-quarters of Pol, including this leucine zipper-like sequence (10). Then, why is the fusion protein dimerization necessary? Or is the N-terminal one-quarter of Pol still sufficient for formation of a Gag-Pol homodimer? As far as we know, there are no convincing data in the literature that show the presence of two fusion protein molecules per intact L-A virion. In our hands, the quantification of coat proteins strongly suggests that intact L-A virions contain a single Gag-Pol molecule per particle.2 Thus we believe that a single fusion polypeptide is responsible for the interactions with the two RNA sites in the encapsidation signal.
The data reported here suggest that the RNA-protein interactions
between the L-A fusion protein and the encapsidation signal are more
complex than those of the RNA bacteriophage. Further fine structural
analyses such as x-ray diffraction or NMR studies would be
indispensable to understand these interactions in detail.
| |
ACKNOWLEDGEMENTS |
|---|
We thank Drs. F. Leal and M. Medarde for valuable comments on the manuscript.
| |
FOOTNOTES |
|---|
* This work was supported by Grant PB97-1121 from Dirección General de Enseñanza Superior (DGES) of the Spanish Ministry of Education.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: Instituto de
Microbiología Bioquímica CSIC, Universidad de
Salamanca, Avda. del Campo Charro s/n Salamanca 37007, Spain. Tel.:
34-923-120673; Fax: 34-923-224876; E-mail address:
tfujimura@www-micro.usal.es.
Published, JBC Papers in Press, August 22, 2000, DOI 10.1074/jbc.M005245200
2 T. Fujimura and R. Esteban, unpublished observations.
| |
ABBREVIATIONS |
|---|
The abbreviations used are: dsRNA, double-stranded RNA; ssRNA, single-stranded RNA; nt, nucleotide; DMS, dimethyl sulfate.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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