Department of Biochemistry, Weill Medical
College of Cornell University, New York, New York 10021
Hepatitis C virus (HCV) infects an estimated 170 million people
worldwide, the majority of whom develop a chronic infection which can
lead to severe liver disease, and for which no generally effective
treatment yet exists. A promising target for treatment is the internal
ribosome entry site (IRES) of HCV, a highly conserved domain within a
highly variable RNA. Never before have the ribosome binding sites of
any IRES domains, cellular or viral, been directly characterized. Here,
we reveal that the HCV IRES sequences most closely associated with 80S
ribosomes during protein synthesis initiation are a series of
discontinuous domains together comprising by far the largest ribosome
binding site yet discovered.
 |
INTRODUCTION |
Nearly all patients infected
with hepatitis C virus (HCV) develop a persistent infection that can
progress to cirrhosis, hepatocellular carcinoma, and liver failure.
Limited therapy is available, and a large number of viral isolates are
resistant. HCV is able to avoid host defenses in part due to the high
variability among different strains and isolates. The development of
better therapies has also been hindered by the lack of an established
in vitro cell culture system and a useful laboratory animal model.
HCV has a positive-strand RNA genome about 9,600 bases long
(12). The most conserved and highly structured portion of
the HCV genome includes the 5' untranslated region (5' UTR) and about 30 bases of coding sequence (14, 15, 32) which have been shown to contain an internal ribosome entry site (IRES) (6, 14). Despite the lack of a 5'-terminal cap, protein synthesis still initiates, although at an AUG codon downstream from several others. IRESs have been found in both viral genomes and cellular mRNAs
(15, 24, 26), yet they fail to share significant primary sequence homology. These IRESs range in size from 300 to 1,500 nucleotides and possess both secondary and tertiary structural elements. Since there is no extensive homology among IRES domains from
viral or cellular mRNAs, higher-order structure along with limited and
widely separated primary sequence elements may combine to create IRES
ribosome recognition sites (13, 14).
Beginning with work by Steitz (31) and others (9,
28), the isolation and sequence analysis of ribosome binding
sites (RBSs) from radioactive polycistronic mRNAs of prokaryotes have been used to define the mRNA sequences primarily responsible for bringing a ribosome into the correct orientation for translation (25). An RBS is the ribosome-protected region of an mRNA
under conditions which would otherwise lead to complete mRNA
solubilization. RBSs remain attached to the ribosome and, thus, are
separated from the bulk of the mRNA molecule by sedimentation after
exhaustive digestion of initiation complexes with RNase (usually
pancreatic RNase A) (23, 25). A similar approach was used
to isolate RBSs from capped monocistronic mRNAs of eukaryotes
(18-22).
Studies on HCV IRES sequences and ribosomal subunits have so far
concentrated on 40S preinitiation complexes (17, 27). No
direct characterization of the 80S RBSs from HCV, or any
IRES-containing mRNA, has yet been reported. We report our discovery
that a series of discontinuous sequences combine to form a ribosome
binding domain many times the conventional size, and we compare these findings to 48S preinitiation complexes.
| |
MATERIALS AND METHODS |
In vitro transcription.
The plasmid pN(1-4728) containing
the first 4.7 kb of HCV sequence adjacent to the phage T7 promoter was
a gift from Stanley Lemon, University of Texas, Galveston. The plasmid
was cleaved by three different restriction enzymes: AatII,
SacII, and BamHI. When these templates were
transcribed in vitro, three 32P-labeled RNAs
spanning bases 1 to 402, 1 to 645, and 1 to 1349 were produced. The
plasmid p
Hb containing rabbit -globin cDNA was a gift from Karen
Browning, University of Texas, Austin. This plasmid was cleaved with
HindIII to produce the proper template for
transcription. The transcription reactions are based on earlier published work from this laboratory (2, 7), and specific activities of 4.13 × 106 or 8.25 × 107 dpm/µg were generally used.
[
-32P]GTP (NEN Life Science Products,
Boston, Mass.) was used in all labeled transcriptions unless noted otherwise.
Ribosome binding.
Ribosome binding reactions followed the
standard translation protocol of Promega (Madison, Wis.). Each sample
contained 35 µl of nuclease-treated rabbit reticulocyte lysate (RRL)
(Promega), 1 µl of amino acid mixture (Promega), and enough water so
that the final reaction volume equaled 50 µl. If necessary, 10 mM
EDTA was added to control reactions at this time (34).
Each reaction mixture was incubated at 30°C, and 100 µM anisomycin
or sparsomycin was added after 1 min. Edeine (1 µM) or aurin
tricarboxylic acid (100 µM) was added after 4.5 min of incubation.
Edeine was a gift from Z. Kurylo-Borowska. After 5 min at 30°C, 1 µg of radiolabeled mRNA was added. The samples were incubated for
another 10 min (15-min total incubation) and then immediately placed on
ice. Pancreatic RNase A was added to each sample (25.6 µg/ml [final concentration] for HCV RNAs and 9.6 µg/ml for -globin RNA)
followed by incubation on ice for 15 min.
Sucrose density gradient analysis.
After 15 min on ice, 250 µl of gradient buffer (25 mM KCl, 10 mM NaCl, 1 mM
MgCl2, 10 mM Tris-HCl [pH 7.5], and 1 mM
dithiothreitol) was added to each reaction and the samples were loaded
onto 5-ml 15 to 30% sucrose gradients using the same gradient buffer.
Gradient buffer similar to that described above, but without
MgCl2 and containing 10 mM EDTA, was used for
both dilution of samples and formation of 5-ml 15 to 30% sucrose
gradients containing EDTA. All samples were then centrifuged for 2 h at 2°C and 45,000 rpm (189,378 × g average).
Two-drop fractions were collected as described before (28,
31), and those corresponding to the appropriate peaks were
pooled. The total volume containing the 80S or 48S peaks was
approximately 1 ml. Protected RNA was recovered from pooled fractions
as described before (20-22). Pooled 80S or 48S peaks were
added to 1 ml of phenol containing 0.5 g of urea, 10 µl of
mercaptoethanol, 10 µl of 10% sodium dodecyl sulfate, and 10 µl of
200 mM EDTA at ambient temperature. The mixture was vortexed, 1 ml of
chloroform was added, and after more vortexing, the phases were
separated by centrifugation. The aqueous layer was collected and
ethanol precipitated.
RNA fingerprinting.
RNA fingerprinting, a technique in which
RNA molecules are digested with RNase T1
(Calbiochem, San Diego, Calif.) and the digestion products are
separated in two dimensions by charge and size, has been described
previously (3).
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RESULTS |
80S ribosomal complex formation on HCV and -globin RNAs.
Internally labeled transcripts containing the complete HCV 5' UTR and
various lengths of HCV mRNA coding sequence were used to form 80S
initiation complexes in RRL. Eukaryotic -globin mRNA was used as a
control since its RBS has already been characterized (21,
22). To interrupt the translation process at the point of first
peptide bond formation, translation inhibitors, anisomycin and
sparsomycin, were added to the reactions (33). At the
concentrations used here, these inhibitors allow the 80S ribosomal
complex to bind to the start codon but prevent translation elongation
on capped mRNAs beyond the dipeptide state, thus permitting a
stable 80S ribosomal-mRNA complex to form (20-22). The
radiolabeled RNAs were incubated in the lysate in the presence of
either anisomycin or sparsomycin, treated with RNase A to solubilize
all parts of the mRNA not protected by ribosomes, and loaded on 15 to
30% sucrose gradients for centrifugation.
RNA sedimentation profiles were obtained for HCV and -globin RNAs
after collecting fractions and determining their radioactivity (Fig.
1). When
the RNAs are centrifuged alone, without any RNase treatment, they both
are found in the top of the gradients (Fig. 1K and L). In the presence
of anisomycin and after treatment with RNase, peaks corresponding to
the 80S ribosomal initiation complex and protected radiolabeled RNA
form and can be seen in the profiles of both HCV and -globin RNAs
(Fig. 1A and B). Similar results were seen when sparsomycin was added
to the reaction (data not shown). These 80S ribosomal complexes cannot
form when the RRL is pretreated with 10 mM EDTA (Fig. 1C and D).
Similarly, these complexes dissociate when centrifuged in sucrose
density gradients containing 10 mM EDTA (Fig. 1E and F). When fractions
from 80S regions obtained with and without EDTA were quantified from a scaled-up preparation, the EDTA-treated samples had only 4 to 5% of
the radioactivity found in those obtained with the complete system.
Thus, recovery of the vast majority of the 80S-protected RNA fragments
requires protein synthesis initiation.
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FIG. 1.
HCV RNAs of 645 bases and -globin RNAs of 602 bases
bind to 80S ribosomal initiation complexes. (A and B) Sucrose gradient
sedimentation profiles of radiolabeled 645-base HCV or 602-base
-globin RNAs incubated in RRL containing anisomycin, treated with
RNase, and centrifuged in sucrose density gradients containing 1 mM
MgCl2. (C and D) Sedimentation profiles of radiolabeled HCV
or -globin RNAs incubated in RRL containing anisomycin and 10 mM EDTA,
treated with RNase, and centrifuged in sucrose density gradients
containing 1 mM MgCl2. (E and F) Sedimentation profiles of
radiolabeled HCV or -globin RNAs incubated in RRL containing
anisomycin, treated with RNase, and centrifuged in sucrose density
gradients containing 10 mM EDTA. (G and H) Sedimentation profiles of
radiolabeled HCV or -globin RNAs incubated in RRL containing
anisomycin and ATA, treated with RNase, and centrifuged in sucrose
density gradients containing 1 mM MgCl2. (I and J)
Sedimentation profiles of radiolabeled HCV or -globin RNAs incubated
in RRL containing edeine, treated with RNase, and centrifuged in
sucrose density gradients containing 1 mM MgCl2. (K and L)
Sedimentation profiles of radiolabeled HCV or -globin RNAs incubated
in RRL and centrifuged in sucrose density gradients containing 1 mM
MgCl2. Both the HCV and -globin RNAs are of
approximately equal lengths, about 600 bases. All profiles show the
counts per minute plotted against the fraction number of the gradient.
The first fraction is taken from the bottom of the gradient, and the
last is taken from the top. The 80S and 48S ribosomal peaks are
indicated.
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Interestingly, the HCV profile of the RNA incubated in RRL containing
anisomycin (Fig. 1A) or sparsomycin (data not shown) also contains a
peak corresponding to a 40S subunit-mRNA complex (fractions 18 to 22)
not observed with -globin mRNA (Fig. 1B) (21, 22).
Furthermore, addition of the translation inhibitor aurin tricarboxylic
acid, which has been shown to block all binding of globin mRNA to
rabbit reticulocyte ribosomes (20-22, 33), abolishes both
globin and HCV 80S ribosome-protected peaks but allows the HCV 40S peak
to persist (Fig. 1G and H). In the presence of edeine, which allows 48S
complexes to accumulate but prevents 60S subunit association, similar
48S peaks are observed for both HCV and -globin mRNAs (Fig. 1I and J).
Analysis of HCV and -globin protected RNA fragments.
The
fractions containing the 80S ribosome peaks were pooled, and the
radioactive, protected RNA comprising the RBSs was isolated (20-22). These RNAs were then electrophoresed on a
denaturing polyacrylamide gel (Fig. 2).
The -globin RNA protected by the 80S ribosomal initiation complex
(Fig. 1B) is four bands approximately 40 to 35 bases in length (Fig. 2,
left lane). In contrast, the protected HCV RNA is up to 30 bands
varying in length from more than 100 bases to approximately 9 bases. It
is evident that many more bases of sequence are protected in the HCV
IRES than in the -globin mRNA. By identifying these IRES sequences,
we will have the first direct indication of how the IRES specifies
ribosome binding.
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FIG. 2.
The 80S ribosomal initiation complex protects many
fragments of HCV RNA of various lengths. The protected RNA fragments
from the 80S peaks of both -globin (left lane) and HCV (1 to
1349) (right lane) mRNAs were isolated, electrophoresed on a 15%
denaturing polyacrylamide gel, and subjected to autoradiography. The
80S ribosomal complexes accumulated after treatment with
sparsomycin. O, origin; XC, xylene cyanol; B , bromophenol.
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The protected RNA fragments were then subjected to RNA fingerprinting
analysis (3). These -globin mRNA fragments and the control intact -globin mRNA were fingerprinted (Fig.
3A and B). The complex fingerprint
pattern of the intact control (Fig. 3A) is greatly simplified for the
ribosome-protected RNA (Fig. 3B), signifying that much of the 600-base
RNA was not protected and is missing from the pattern. The spots from
the 80S-protected -globin mRNA fingerprint were eluted, and
secondary RNase digestions were performed to determine the sequence of
each RNase T1-resistant oligonucleotide. The
presence of the previously reported 80S ribosome-protected sequence
(21, 22) was thus confirmed and found to contain bases 43 to 82 of rabbit -globin mRNA, a sequence 40 bases in length (see
Fig. 5A). The shorter bands seen in the
left lane of Fig. 2 contain this same sequence minus a few bases from
either end, a "frayed" end. The shortest protected band is defined
as the "core" sequence. The intact HCV mRNA (Fig. 3C) and the 80S ribosome-protected fragments (Fig. 3D) were also fingerprinted. A
simpler pattern is seen in the 80S ribosome-protected HCV fingerprint.
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FIG. 3.
The RNA fingerprints of the 80S ribosomal
complex-protected -globin and HCV mRNAs are much simpler. (A) Intact
-globin mRNA was subjected to RNA fingerprinting. The radiolabeled
RNA was subjected to exhaustive RNase T1 digestion. The
resulting oligonucleotides then were separated in the first dimension
by charge (from right to left) and in the second dimension by size
(from bottom to top). (B) The protected -globin RNA fragments were
isolated from the 80S ribosomal initiation complex peak (like that of
Fig. 1B) and subjected to RNA fingerprinting. The 80S
ribosome-protected mRNA produces a simpler pattern than does the intact
control mRNA. (C) Control HCV mRNA containing bases 1 to 1349 was
fingerprinted. (D) HCV mRNA (bases 1 to 1349) from the 80S ribosomal
peak (like that of Fig. 1A) was fingerprinted. This fingerprint pattern
is much simpler than the control shown in panel C.
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FIG. 4.
The 80S-protected fragments of HCV mRNA are
discontinuous and noncontiguous. The HCV RNA genome is depicted here as
a single thick black line. The recovered, protected fragments are
depicted above the genome as thin black lines. All protected fragments
lie within bases 124 to 392. No protected fragments were found between
bases 191 and 202.
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The HCV RBS contains a series of discontinuous fragments.
The
gel pattern for the HCV RBS (Fig. 2, right lane) is reproducible, and
each band was analyzed in detail. On two occasions, separate HCV or
-globin mRNA transcripts labeled with
[-32P]GTP or
[-32P]CTP were analyzed. In the case of the
HCV RBS, at least 12 gel band sequences (Fig. 2, right lane) were
characterized in each of the four preparations. Each of the individual
gel bands of HCV protected RNA was eluted and fingerprinted (data not
shown). Using secondary RNase digestions of the RNase
T1-resistant oligonucleotides of each fingerprint
(1, 3), the sequence of each purified 80S
ribosome-protected fragment was determined. The termini of these
sequences identified points of RNase cleavage within, or at the borders
of, HCV RBS elements. The longest protected sequence contains 128 bases, and the shortest contains 9 bases. All of the protected
fragments fall between bases 124 and 392 of the HCV map (Fig. 4). The
largest fragment covers bases 204 to 331, which contain the right side
of stem-loop III and much of the pseudoknot region. Another large
fragment contains the left side of stem-loop III from bases 124 to 191. Another fragment contains the start codon and 36 bases of downstream
coding sequence. The sequences recovered four times or more from the
HCV IRES, either alone or as part of a larger fragment, are defined
here as core sequences. These protected fragments must interact to form
a specific ribosome binding domain.
The RBS of the approximately 600-base -globin mRNA is 40 bases in
length (blue box) with the start codon located at bases 56 to 58 (Fig.
5A).
The core sequence (orange box), the smallest protected fragment, is 35 bases in length. In contrast, the RBS of the HCV genomic mRNA, drawn to
a similar scale, comprises two segments 68 and 189 bases long,
respectively (blue boxes), separated by a 12-base region of unprotected
RNA. The start codon is located at bases 342 to 344. The core elements
of the HCV RBS, the most frequently protected sequences, consist
of five noncontiguous RNA segments (orange boxes) which contain a
total of 108 bases, as follows: 128 to 138 (Fig. 5A, domain 1),
145 to 156 (Fig. 5A, domain 2), 237 to 249 (Fig. 5A, domain 3), 286 to
324 (Fig. 5A, domain 4), and 342 to 373 (Fig. 5A, domain 5). Each core
is substantially or completely protected from RNase digestion and was
recovered both alone and covalently linked to additional RNA segments
(Fig. 4). Two kinds of noncore elements are recovered only as part of larger segments containing at least one core element. Noncore elements
are sometimes cleaved by RNase A during RBS isolation, although much
less frequently than the rest of the HCV mRNA. One subset of HCV
noncore elements has not been observed to undergo cleavage. The three
segments comprising this group are indicated by pairs of vertical
arrows connected by horizontal dashed lines (Fig. 5A).
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FIG. 5.
The RBS of HCV mRNA is large and discontinuous.
(A) The RBSs of -globin and HCV mRNAs are shown as blue boxes. The
core regions are represented by orange boxes. HCV mRNA has five core
regions which are discontinuous. The three segments comprising noncore
elements which have not been observed to undergo cleavage are indicated
by vertical arrows connected by horizontal dashed lines. (B) The HCV
RBS domains are mapped on the secondary structure of the HCV IRES. The
protected regions are shown in blue, and the core sequences are shown
in orange.
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The protected regions of the HCV IRES are shown in blue, and the core
sequences are shown in orange (Fig. 5B). Core domain 1+4 contains the
pseudoknot of the HCV IRES, core domain 2+3 is located in the
base-paired region below stem-loops IIIa and IIIc, and core domain 5 contains the initiator AUG codon and is the most similar to the globin
mRNA core domain. Arrows indicate termini of noncore elements lacking
cleavage sites (Fig. 5).
As previously described in the literature (21), the
edeine-induced 48S ribosomal complex protected a larger region of
-globin mRNA of approximately 60 bases in length (Fig.
6, lanes 1 and 3). The 48S-protected HCV
RNA fragments, like the 80S-protected RNA fragments, range in length
from 10 bases to over 100 bases (Fig. 6, lane 4). These protected
fragments were isolated and characterized in a manner similar to that
described for the 80S-protected fragments (data not shown). The largest
protected fragment lies between bases 259 and 355. Another large
fragment covers bases 125 to 192. The 48S-protected region is very
similar to that protected by the entire 80S ribosomal complex. However,
in contrast to the RBS, the most 3' protected base is nucleotide 355, which corresponds with the end of stem-loop IV. No other downstream
nucleotides are protected. Otherwise, the 48S-protected region, like
the RBS, contains stem-loop III minus the apical loop, the pseudoknot
region, and stem-loop IV.
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FIG. 6.
The 48S ribosomal initiation complex also protects many
fragments of HCV RNA of various lengths. The protected fragments from
the anisomycin-induced 80S peaks of both -globin (lane 1) and HCV
(bases 1 to 645) (lane 2) mRNAs were electrophoresed on a 15%
denaturing polyacrylamide gel and subjected to autoradiography. The
edeine-induced 48S ribosomal complexes of -globin (lane 3) and HCV
(bases 1 to 645) (lane 4) mRNAs are also shown. XC, xylene
cyanol; B, bromophenol.
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DISCUSSION |
We report here the initial results of our studies involving direct
analysis of the interaction between mammalian ribosomes and the HCV
IRES using the conventional capped -globin mRNA for comparison.
Under conditions of pancreatic RNase digestion which lead to complete
solubilization of both HCV mRNA and the control globin mRNA species, we
find, in both cases, specific protection of RBS domains by initiating
80S ribosomes. The globin mRNA RBS, under the translation-blocked
conditions used here, is typical of a conventional capped mRNA
(18-22), comprising a 40-base region with a 35-base core
domain containing the AUG start codon (Fig. 5A). In contrast, the RBS
sequences of the HCV IRES contain substantially more bases (257) than
those of the globin mRNA RBS, including five core elements totaling 108 bases. Unlike globin RNA, in which the start codon is present in all
RNase-protected fragments, many of the HCV IRES-protected fragments do
not contain the initiator AUG codon.
It is clear that, even when we adopt the conventional
secondary-structure folding pattern for the HCV IRES (Fig. 5B)
(4, 10, 11), we see three separate core domains: 1+4,
containing the pseudoknot structure; 2+3, a duplex stem between
stem-loop IIId and stem-loops IIIb and IIIc; and 5, which contains the
AUG start codon and the first 30 bases of coding sequence. These
results suggest to us a minimum of three different sites on the 80S
ribosome which protect substantial IRES core sequences while ensuring
accurate initiation. In this regard, it is likely that core sequence 5 occupies the ribosomal mRNA binding groove with its AUG triplet precisely aligned with initiator tRNA in the P site. HCV IRES core
domain 5 would thus occupy the position analogous to that of the RBS of
a capped mRNA.
Which factors or ribosomal proteins are responsible for protection of
such a large amount of the HCV IRES RNA? The HCV IRES has a distinctive
mechanism for ribosomal complex formation (27) and,
therefore, may require novel factors and/or a heretofore unknown order
of assembly; in this sense, the HCV-80S ribosomal initiation complex
may be unlike typical 80S ribosomal complexes with capped mRNAs and may
contain unknown factors or factors normally not present after ribosomal
complex initiation. Since our 80S ribosomal complexes do not include
purified components and are formed in RRL, all cellular factors or
proteins are available for the perhaps unique HCV-80S ribosomal
initiation complex formation. In this light, perhaps the ribosomal
entity responsible for protecting core domain 2+3 could be eukaryotic
initiation factor 3 (eIF3). While this initiation factor is thought to
exit the ribosome upon 60S subunit addition in the binding of
conventional capped mRNAs (8), the HCV IRES shows a strong
and direct binding reaction to purified eIF3 (5, 17, 27,
29). Therefore, an HCV IRES domain remaining bound to eIF3 could
lead to a delayed exit of this factor from 80S ribosomes, resulting in
the protection of core domain 2+3 observed here.
The protection from RNase of our core domain 1+4, which contains the
HCV IRES pseudoknot region, leads us to postulate the involvement of a
third ribosomal domain. We have found in collaboration with J. Gomez
and colleagues of Barcelona, Spain, that the host pre-tRNA processing
enzyme RNase P can cleave HCV RNA at two sites, one of which is located
in the HCV IRES near the initiator AUG codon (A. Nadal, M. Martell,
J. R. Lytle, H. D. Robertson, B. Cabot, J. I. Esteban,
R. Esteban, J. Guardia, and J. Gomez, submitted for
publication). Since RNase P, in the absence of external guide sequences, cleaves only tRNA-like structures, this result suggests that
a tRNA-like structure exists within the HCV IRES at a site near our
pseudoknot-containing core domain 1+4. Ribosomes bind tRNA at three
sites: the A site, the P site, and the E site. We suggest that this
tRNA-like domain of the HCV IRES, and perhaps analogous domains in
other IRESs, could occupy one of the tRNA binding sites of the
initiating 80S ribosome.
Our work here focuses on the HCV RNA protected by 80S ribosomal
initiation complexes, while work done by others has determined locations of HCV RNA involved in 40S preinitiation complexes (with or
without additional factors) (17, 27). Significantly,
several of our domains have already been shown to be important regions for binding. Core domain 3 has been shown to be involved in eIF3 binding to the HCV IRES (27). Similarly, bases from our
core domains 4 and 5 have been shown to interact with the 40S subunit alone using the indirect toeprinting assay to measure 40S subunits' ability to block primed DNA synthesis (27). Several
laboratories have determined stem-loop IIId to be important for
translation (16, 17), and we find these bases in a
protected, but not core, domain. This region may be more important for
an earlier initiation step in protein synthesis and not as strongly
protected at the peptide formation stage.
The HCV RNA sequences protected by the 48S preinitiation complex (Fig.
1I and J) are very similar to those protected by the 80S ribosomal
complex. This result is similar to that described previously for
-globin mRNA: the 48S-protected region of -globin mRNA contains
the entire RBS sequence but also additional 5' sequence from the 5' UTR
(21). Initially, the large amount of 48S-protected HCV RNA
may seem surprising. However, the recent cryoelectron microscopy map
made by Spahn et al. (30) of the HCV IRES complexed with
the 40S ribosomal subunit shows that almost all of the HCV RNA binds to
the 40S subunit on the side opposite where the 60S subunit associates.
Only stem-loop II wraps around the 40S subunit to the 60S side, and
this domain has so far not been shown to be protected from digestion by
either the 48S or the 80S complex. This finding implies that the 60S
subunit would not afford any additional protection from RNase and that
the protection of the HCV IRES by the 48S preinitiation complex could
indeed be very similar to that provided by the entire 80S ribosomal complex.
The HCV RBS is the largest RBS found to date. The generality of this
HCV RBS paradigm for other cellular and viral IRESs remains to be seen.
However, the discontinuous nature and considerable size of the HCV RBS
are likely to be shared by related, viral IRESs. Confirmation of a
pattern of common structural features will help explain the action and
specificity of IRESs.
This work was supported in part by grant U35-8010 from the New
York State Science and Technology Foundation's Centers for Advanced
Technology Program and by Public Health Service grant DK-56424 from NIH.
We thank S. Genus and P. Mercado for excellent technical assistance, E. Kanner for help in growing DNA plasmids, K. Browning and S. Lemon for
gifts of plasmid DNA, Z. Kurylo-Borowska for the gift of edeine, and J. Gomez for helpful discussions.
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Abstract
Introduction
