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. 1998 Jun;72(6):4775-82.
doi: 10.1128/JVI.72.6.4775-4782.1998.

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

D V Sizova  1 , V G Kolupaeva, T V Pestova, I N Shatsky, C U Hellen
Affiliations

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

D V Sizova et al. J Virol. 1998 Jun.

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.

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Figures

FIG. 1
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 V1 and 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.
FIG. 2
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.
FIG. 3
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.
FIG. 4
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 .
FIG. 5
FIG. 5
Primer extension analysis of the dependence of eIF3-HCV IRES ribonucleoprotein complex formation on the presence of hairpins IIIb and IIIc in domain III of the IRES. HCV nt 1 to 342 CAT mRNA (lanes 1 and 2), HCV nt 1 to 342(Δ172–227) CAT mRNA (lanes 3 and 4), and HCV nt 1 to 342(Δ229–238) CAT mRNA (lanes 5 and 6) were incubated with (lanes 2, 4, and 6) or without (lanes 1, 3, and 5) eIF3 under standard conditions. Primer 5′-CGCAAGCACCCTATC-3′ was annealed to HCV nt 295 to 309 of these mRNAs and extended with avian myeloblastosis virus RT. The full-length cDNA extension products derived from reverse transcription of HCV nt 1 to 342 CAT, nt 1 to 342(Δ229–238) CAT, and nt 1 to 342(Δ172–227)- CAT mRNAs are marked E, E′, and E", respectively. The cDNA products labelled A243 and A244 on the right terminated at these nucleotides. The positions of HCV nucleotides are indicated on the left.
FIG. 6
FIG. 6
UV cross-linking in vitro of eIF3 and [32P]UTP-labelled HCV nt 40 to 373 RNA. Lanes: 1, Coomassie blue-stained gel of native eIF3; 2, autoradiograph of cross-linked eIF3. The cross-linked sample was digested with cobra venom nuclease and RNases A and T1. Polypeptides were separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis on an SDS–12% polyacrylamide gel. The designation of individual subunits is based on their electrophoretic mobility and that of known standards, except for p170, which was also identified by Western blotting.
FIG. 7
FIG. 7
Enzymatic footprinting and UV cross-linking analysis of ternary eIF3-40S subunit-IRES complexes. (A) Polyacrylamide-urea gel fractionation of cDNA products obtained after primer extension showing the sensitivity of HCV RNA (1 pmol) upstream of nt 273 to cleavage by RNase V1 (lanes 1 to 4) either alone (lane 1), with 3 pmol of eIF3 (lane 2), with 3pmol of eIF3 and 3pmol of 40S subunits (lane 3), or with 3 pmol of eIF3 and 9 pmol of 40S subunits (lane 4). (B) Polyacrylamide-urea gel fractionation of cDNA products obtained after primer extension showing the sensitivity of CSFV RNA (1 pmol) upstream of nt 232 to cleavage by RNase V1 (lanes 1 to 3) either alone (lane 3), with 9 pmol of 40S subunits (lane 2), or with 3 pmol of eIF3 and 9 pmol of 40S subunits (lane 1). cDNA products obtained after primer extension of untreated HCV and CSFV RNA are shown in lane 5 of panel A in lane 4 of panel B, respectively. 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 or CSFV nucleotides are indicated to the left of each panel. (C) Autoradiograph of [32P]UTP-labelled CSFV nt 1 to 442 RNA (1 pmol) after UV cross-linking to 3 pmol of eIF3 (lanes 1 to 4), 9 pmol of eIF3 (lane 6), or 15 pmol of eIF3 (lane 7) and either 3 pmol of 40S subunits (lanes 2 and 5 to 7), 9 pmol of 40S subunits (lane 3), or 15 pmol of 40S subunits (lane 4), followed by RNase digestion and electrophoretic separation of polypeptides by SDS-polyacrylamide gel electrophoresis on an SDS–12% polyacrylamide gel.
FIG. 7
FIG. 7
Enzymatic footprinting and UV cross-linking analysis of ternary eIF3-40S subunit-IRES complexes. (A) Polyacrylamide-urea gel fractionation of cDNA products obtained after primer extension showing the sensitivity of HCV RNA (1 pmol) upstream of nt 273 to cleavage by RNase V1 (lanes 1 to 4) either alone (lane 1), with 3 pmol of eIF3 (lane 2), with 3pmol of eIF3 and 3pmol of 40S subunits (lane 3), or with 3 pmol of eIF3 and 9 pmol of 40S subunits (lane 4). (B) Polyacrylamide-urea gel fractionation of cDNA products obtained after primer extension showing the sensitivity of CSFV RNA (1 pmol) upstream of nt 232 to cleavage by RNase V1 (lanes 1 to 3) either alone (lane 3), with 9 pmol of 40S subunits (lane 2), or with 3 pmol of eIF3 and 9 pmol of 40S subunits (lane 1). cDNA products obtained after primer extension of untreated HCV and CSFV RNA are shown in lane 5 of panel A in lane 4 of panel B, respectively. 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 or CSFV nucleotides are indicated to the left of each panel. (C) Autoradiograph of [32P]UTP-labelled CSFV nt 1 to 442 RNA (1 pmol) after UV cross-linking to 3 pmol of eIF3 (lanes 1 to 4), 9 pmol of eIF3 (lane 6), or 15 pmol of eIF3 (lane 7) and either 3 pmol of 40S subunits (lanes 2 and 5 to 7), 9 pmol of 40S subunits (lane 3), or 15 pmol of 40S subunits (lane 4), followed by RNase digestion and electrophoretic separation of polypeptides by SDS-polyacrylamide gel electrophoresis on an SDS–12% polyacrylamide gel.
FIG. 7
FIG. 7
Enzymatic footprinting and UV cross-linking analysis of ternary eIF3-40S subunit-IRES complexes. (A) Polyacrylamide-urea gel fractionation of cDNA products obtained after primer extension showing the sensitivity of HCV RNA (1 pmol) upstream of nt 273 to cleavage by RNase V1 (lanes 1 to 4) either alone (lane 1), with 3 pmol of eIF3 (lane 2), with 3pmol of eIF3 and 3pmol of 40S subunits (lane 3), or with 3 pmol of eIF3 and 9 pmol of 40S subunits (lane 4). (B) Polyacrylamide-urea gel fractionation of cDNA products obtained after primer extension showing the sensitivity of CSFV RNA (1 pmol) upstream of nt 232 to cleavage by RNase V1 (lanes 1 to 3) either alone (lane 3), with 9 pmol of 40S subunits (lane 2), or with 3 pmol of eIF3 and 9 pmol of 40S subunits (lane 1). cDNA products obtained after primer extension of untreated HCV and CSFV RNA are shown in lane 5 of panel A in lane 4 of panel B, respectively. 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 or CSFV nucleotides are indicated to the left of each panel. (C) Autoradiograph of [32P]UTP-labelled CSFV nt 1 to 442 RNA (1 pmol) after UV cross-linking to 3 pmol of eIF3 (lanes 1 to 4), 9 pmol of eIF3 (lane 6), or 15 pmol of eIF3 (lane 7) and either 3 pmol of 40S subunits (lanes 2 and 5 to 7), 9 pmol of 40S subunits (lane 3), or 15 pmol of 40S subunits (lane 4), followed by RNase digestion and electrophoretic separation of polypeptides by SDS-polyacrylamide gel electrophoresis on an SDS–12% polyacrylamide gel.
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