Specific interaction of eukaryotic translation initiation factor 3 with the 5' nontranslated regions of hepatitis C virus and classical swine fever virus RNAs.

Abstract

Translation of hepatitis C virus (HCV) and classical swine fever virus (CSFV) RNAs is initiated by cap-independent attachment (internal entry) of ribosomes to the approximately 350-nucleotide internal ribosomal entry segment (IRES) at the 5' end of both RNAs. Eukaryotic initiation factor 3 (eIF3) binds specifically to HCV and CSFV IRESs and plays an essential role in the initiation process on them. Here we report the results of chemical and enzymatic footprinting analyses of binary eIF3-IRES complexes, which have been used to identify the eIF3 binding sites on HCV and CSFV IRESs. eIF3 protected an internal bulge in the apical stem IIIb of domain III of the CSFV IRES from chemical modification and protected bonds in and adjacent to this bulge from cleavage by RNases ONE and V1. eIF3 protected an analagous region in domain III of the HCV IRES from cleavage by these enzymes. These results are consistent with the results of primer extension analyses and were supported by observations that deletion of stem-loop IIIb or of the adjacent hairpin IIIc from the HCV IRES abrogated the binding of eIF3 to this RNA. This is the first report that eIF3 is able to bind a eukaryotic mRNA in a sequence- or structure-specific manner. UV cross-linking of eIF3 to [32P]UTP-labelled HCV and CSFV IRES elements resulted in strong labelling of 4 (p170, p116, p66, and p47) of the 10 subunits of eIF3, 1 or more of which are likely to be determinants of these interactions. In the cytoplasm, eIF3 is stoichiometrically associated with free 40S ribosomal subunits. The results presented here are consistent with a model in which binding of these two translation components to separate, specific sites on both HCV and CSFV IRESs enhances the efficiency and accuracy of binding of these RNAs to 40S subunits in an orientation that promotes entry of the initiation codon into the ribosomal P site.

Full text

Page 1

JOURNAL OF VIROLOGY,
0022-538X/98/$04.00?0
Copyright © 1998, American Society for Microbiology
June 1998, p. 4775–4782 Vol. 72, No. 6
Specific Interaction of Eukaryotic Translation Initiation Factor 3
with the 5? Nontranslated Regions of Hepatitis C Virus
and Classical Swine Fever Virus RNAs
DARIA V. SIZOVA,1VICTORIA G. KOLUPAEVA,1,2TATYANA V. PESTOVA,1,2
IVAN N. SHATSKY,1AND CHRISTOPHER U. T. HELLEN2*
A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, 119899 Moscow,
Russia,1and Department of Microbiology and Immunology, Morse Institute for
Molecular Genetics, State University of New York Health Science
Center at Brooklyn, Brooklyn, New York 11203-20982
Received 9 October 1997/Accepted 12 February 1998
Translation of hepatitis C virus (HCV) and classical swine fever virus (CSFV) RNAs is initiated by cap-
independent attachment (internal entry) of ribosomes to the ?350-nucleotide internal ribosomal entry seg-
ment (IRES) at the 5? end of both RNAs. Eukaryotic initiation factor 3 (eIF3) binds specifically to HCV and
CSFV IRESs and plays an essential role in the initiation process on them. Here we report the results of
chemical and enzymatic footprinting analyses of binary eIF3-IRES complexes, which have been used to identify
the eIF3 binding sites on HCV and CSFV IRESs. eIF3 protected an internal bulge in the apical stem IIIb of
domain III of the CSFV IRES from chemical modification and protected bonds in and adjacent to this bulge
from cleavage by RNases ONE and V1. eIF3 protected an analagous region in domain III of the HCV IRES from
cleavage by these enzymes. These results are consistent with the results of primer extension analyses and were
supported by observations that deletion of stem-loop IIIb or of the adjacent hairpin IIIc from the HCV IRES
abrogated the binding of eIF3 to this RNA. This is the first report that eIF3 is able to bind a eukaryotic mRNA
in a sequence- or structure-specific manner. UV cross-linking of eIF3 to [32P]UTP-labelled HCV and CSFV
IRES elements resulted in strong labelling of 4 (p170, p116, p66, and p47) of the 10 subunits of eIF3, 1 or more
of which are likely to be determinants of these interactions. In the cytoplasm, eIF3 is stoichiometrically
associated with free 40S ribosomal subunits. The results presented here are consistent with a model in which
binding of these two translation components to separate, specific sites on both HCV and CSFV IRESs enhances
the efficiency and accuracy of binding of these RNAs to 40S subunits in an orientation that promotes entry of
the initiation codon into the ribosomal P site.
Hepatitis C virus (HCV) is the etiologic agent of most cases
of posttransfusion hepatitis (1). It is a member of the genus
Flaviviridae and is related to pestiviruses such as classical swine
fever virus (CSFV) and bovine viral diarrhea virus. Members
of this genus are enveloped positive-stranded RNA viruses.
Their genomes encode a single polyprotein that is cleaved by
virus-encoded and cellular proteinases to yield the various viral
capsid and nonstructural proteins.
Initiation of translation of HCV and pestivirus polyproteins
occurs as a result of ribosomal attachment to the ?350-nucle-
otide (nt) internal ribosomal entry segment (IRES) (23, 28,
33). HCV and CSFV IRESs comprise almost the entire 5? un-
translated region and up to 30 nt of coding sequence. Although
the sequences of these two IRESs differ at almost 50% of base
positions, many of these nucleotide differences are covariant
substitutions, indicative of conserved secondary and/or tertiary
structures. HCV and CSFV IRESs contain four major struc-
tural domains, designated I to IV (see Fig. 1), and a pseudo-
knot upstream of the initiation codon (5, 11, 34). The struc-
tural integrity of the pseudoknot and of these domains is
important for IRES function, but the roles that they play in
internal initiation have not been defined (10, 11, 22, 25, 26, 28,
34, 35).
Many other aspects of the mechanism of IRES-mediated
initiation are also obscure. To identify the factors that are in-
volved in initiation on HCV and CSFV IRESs and to charac-
terize their functions in it, we reconstituted this process in vitro
up to the stage of 48S preinitiation complex formation from
individual purified translation components (mRNA, aminoacy-
latedinitiatortRNA,40Ssubunits,andeukaryoticinitiationfac-
tors [eIFs]) (22). These 48S complexes are competent to com-
plete the remaining steps in initiation of translation, including
joining with the large (60S) ribosomal subunits to form 80S
complexes and formation of the first peptide bond. 40S ribo-
somal subunits bind directly to these IRESs without additional
factors to form binary complexes and require only eIF2, GTP
and aminoacylated initiator tRNA to assemble into a 48S com-
plex at the authentic initiation codon. The process of 48S com-
plex formation on HCV and CSFV IRESs is exceptional be-
cause it does not involve the canonical initiation factors eIF4A,
eIF4B, or eIF4F. eIF3 enhances 48S complex formation and is
absolutely required for 80S complex formation on these IRESs.
Assembly of ribosomal complexes was assessed in these exper-
iments by primer extension inhibition (toeprinting) and su-
crose density gradient centrifugation. In the course of these
experiments, we noted that eIF3 arrested primer extension at
AA243–244 on the HCV IRES and at AC250–251 and U304
on the CSFV IRES. These stops are all within domain III. Toe-
printing involves cDNA synthesis with reverse transcriptase
* Corresponding author. Mailing address: Department of Microbi-
ology and Immunology, Morse Institute for Molecular Genetics, State
University of New York Health Science Center at Brooklyn, 450 Clark-
son Ave., Box 44, Brooklyn, NY 11203-2098. Phone: (718) 270-1034.
Fax: (718) 270-2656. E-mail: chellen@netmail.hscbklyn.edu.
4775

Page 2

(RT) on a template RNA to which a ribosome or protein is
bound. cDNA synthesis is arrested either directly by the bound
complex, yielding a stop or toeprint at its leading edge, or in-
directly by stabilization of adjacent helices (3, 9). eIF3 there-
fore binds to HCV and CSFV IRESs, most probably in the
large central domain III, but toeprinting did not permit the
binding site to be determined precisely.
We have now investigated the interaction of eIF3 with HCV
and CSFV IRES elements in detail by using UV cross-linking
and chemical and enzymatic footprinting techniques. Our re-
sults show that eIF3 bound specifically and exclusively to the
apical region of domain III of both HCV and CSFV, resulting
in the formation of stable ribonucleoprotein complexes. UV
cross-linking showed that this interaction involves the p170,
p116, p66, and p47 subunits. This is the first time that a specific
interaction of eIF3 with an eukaryotic mRNA has been de-
scribed. The role of this and other interactions of HCV and
CSFV IRESs with translation components in IRES-mediated
initiation is discussed.
MATERIALS AND METHODS
Plasmids. Plasmids pHCV(40–372).NS? and pCSFV(1–442).NS? contain HCV
and CSFV sequences, respectively, linked to a truncated influenza virus NS?
reporter (22, 24). p?B-CAT, p?E-CAT, and p?F-CAT were derived by deletion
of HCV nt 26 to 67, 172 to 227, and 229 to 238, respectively, from plasmid
pWT-CAT, which contains the full HCV 5? untranslated region linked to a
chloramphenicol acetyltransferase (CAT) reporter cistron (26).
Transcription of HCV and CSFV RNAs. HCV and CSFV RNAs were tran-
scribed in vitro with T7 RNA polymerase and either with or without [32P]UTP
(?3,000 Ci/mmol; ICN Radiochemicals, Irvine, Calif.) from plasmids that had
been linearized at appropriate sites. These restriction sites included the EcoRI
site after the NS? cistron in the HCV-NS? and CSFV-NS? plasmids, the BamHI
site at the junction of the HCV and NS? sequences in the HCV-NS? plasmid, and
the HindIII site after the CAT cistron in the HCV-CAT plasmids. RNA was
purified with Nuc-Trap columns (Stratagene, La Jolla, Calif.) as described pre-
viously (19). Radiolabeled RNAs had specific activities of ?400,000 cpm/?g.
Purification of eIF3. eIF3 was purified from rabbit reticulocyte lysate (RRL;
Green Hectares, Oregon, Wis.) by established procedures (15). The purity and
quality of this factor (see Fig. 6) were equal to or greater than those described by
us previously (20–22).
Assembly and toeprint analysis of eIF3-HCV IRES complexes. Complexes
between eIF3 (7.2 pmol) and HCV RNA (2.4 pmol) were allowed to form during
incubation for 5 min at 30°C in binding buffer (20 mM Tris-HCl [pH 7.4], 100
mM KCl, 2 mM magnesium acetate, 1 mM dithiothreitol). These complexes were
analyzed by primer extension with primer 5?-CGCAAGCACCCTATC-3? (com-
plementary to HCV nt 295 to 309) and avian myeloblastosis RT (Promega,
Madison, Wis.) as described previously (20), except that [?-32P]dATP was not
present in the extension reaction mixtures and primers were instead first end
labelled with [?-32P]ATP (?6,000 Ci/mmol; ICN Radiochemicals) by using T4
polynucleotide kinase.
Chemical and enzymatic footprint analysis of binary eIF3-IRES and ternary
eIF3-40S subunit-IRES complexes. RNP complexes were assembled from eIF3,
40S subunits, and HCV or CSFV RNAs as described above, with the amounts of
these translation components described below. Free or protein-bound IRES-
specific RNAs in binding buffer were digested enzymatically by incubation with
either RNase V1(Pharmacia, Piscataway, N.J.) or RNase ONE (Promega) or
were modified chemically either at N-1 of adenines by incubation with dimethyl
sulfate (DMS) or at N-3 of uracils and at N-1 of guanines by incubation with
1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluene sulfate
(CMCT), as described previously (13). Cleaved or chemically modified RNAs
were hybridized to appropriate end-labelled DNA primers, and primer extension
was done exactly as described previously (13). Primers 5?-CTCGTTTGCGGAC
ATGCC-3? and 5?-GGGATTTCTGATCTCGGCG-3? (complementary to dif-
ferent parts of the NS? coding sequence), 5?-GCAACTGACTGAAATGCC-3?
(complementary to part of the CAT coding sequence) and 5?-CGCAAGCACC
CTATC-3? (complementary to HCV nt 295 to 309) were used for analysis of
complexes formed on CSFV-NS?, HCV-NS?, and HCV-CAT mRNAs, as appro-
priate. cDNA products were analyzed by electrophoresis on 10% polyacryl-
amide–8 M urea gels.
UV cross-linking. UV cross-linking of binary IRES-eIF3 complexes, binary
IRES-40S subunit complexes, and ternary IRES-eIF3-40S subunit complexes was
done essentially as described previously (19, 22), with [32P]UTP-labelled CSFV
nt 1 to 442 and HCV nt 40 to 372 RNAs, as appropriate.
RESULTS
Probing the structure of the CSFV IRES. Models of the
secondary and tertiary structures of the CSFV and HCV IRES
have been proposed on the basis of minimum free-energy
calculations, phylogenetic comparison, and genetic analysis (5,
FIG. 1. Summary of sites within nt 127 to 390 of the CSFV IRES that are modified by DMS and CMCT (A) or cleaved by RNase V1and RNase ONE (B). These
chemical and enzymatic probes are indicated by symbols at the upper right of each panel. The results are displayed on a structure that is based on previous proposals
(5, 34) and that takes into account the results presented here. The nomenclature used to describe CSFV domains and hairpins is adapted from proposals (11) for the
HCV IRES.
4776SIZOVA ET AL.J. VIROL.

Page 3

11, 28, 30, 34). These models suggest that both RNAs are
extensively base paired and consist of four major structural
domains. Domain III consists of a basal pseudoknot and large
irregular helix with several branching hairpins designated IIIa
to IIIe. Domain IV of HCV contains the initiation codon
within a single hairpin (11). The structural model of the HCV
IRES has been experimentally verified and in part revised in
light of the results of analysis with structure- and sequence-
specific chemical and enzymatic probes (5, 10, 34). Models for
the CSFV IRES have not been experimentally verified, and we
therefore undertook a similar structural analysis of CSFV do-
mains III and IV. In these experiments, we used RNase V1
from cobra venom, which cleaves double-stranded or other
base-paired regions (6); RNase ONE from Escherichia coli,
which cleaves single-stranded RNA without base specificity
(14); DMS, which reacts with N-1 of adenine residues and to a
lesser extent with N-3 of cytosine residues; and CMCT, which
reacts with N-3 of uracil and N-1 of guanine residues (17).
Chemical modification at these positions is inhibited by base
pairing. Modification of these positions and strand scission
both arrest cDNA elongation by RT and can therefore be
analyzed by primer extension. Chemical modification results in
arrest of primer extension at the nucleotide immediately 3? to
the modified position, and enzymatic cleavage results in arrest
of primer extension at the nucleotide on the 3? side of the
cleaved bond. Numbering of the residues below indicates ei-
ther the chemically modified base or the nucleotide on the 3?
side of the cleaved bond.
The CSFV IRES was extensively modified by DMS and
CMCT (Fig. 1A). Residues that were modified by these chem-
icals were mostly clustered downstream of the pseudoknot,
within the long interhelical strand of the pseudoknot, and at
the apices of each of the proposed hairpins IIIa to IIIe in
domain III. These results are almost wholly consistent with
models of domain III and of the pseudoknot that have been
proposed on the basis of phylogenetic comparisons (5, 34).
However, they do not support the proposed structure of do-
main IV and instead indicate that this regions is almost entirely
single stranded under ionic conditions that are used for effi-
cient CSFV IRES-mediated translation in vitro.
The pseudoknot and the adjacent hairpins IIId2 and IIIe are
resistant to cleavage by both RNase ONE and RNase V1(Fig.
1B). The low reactivity of many DMS- and CMCT-insensitive
residues in helices to RNase V1and of DMS- and CMCT-
sensitive regions in unpaired regions to RNase ONE suggests
that the pseudoknot adopts a very compact structure and is
thus sterically inaccessible to enzymatic cleavage. In contrast,
the upper half of domain III was cleaved extensively by RNase
V1in a manner that is wholly consistent with the model pro-
posed by Brown et al. (5). The small number of bonds that
FIG. 2. Chemical and enzymatic footprinting of the eIF3-CSFV IRES complex. Polyacrylamide-urea gel fractionation of cDNA products obtained after primer
extension shows the sensitivity of CSFV RNA upstream of nt 265 to cleavage by RNase V1(lanes 1 and 2) either alone (lane 1) or complexed with eIF3 (lane 2) (A),
the sensitivity of CSFV RNA upstream of nt 259 to cleavage by RNase ONE (lanes 1 and 2) either alone (lane 2) or complexed with eIF3 (lane 1) (B), and the reactivity
of CSFV RNA upstream of nt 252 to modification by CMCT (lanes 1 and 2) either alone (lane 2) or complexed with eIF3 (lane 1) (C). cDNA products obtained after
primer extension of untreated CSFV RNA are shown in lanes 3 of panels A and B. A dideoxynucleotide sequence generated with the same primer was run in parallel
on each gel. The positions of protected residues are indicated to the right of each panel, and the positions of CSFV nucleotides at 50-nt intervals are indicated to the
left of each panel.
VOL. 72, 1998INTERACTION OF eIF3 WITH HCV AND CSFV IRES ELEMENTS4777

Page 4

were cleaved by RNase ONE are clustered at the junction
between domains II and III, in two internal bulges within the
stem of hairpin IIIb, and in the apical loop of hairpin IIId1.
The observation that these unpaired regions and some helical
regions in the upper half of domain III are readily accessible to
enzymatic cleavage indicates that they must be exposed.
Specific interaction of eIF3 with domain III of the CSFV
IRES. To localize eIF3 binding sites on the CSFV IRES, ri-
bonucleoprotein complexes were assembled from these two
moieties under buffer conditions and at a temperature similar
to those that are used normally for translation of CSFV RNA
(22). eIF3 was present in a threefold molar excess over IRES-
containing RNA. We first used RNase V1to identify cleavage
sites within the CSFV IRES that are occluded by the binding
of eIF3, because this enzyme cleaves the IRES at numerous
sites in close proximity to the toeprint at AC250–251 that is
caused by binding of eIF3 (22). eIF3 protected residues
GUG198–200; CGA216–218, A225, C232, and ACA248–250
from cleavage by RNase V1(Fig. 2A, lane 2). These protected
regions flank two internal bulges in hairpin IIIb that are sen-
sitive to cleavage by RNase ONE, and we therefore next as-
sayed the protection of this region of the CSFV IRES by eIF3
against cleavage by this enzyme. These two bulges were bound
by eIF3: residues GU227–228 and, particularly, AUG220–222
were protected by eIF3 from cleavage by RNase ONE (Fig. 2B,
lane 1).
Binary CSFV IRES-eIF3 complexes were next footprinted
with the base-specific chemical probes CMCT and DMS. Bind-
ing of eIF3 to CSFV RNA protected U221 from modification
by CMCT (Fig. 2C, lane 1) and A218 and A220 from modifi-
cation by DMS (data not shown). Residues within the apical
loops of hairpins IIIa, IIIb, IIIc, IIId1, and IIId2 that are
susceptible to chemical modification were not protected from
such modification by eIF3. Bound eIF3 did not protect resi-
dues in CSFV domains I, II, or IV from enzymatic cleavage or
chemical modification (data not shown).
The results of chemical and enzymatic footprinting are sum-
marized in Fig. 5B. They indicate that eIF3 binds strongly and
specifically to the apical region of domain III of the CSFV
IRES. These results strongly support the results of primer
extension analysis; the toeprint at AC250–251 caused by bound
eIF3 probably corresponds to the leading edge of this factor,
whereas the toeprint at U304 that is strengthened by the bind-
ing of eIF3 occurs within an adjacent helix that is stabilized by
this interaction.
Specific interaction of eIF3 with domain III of the HCV
IRES. To localize the eIF3 binding site on the HCV IRES, we
assembled RNP complexes from these two moieties as de-
scribed above for CSFV and then used RNases V1and ONE as
probes to identify sites in the IRES that are occluded from
cleavage by the binding of eIF3. A single prominent RNase V1
cleavage site at U220 was occluded by binding of eIF3 (Fig. 3A,
lane 2; also see Fig. 7A, lane 2). A number of other sites in the
HCV IRES that appear to be protected by eIF3 from cleavage
coincide with strong stops formed during primer extension on
this highly structured RNA (Fig. 3, lanes 3). Protection of all
but one of these cleavage sites is therefore equivocal and is not
discussed below. The exception is the cleavage site at U212,
which in Fig. 7A (lane 2) was clearly occluded by eIF3. In ad-
dition, RNase ONE cleavage at C204, A214, A215, and U216
was impaired by the binding of eIF3 (Fig. 3B, lane 2). These
five protected sites are located close together within the irreg-
FIG. 3. Enzymatic footprinting of the eIF3-HCV IRES complex. Polyacrylamide-urea gel fractionation of cDNA products obtained after primer extension shows
the sensitivity of HCV RNA upstream of nt 225 to cleavage by RNase V1(lanes 1 and 2) either alone (lane 1) or complexed with eIF3 (lane 2) (A) and the sensitivity
of HCV RNA upstream of nt 277 to cleavage by RNase ONE (lanes 1 and 2) either alone (lane 1) or complexed with eIF3 (lane 2) (B). cDNA products obtained after
primer extension of untreated HCV RNA are shown in lanes 3. A dideoxynucleotide sequence generated with the same primer was run in parallel on each gel. The
positions of protected residues are indicated to the right of each panel, and those of HCV nucleotides are indicated to the left of each panel.
4778SIZOVA ET AL.J. VIROL.

Page 5

ular apical hairpin IIIb (Fig. 4A). The identification of this
region of the HCV IRES as the eIF3 binding site strongly
supports our earlier identification by toeprinting of an inter-
action between eIF3 and the apical half of domain III of this
RNA (22). Bound eIF3 did not protect residues elsewhere in
the HCV IRES from enzymatic cleavage (data not shown).
Abrogation of interaction of eIF3 with the HCV IRES by
deletion of hairpins IIIb and IIIc. The results of chemical and
enzymatic footprinting analysis described above indicate that
eIF3 binds to the apical stem of a cloverleaf-like structure that
constitutes the apical half of domain III of the HCV IRES. To
confirm that elements of this structure are required for this
interaction to occur, we used toeprinting to assess the binding
of eIF3 to the wild-type (wt) HCV IRES and to mutant HCV
IRES transcripts that lack either the IIIb or IIIc hairpin (26).
eIF3 strongly enhanced the arrest of primer extension at
A243 and yielded a new stop at A244 on wt HCV nt 1 to 341
CAT RNA (Fig. 5, lane 2), as reported previously (22). How-
ever, eIF3 did not arrest primer extension on corresponding
mRNAs that lacked either the IIIb hairpin or the IIIc hairpin
(lanes 4 and 6). These results show that eIF3 did not bind stab-
ly to these two mutant IRES elements. They confirm that the
interaction of eIF3 with the wt HCV IRES is highly specific
and indicate that the IIIb and IIIc hairpins are important
determinants of this interaction.
Identification of eIF3 subunits that bind to CSFV and HCV
IRES elements. The mammalian eIF3 complex consists of at
least 10 subunits: p170, p116, p110, p66, p48, p47, p44, p40,
p36, and p35 (16). These subunits are shown in Fig. 6 (lane 1).
Binary eIF3-HCV IRES complexes were assembled with [32P]
UTP-labelled HCV nt 40 to 372 RNA, and UV cross-linking
was then used to identify which of these subunits are in direct
contact with the HCV IRES. The p116 and p66 subunits be-
came prominently labelled after UV irradiation of this com-
plex; strong labelling of p170 and p47 was also apparent (lane
2). The other labelled bands shown in this lane are likely to be
degradation products of p170 and p116. None of the bands
described above was detected if eIF3 was omitted from the
reaction mixtures (data not shown).
In a parallel series of experiments, UV cross-linking of bi-
nary complexes formed by binding eIF3 to [32P]UTP-labelled
CSFV nt 1 to 442 RNA transcripts also resulted in strong la-
belling of the p170, p116, p66, and p47 subunits of eIF3 (see
Fig. 7C, lane 1).
Simultaneous binding of eIF3 and 40S ribosomal subunits
to CSFV and HCV IRES elements. Initiation factor eIF3 is
stoichiometrically associated with native 40S ribosomal sub-
units in the cytoplasm (31). We have previously reported that
40S subunits can bind directly to HCV and CSFV IRESs to
form stable binary complexes (22). In light of the results pre-
sented above, which show that eIF3 also binds specifically to
these IRESs, it was interesting to examine whether eIF3 and
40S subunits could bind simultaneously to the same IRES or
whether binding of one to the IRES precluded binding of the
other.
Inclusion of eIF3 in reaction mixtures in a threefold molar
excess over the HCV IRES resulted in protection of U212 and
U220 from cleavage by RNase V1(Fig. 7A, lanes 1 and 2).
Protection of U212 and U220 by eIF3 was not affected by
inclusion of 40S subunits in reaction mixtures in equimolar
amounts with eIF3 or in a threefold molar excess over eIF3
(lanes 2 to 4). 40S subunits bound to the HCV IRES in these
conditions as shown by sucrose density gradient centrifugation
analysis (22).
In similar footprinting experiments, inclusion of 40S sub-
units in a threefold molar excess over eIF3 did not prevent the
binding of eIF3 to the CSFV IRES: under these conditions,
the presence of eIF3 in threefold molar excess over the RNA
FIG. 4. Summary of the sites within the HCV (A) and CSFV (B) IRESs that are protected from modification by CMCT and DMS and from cleavage by the
single-strand-specific RNase ONE and the double-strand-specific RNase V1. These chemical and enzymatic probes are indicated by symbols at the upper left. The
results are displayed on secondary-structure models of the upper half of domain III that are based on previous proposals (5) (A) and on the results shown in Fig. 1
(B). Domains and subdomains are described according to the nomenclature in reference 11.
VOL. 72, 1998 INTERACTION OF eIF3 WITH HCV AND CSFV IRES ELEMENTS4779