Fig. 5. Purification of the viral RNA binding proteins. (A) Purification scheme (for details, see Materials and methods). (B) Cross-link assays performed with the labeled BVDV 3′NTR probe and cytoplasmic extract, the heparin–Sepharose flowthrough and fractions that derived from the final MonoQ chromatography at ∼350–420 mM KCl, respectively. The viral RNA binding proteins are indicated.

Fig. 8. The [NF90/NFAR-1, NF45, RHA] complex supports the interaction of both termini of the viral RNA genome. (A) 5′–3′coprecipitation assay. The top panel shows a schematic representation of the assay carried out with biotinylated 5′NTR and ³²P-labeled 3′NTR (see Materials and methods). Bottom panel: precipitation of the 3′NTR (as indicated) was performed with the naked RNAs, in the presence of the [NF90/NFAR-1, NF45, RHA] complex ([]) or in the presence of bovine serum albumin (BSA) (20-fold molar excess with respect to the complex). (B) Model of a circularized viral RNA mediated by [NF90/NFAR-1, NF45, RHA]. The replication complex, which is supposed to form at the immediate 3′-terminus of the viral RNA, is depicted in light gray; translating ribosomes are represented in shaded black. The [NF90/NFAR-1, NF45, RHA] complex is schematized by four differently shaded circles (see text for details).

Fig. 4. The same set of cellular proteins binds specifically to each of the BVDV non-translated regions. (A) Scheme of the secondary/tertiary structure of the BVDV 5′NTR (Pestova and Hellen, 1999). The minimal IRES element is boxed. The position of ‘mutation 4’ which contains four nucleotide changes at positions 2–5 of the BVDV genome (Yu et al., 2000) is indicated. The positions of the AUG translation initiation codon and of restriction sites (EagI, PstI) used to generate different run-off transcripts are also indicated. (B) Cross-linking and competition experiments with BVDV 5′NTR transcripts (conditions as in Figures 2 and 3). The left panel shows cross-linking assays with the following probes: control RNA, BVDV 3′NTR, BVDV 5′NTR (PstI) and BVDV 5′NTR (EagI). The middle panel shows a side-by side experiment performed with wt 5′NTR probe and a 5′NTR probe (mut4) that does not form hairpin Ia. The right panel shows assays with the labeled BVDV 3′NTR probe performed in the absence and presence of a 50- and 200-fold molar excess of unlabeled 5′NTR competitor, respectively.

Fig. 2. A set of cellular proteins associates specifically with the pestiviral 3′NTR. (A) UV cross-linking/label transfer experiments performed with [³²P]UTP-labeled RNA probes (∼10 ng) and extracts of BHK-21 cells. A non-related control RNA with the same specific activity was compared side-by-side with a transcript corresponding to the BVDV 3′NTR. The control RNA derived from transcription of the Bluescript polylinker; the BVDV transcript was described previously (Yu et al., 1999). Cytoplasmic extracts (20 µg protein/assay) of mock-transfected and BVDV DI9c transfected (+) BHK-21 cells were used as protein sources; all reactions were supplemented with 20 µg of tRNA. Proteins found to be charged with labeled RNA were named according to their molecular weight as they appeared on the analytical SDS–PAGE. PTB, which was found to interact with the control RNA, is also indicated; other control RNAs yielded a blank gel (not shown). Note that [³²P]GTP-labeled transcripts yielded identical results (not shown). (B) Competition experiment with different pestiviral 3′NTR transcripts. UV cross-linking was performed as described above with labeled control RNA probe (lanes 1–3) and labeled BVDV 3′NTR probe (lanes 4–7) and in the absence or presence of unlabeled competitor RNA. Lanes 1 and 4, without competitor; lanes 2 and 5, in the presence of a 200-fold molar excess of unlabeled control RNA competitor; lanes 3 and 6, ∼200-fold molar excess of BVDV 3′NTR competitor; lane 7, ∼200-fold molar excess of CSFV 3′NTR competitor.

Fig. 1. Organization of genomic and subgenomic BVDV RNAs, and secondary structure of the BVDV DI9c 3′NTR. (A) Physical map of the full-length BVDV genome and the subgenomic replicon ‘DI9c’. The NTRs are indicated as single lines; the genetic units encoding the viral proteins are represented as shaded boxes. Enzymatic activities associated with the viral proteins are indicated (Lindenbach and Rice, 2001). The proteolytic cleavage sites in the polyprotein are marked as follows: arrow, cleavage by NS3/NS4A; circle, cleavage by signal peptidase; A, autoproteolytic activity; ?, uncertain. (B) Structure of the BVDV DI9c 3′NTR. The depicted region corresponds to nucleotides 12 090–12 281 of the genome of the DI9c ancestor BVDV CP7 (Meyers et al., 1996); numbering starts at the translational stop codon (italics). The UGA segments are boxed. An arrow marks the border between the variable 3′V and conserved 3′C region. Residues that are 100% conserved in all pestivirus strains are underlined. The depicted secondary structure summarizes the results of published experimental probing data on the structure of SLI, SS and SLII (Yu et al., 1999) and novel data on SL_stop. They were superimposed on an mfold-aided structure prediction of the BVDV DI9c 3′NTR at lowest ΔG_max. Nucleotides that were highly exposed to single-strand specific RNases and/or chemical modification are indicated in dark gray; slightly exposed nucleotides are indicated in light gray. Residues in SL_stop that were specifically accessible to RNases are marked by arrows: white (weak); black (strong). The partial accessibility of the A-U-rich stem of SL_stop (especially its upstream portion) to nucleases and chemicals was explained by its low stability (see text; O.Isken, C.W.Grassmann, H.Yu and S.-E.Behrens, in preparation). Representative examples of chemical and enzymatic probing experiments of the BVDV 3′NTR are included in the Supplementary data.

Fig. 6. The viral RNA binding proteins correspond to the ‘NFAR group of proteins’. (A) RMSA assay with αNF90. The experiment was performed as described in Materials and methods using ³²P-labeled BVDV 3′NTR RNA. Except for the control lane (right), the RNA was incubated with increasing amounts of cytoplasmic extract (right to left: 5, 20 and 50 ng) and a constant amount of αNF90 or control antiserum. (B) PolyU pulldown experiment. The experiment was carried as described in Materials and methods using polyadenylated BVDV 3′NTR (spec.) and polyadenylated control RNA (non spec.). The binding behavior of in vitro translated [³⁵S]-labeled NF90/NFAR-1 was compared with that of a control protein (luciferase). (C) Poly(I):(C) purification of the [NF90/NFAR-1, NF45, RHA] complex following the protocol described (Liao et al., 1998). The top panel shows aliquots of total HeLa cytoplasm and of fractions that eluted at high KCl (left to right ∼0.6–1 M) from poly(I):(C) analyzed by SDS–PAGE and silver staining. RHA, NF90/NFAR-1 and NF45 are indicated. The bottom panel (left and middle) shows the UV cross-linking assay with HeLa cytoplasmic extract (20 µg of protein) performed side-by-side with an aliquot (50 ng of protein) of the poly(I):(C) fraction 11. The autoradiograph of the experiment with fraction 11 was contrasted differently to reveal the under-represented band of RHA as well. The right bottom panel shows western blot analysis of fraction 11 performed with antisera against all three proteins. (D) αNF90 antiserum cross-reacts with proteins that exhibit essentially the same retardation factor as p110 and p64 on SDS–PAGE. The figure shows a side-by-side projection of a cross-link experiment (left) and a western blot experiment (right), each performed with total HeLa cytoplasmic extract. The western blot analysis was performed with a mixture of αNF45 and αNF90 (Kao et al., 1994); αNF90 alone stained the same pattern of proteins except for NF45 (not shown).

Fig. 3. The cellular proteins bind to the 3′V portion of the 3′NTR. (A) UV cross-linking assay performed with shorter versions of the BVDV 3′NTR (conditions as described in Materials and methods and Figure 2). The upper panel shows the scheme of the BVDV 3′NTR transcript (Yu et al., 1999) (the black box represents a residual part of the ORF) indicating the positions of restriction sites (AatII, DraI) used to generate different run-off versions (for numbering see Figure 1B). The lower panel shows the cross-linking assay with the different transcripts. Note that experiments with transcripts that comprised only the 3′C portion of the 3′NTR yielded blank gels (not shown). (B) Conserved sequence elements (‘UGA boxes’) in the pestiviral 3′V region. The table shows an alignment of the sequence elements and their position in the genomic 3′V region of representative virus strains (for numbering see Figure 1B). The consensus sequence is depicted below. One hundred percent conserved nucleotides are underlined or marked in gray. Some strains contain several UGA box elements; in these cases, the UGA_pos.cons box is indicated by an asterisk (see text). (C) Viral RNA–cellular protein interaction studies with RNA probes encoding individual UGA box elements. The upper panel shows schemes of the UGA1 and UGA2 transcripts (for numbering see Figure 3B). The left lower panel shows cross-linking experiment performed with identical molar amounts of the following probes: control RNA (lane 1), BVDV 3′NTR (lane 2), UGA2 (lane 3) and UGA1 (lane 4). The right lower panel shows competition experiments carried out with the BVDV 3′NTR probe (lanes 5–9) and the UGA2 probe (lanes 10–14). Lanes 5 and 10, without competitor; lanes 6 and 11, in the presence of a 200-fold molar excess of unlabeled control RNA competitor; lanes 7 and 12, ∼200-fold excess of BVDV 3′NTR competitor; lanes 8 and 13, ∼200-fold excess of UGA2 competitor; lanes 9 and 14, ∼200-fold excess of UGA1 competitor.

Fig. 7. Importance of the NFAR proteins for BVDV replication. (A) Mutations in the 3′V region inhibit protein binding and RNA replication. The top panel shows the structure of the upstream 62 residues of the wild type (wt) BVDV DI9c 3′V region compared with a mutant that contains nucleotide changes (marked by asterisks) at three conserved positions in the 5′UGA box and the UGA_pos.cons. box, respectively. The mutant RNA was shown by chemical modification (see Supplementary data) to form a different hairpin structure; the accessible residues are indicated as in Figure 1B. The structure of the residual 3′NTR was found unchanged (not shown). The bottom left panel shows a cross-linking assay performed with wt and mutant BVDV 3′NTR transcripts. The bottom right panel shows the replication capacity of the wt and the mutant replicon RNA. Replication was monitored by RNase protection of progeny positive-strand RNA at 9, 11 and 17 h post transfection into MDBK cells (for experimental details, see Behrens et al., 1998). The replication capacity of the mutant is compared with that of the wt (considered 100%). The diagram shows average values of three transcription/transfection experiments. (B) siRNA-mediated depletion of cytoplasmic RHA inhibits BVDV replication. The top left panel shows a representative western blot experiment analyzing the amount of RHA in the cytoplasm of Huh-7 cells at days 3 and 4 after the first round of transfection with a control siRNA and an anti-RHA siRNA, respectively. The top right panel shows average values of the concentration of RHA at day 4 as determined in three independent siRNA transfection experiments; the error bar indicates the mean deviation. Note that the level of depletion was found to be in the same range at days 3 and 4. The bottom left panel shows a representative RNase protection experiment measuring the replication capacity of BVDV DI9c RNA in control and anti-RHA siRNA transfected cells, respectively. Left, original RNA probe used for protection: lanes 1 and 2, protected progeny positive-strand RNA at 18 h post transfection of BVDV RNA (i.e. at day 3 after the first round of siRNA transfection); lanes 3 and 4, at ∼36 h post transfection of BVDV RNA (at day 4 after the first round of siRNA transfection). Transfection experiments with replication-defective BVDV RNA (Behrens et al., 1998) resulted in a blank gel (not shown). Right: diagram summarizing the data of three independent replication experiments; the error bar indicates the mean deviation. Note that analogous effects on the level of cytoplasmic RHA and viral replication were observed with two different anti-RHA siRNAs (see Materials and methods) and that, compatible with the idea of a limiting host factor, replication was inhibited in the same way with higher amounts of transfected BVDV RNA (e.g. 5 pmol instead of 1 pmol per 3 × 10⁶ cells, not shown).