Autogenous translational regulation of the Borna disease virus negative control factor X from polycistronic mRNA using host RNA helicases

Abstract

Borna disease virus (BDV) is a nonsegmented, negative-strand RNA virus that employs several unique strategies for gene expression. The shortest transcript of BDV, X/P mRNA, encodes at least three open reading frames (ORFs): upstream ORF (uORF), X, and P in the 5' to 3' direction. The X is a negative regulator of viral polymerase activity, while the P phosphoprotein is a necessary cofactor of the polymerase complex, suggesting that the translation of X is controlled rigorously, depending on viral replication. However, the translation mechanism used by the X/P polycistronic mRNA has not been determined in detail. Here we demonstrate that the X/P mRNA autogenously regulates the translation of X via interaction with host factors. Transient transfection of cDNA clones corresponding to the X/P mRNA revealed that the X ORF is translated predominantly by uORF-termination-coupled reinitiation, the efficiency of which is upregulated by expression of P. We found that P may enhance ribosomal reinitiation at the X ORF by inhibition of the interaction of the DEAD-box RNA helicase DDX21 with the 5' untranslated region of X/P mRNA, via interference with its phosphorylation. Our results not only demonstrate a unique translational control of viral regulatory protein, but also elucidate a previously unknown mechanism of regulation of polycistronic mRNA translation using RNA helicases.

Figure 1. Autogenous regulation of BDV X translation from the X/P polycistronic mRNA.

(A) A schematic representation of the 0.8 kb BDV polycistronic X/P mRNA. The numbers indicate nucleotide positions in the X/P mRNA. (B) COS-7 cells and OL cells were transfected with pX/Pwt, and the total cell lysates were used for Western blotting probed with anti-BDV P and X antibodies at 24 and 48 h post-transfection. The expression ratio of X to P was determined after quantitation of band intensities by ImageJ software. (C) OL cells were transfected with pX/Pwt (0.5 µg) and the subcellular localization of X and P was determined by immunofluorescence assay. Arrows indicate cytoplasmic localization of X and P at 72 h post-transfection.

Figure 2. The uORF influences translation of the X and P ORFs.

(A) Schematic representation of mutants of the X/P expression plasmid. The nucleotide sequences substituted in the wt plasmid are indicated. (B) OL cells were transfected with 0.8 µg of each plasmid and at 12 h after transfection cells were harvested and subjected to Western blotting using anti-BDV P and X antibodies. (C) Fold-activation of X and P expression in the cells transfected with mutant plasmids was determined after quantitation of band intensities by ImageJ software. The mean plus S.D. of three independent experiments are shown. **P<0.01, *P<0.05 (Student's t test).

Figure 3. Expression of BDV P enhances translation of the X ORF.

(A) OL cells were transfected with 0.8 µg of the plasmids indicated and, 48 h after transfection, expression of the X and uORF-X fusion proteins was detected by Western blotting using anti-BDV P and X antibodies. The asterisk in the puORF-X/P-transfected panel indicates a non-specific reaction. (B) OL cells were cotransfected with pX/PΔP or puORF-X/PΔP (0.4 µg) and a four-fold dilution series of BDV N (pcN) or P (pcP) expression plasmid (0.00625, 0.025, 0.1 and 0.4 µg). The empty plasmid was used for equilibration of the total amount of transfected plasmid. The cells were harvested at 48 h post-transfection and subjected to Western blotting. The expression of BDV N was detected by anti-BDV N monoclonal antibody. (C) OL cells were transfected with 0.8 µg of the plasmids indicated in the presence or absence of P and, 48 h after transfection, expression of the P and X was detected by Western blotting using anti-BDV P and X antibodies. Relative expression levels of detected proteins were determined after quantitation of band intensities by ImageJ software. The mean plus S.D. of three independent experiments are shown. **P<0.01, *P<0.05 (Student's t test).

Figure 4. Nuclear proteins suppress translation of the X ORF via the 5′ UTR of the mRNA.

(A) A nuclear extract inhibits the initiation of translation of the X ORF. The extracts (1 µg) from OL cells were incubated with in vitro transcribed X- or P-Luciferase fusion RNA (X-Luc, P-Luc) in 20 µl of binding buffer. After the reaction, in vitro translation was performed using a reticulocyte lysate mixture. After the incubation, 10 µl of the mixture was subjected to luciferase assay. Δ5′ UTR indicates luciferase plasmids lacking the 5′ UTR of X/P mRNA. (B) The nuclear factors interact with the X/P UTR. A serial amount of decoy RNA (X/P and M/G UTR) was incubated with the nuclear extract (4 µg) prior to incubation with X-Luc RNA, and then in vitro translation was performed using the reticulocyte lysate mixture. Relative luciferase activities of the X fusion protein were determined. The mean plus S.D. of three independent experiments are shown. **P<0.01, *P<0.05 (Student's t test). (C and D) RNA EMSAs were performed using nuclear extracts of OL cells as described in Methods. For competition, serial amounts of non-labeled own (C) or M/G UTR probes (D) were incubated with the nuclear extract. Arrows indicate shifted bands produced by incubation with the nuclear extract. Bound complexes were resolved from free RNA by electrophoresis in 4% native polyacrylamide gels.

Figure 5. Identification of the 5′ UTR-binding proteins.

(A) Silver staining of sequential RNA column purified proteins is shown. The bands specific for the X/P UTR probe (X/P) are indicated. M/G represents a control RNA column using the 5′ UTR of M/G mRNA. (B) The specificity of identified proteins was determined by Western blotting using antibodies specific to each protein (see Materials and Methods).

Figure 6. Interaction among the 5′ UTR-binding proteins.

(A and B) Interaction of endogenous DDX21 (A) and nucleolin (B) with Flag-tagged UBPs. OL cells were transfected with 10 µg of each Flag-tagged UBP expression plasmid and lysates were immunoprecipitated with anti-FLAG antibody. Western blot analysis was performed using anti-Flag and anti-DDX21 (A) or anti-nucleolin (B) antibodies. (C and D) Pull-down analyses of Flag- and HA-tagged UBPs. OL cells were co-transfected with a combination of 5 µg each of the Flag-tagged and HA-tagged UBPs expression plasmids indicated. At 36 h post-transfection, cell lysates were immunoprecipitated with anti-FLAG antibody. Western blot analysis was performed using anti-HA antibody.

Figure 7. DDX21 is a core protein interacting with the 5′ UTR of X/P mRNA.

(A) Immunoprecipitation RT-PCR analysis of UBPs in BDV-infected cells. BDV-infected OL cells were transfected with Flag-tagged UBP expression plasmids. Cell lysates were prepared with RIPA buffer including ribonuclease inhibitor and immunoprecipitated with anti-Flag antibody. The co-purified RNAs in the immunoprecipitates were recovered in TE buffer as described in the Methods section and RT-PCR analysis was performed using a specific primer set for X/P mRNA. (B) RNA EMSA was performed by incubating ³²P-labelled X/P UTR or M/G UTR riboprobe with GST-tagged DDX21 or nucleolin [Nuc (1234R)]. For competition, non-labeled probes were incubated with each recombinant protein. Bound complexes were resolved from free RNA by electrophoresis in 4% native polyacrylamide gels.

Figure 8. DDX21 causes structural alterations of X/P UTR.

(A and B) In vitro RNA folding assays were performed with 1.0 pmol of ³²P-labeled X/P UTR riboprobe. The labeled riboprobe was incubated with 5 pmol of GST-tagged DDX21 and the folding reactions were detected as described in the Methods section. After the incubation, the reaction mixtures were applied to a 12% native polyacrylamide gel. After electrophoresis, the gel was exposed to X-ray film overnight at −80°C. Arrowheads indicate the X/P UTR riboprobe without boiling. Arrows and asterisks represent the extended and folded RNAs on native gels, respectively. Double-asterisks in panel (B) indicate the migration of the riboprobes restored by the re-boiling of the reaction mixture after incubation with DDX21.

Figure 9. Interaction between BDV P phosphorylation and translation of the X ORF.

(A) In vitro RNA binding and translation assay of DDX21 and X/P mRNA. In vitro transcribed X/P mRNA was incubated with recombinant DDX21 and then in vitro translation was performed using a rabbit reticulocyte lysate, according to the manufacturer's recommendations. After incubation, 10 µl of the mixture was subjected to SDS-PAGE and Western blotting using anti-P and -X antibodies. (B) BDV P does not influence the expression of UBPs. OL cells were transfected with plasmids expressing BDV N, P or P^S26/28A, and 48 h post-transfection the cells were lysed with sample buffer and then subjected to Western blotting using the antibodies indicated. (C) Expression of P reduces phosphorylation levels of DDX21 and nucleolin. Flag-tagged DDX21 or nucleolin was cotransfected with BDV N, P or P^S26/28A into OL cells. Forty-eight h after transfection, the cell lysates were immunoprecipitated by anti-Flag antibody and the immunoprecipitants were detected by anti-Flag and anti-phosphoserine antibodies. (D) Expression of P reduces the RNA-binding activity of DDX21 and nucleolin. Flag-tagged recombinant DDX21 and nucleolin were obtained from lysates of OL cells transfected with either empty (E), wt P (P) or mutant P (P^S26/28A) expression plasmid, and in vitro RNA binding assay was performed with ³²P-labeled X/P UTR riboprobe and purified recombinant proteins as described in the Methods section. Each value represents the mean plus S.D. of at least three independent experiments. **P<0.01, (Student's t test). (E) BDV P, but not the P^S26/28A mutant, enhances translation of X ORF. OL cells were cotransfected with 0.4 g of pX/PΔP and a serially diluted P or P^S26/28A plasmid (4 fold dilution; 0.00625, 0.025, 0.1, 0.4 µg). The expression of BDV X, P and P^S26/28A was detected by Western blotting. The relative expression level of X is shown. Each value represents the mean plus S.D. of three independent experiments. E: empty plasmid-transfected. **P<0.01, *P<0.05 (Student's t test).

Figure 10. Possible mechanism of autogenous regulation of BDV polycistronic mRNA translation.

During the early stage of BDV replication, the phosphorylated DDX21 and other UBPs, including nucleolin, interact with the 5′ UTR of X/P mRNA (left arrow). The interaction may facilitate dissociation or impede X-AUG recognition of ribosomes at the overlapping stop-start codon, leading to inefficient termination-coupled reinitiation, by ribosomes which have translated the uORF, at the X ORF. The DDX21 may be dissociated from the 5′ UTR in the cytoplasm. BDV P accumulation in BDV-infected cells may interfere with phosphorylation of DDX21 and UBPs (right arrow), resulting in the detachment of the RNA helicase complex from the 5′ UTR. The free 5′ UTR may increase the reinitiation processes of ribosomes at X-AUG.