FUSE binding protein 1 interacts with untranslated regions of Japanese encephalitis virus RNA and negatively regulates viral replication

Abstract

The untranslated regions (UTRs) located at the 5' and 3' ends of the Japanese encephalitis virus (JEV) genome, a positive-sense RNA, are involved in viral translation, the initiation of RNA synthesis, and the packaging of nascent virions. The cellular and viral proteins that participate in these processes are expected to interact with the UTRs. In this study, we used biotinylated RNA-protein pulldown and liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) analyses to identify that the far upstream element (FUSE) binding protein 1 (FBP1) binds with JEV 5' and 3' UTRs. The impact of FBP1 on JEV infection was determined in cells with altered FBP1 expression. JEV replication was enhanced by knockdown and reduced by the overexpression of FBP1, indicating a negative role for FBP1 in JEV infection. FBP1, a nuclear protein, was redistributed to the perinuclear region and appeared as cytoplasmic foci that partially colocalized with JEV RNA in the early stage of JEV infection. By using a JEV replicon reporter assay, FBP1 appeared to suppress JEV protein expression mediated by the 5' and 3' UTRs. Thus, we suggest that FBP1 binds with the JEV UTR RNA and functions as a host anti-JEV defense molecule by repressing viral protein expression.

Fig. 1.

Interaction of FBP1 with JEV UTRs. (A) Identification of the cellular proteins interacting with JEV UTRs by pulldown assay as described in Materials and Methods. Mouse N18 cell extracts (200 μg) were incubated with UTR-Luc or biotin-UTR-Luc RNA (6.5 μg), and then streptavidin beads were used to capture the biotinylated RNA. After the beads were washed, the bound proteins were eluted and resolved using 12% SDS-PAGE and stained with SYPRO Ruby. The extra protein bands, which bound to biotin-UTR-Luc but not to the unbiotinylated control (UTR-Luc), were excised for in-gel trypsin digestion and analyzed by LC-MS/MS. The protein bands identified as FBP1 and hnRNP A2/B1 are indicated by arrows. (B) The interaction of JEV UTR with FBP1 was confirmed by anti-FBP1 antibody. The pulldown assay was performed as described above, and then the proteins were separated on SDS-PAGE and subjected to immunoblotting with anti-FBP1 antibody. An unrelated RNA, UTR-GAPDH, with biotin label, was included as a negative control (lane 2). (C) Human FBP1 from HeLa cells also interacted with JEV UTRs. HeLa cell extracts (200 μg) were incubated with 6.5 μg of unbiotinylated or biotinylated UTR-Luc RNA (lanes 1 and 2) or with 4 μg of biotinylated 5′ UTR or 3′ UTR (lanes 3 and 4). The pulldown proteins then were analyzed by immunoblotting with anti-FBP1 antibody, with the HeLa cell lysate as the protein control (lane 5). (D) JEV RNA was pulled down with FBP1 from JEV-infected cell extracts. HeLa cells were infected by JEV (MOI, 10) for 4 h before cell extract collection. The mouse anti-FBP1 (αFBP1; lane 1), mouse anti-Bst2 (αBst2; lane 2), or normal mouse IgG control (lane 3) was incubated with 200 μg of JEV-infected cell extracts, and then protein G/A-agarose beads were added to each sample to pull down the immune complexes. Following the washing step, the RNA was extracted and subjected to RT-PCR with JEV 3′ UTR-specific primers. The cDNA was separated by agarose gel electrophoresis, and the expected band of 585 bp is indicated by an arrow.

Fig. 2.

Reduction of FBP1 expression enhanced JEV protein expression and viral production. (A) HeLa cells transduced with lentivirus carrying shRNA-targeting FBP1 (shFBP1; TRCN0000013297) or LacZ (shLacZ) were selected by puromycin (5 μg/ml). The FBP1 knockdown effect was verified by immunoblotting with anti-FBP1 antibody at the protein level (left) and by RT-PCR with FBP1-specific primers at the RNA level (right). The band intensities were quantified with Adobe Photoshop, and the relative ratios of FBP1 to actin were calculated and are shown at the bottom. (B) HeLa cells carrying shFBP1 or control shLacZ were infected with JEV (MOI, 0.1), and at 24, 30, 48, and 72 h postinfection (p.i.) culture supernatants were harvested for plaque assays. The virus titers (PFU/ml) at the same time points from three independent samples, shown here as averages and standard deviations (SD), were compared using a two-tailed Student's t test (n = 3), and the results are shown (**, P < 0.001; ***, P < 0.0005). (C) Extracts from HeLa-shFBP1 or control shLacZ cells infected with JEV (MOI, 5) were collected at 8, 10, 12, and 24 h p.i. and subjected to immunoblotting using antibodies against JEV NS3 and actin as indicated.

Fig. 3.

DEN-2 protein expression and viral production were increased in cells with reduced FBP1 expression. (A) Extracts from HeLa-shFBP1 or control shLacZ cells infected with DEN-2 (MOI, 5) were collected at 18, 25, and 30 h p.i. and subjected to immunoblotting using antibodies against DEN-2 NS3 and actin as indicated. (B) HeLa cells carrying shFBP1 or control shLacZ were infected with DEN-2 (MOI, 0.1), and at 24, 30, 48, and 72 h p.i. culture supernatants were harvested for plaque assays. The virus titers at the same time points from three independent samples were compared using a two-tailed Student's t test (n = 3), and the results are shown (**, P < 0.001; ***, P < 0.0005).

Fig. 4.

Rescue of FBP1 expression in HeLa-shFBP1 cells repressed JEV production. (A) The sequences of shRNA targeting human FBP1 (TRCN0000013297), the relative region of human and mouse FBP1, and shFBP1-resistant human FBP1 are shown. The different nucleotides are underlined and presented as italic letters. The amino acids encoded by this region also are indicated at the top. (B) HeLa-shFBP1 cells transfected with a vector control (pcDNA3.1) or a V5-tagged mouse FBP1-expressing plasmid (M-FBP1) were infected with JEV (MOI, 5). Cells lysates were harvested at 10, 12, and 24 h p.i. and subjected to immunoblotting with antibodies against JEV NS3, V5-tag (for FBP1), and actin as indicated. (C) HeLa-shFBP1 cells carrying a neomycin-resistant gene-containing JEV replicon (HeLa-shFBP1/JEV replicon) were transfected with pcDNA3.1 (vector) or a V5-tagged human FBP1 wobble mutant that is resistant to the targeting of shFBP1 (Hr-FBP1). At 10, 18, and 24 h posttransfection (p.t.), cells lysates were harvested for immunoblotting with antibodies against JEV NS3, V5-tag (for FBP1), and actin as indicated.

Fig. 5.

Overexpression of FBP1 reduced JEV protein expression and viral production. (A) HeLa cells transduced with lentivirus expressing EGFP, V5 tagged-FBP1, or vector control were infected with JEV (MOI, 5). After 24 h of infection, cells were fixed for immunofluorescence assays with anti-JEV NS3 (red) (A) or anti-dsRNA antibody (red) (B). FBP1 expression was detected by anti-V5 tag antibody (green), and nuclei were stained with DAPI (blue). The signals were photographed using a fluorescence microscope. (C) Cells were infected with JEV (MOI, 5), and cell lysates were collected at 8, 10, 12, and 24 h p.i. for immunoblotting with antibodies against JEV NS3, JEV NS1, V5-tag (for FBP1), EGFP, and actin as indicated. (D) At 24 h p.i., culture supernatants were harvested for virus titration by plaque-forming assays. The virus titers (PFU/ml), shown as averages and standard deviations from three independent samples, were compared by two-tailed Student's t test (n = 3).

Fig. 6.

Cellular localization of FBP1 and dsRNA. HeLa cells with V5-tagged FBP1 stable expression (HeLa-FBP1) were infected with JEV (MOI, 5) for 2, 4, 10, and 24 h and stained with antibodies against dsRNA (red), V5-tag (green), and DAPI (blue). The samples were observed and photographed by using a confocal microscope (Zeiss LSM 510 META).

Fig. 7.

FBP1 negatively regulated JEV UTR-dependent protein expression measured by a JEV reporter replicon. (A) Schematic representation of the J-R2A replicon. The construct contains an SP6 promoter upstream of the JEV 5′ UTR, followed by the first 34 amino acids of JEV C protein (C_96-197), Renilla luciferase, the foot-and-mouth disease virus 2A self-cleaving protease (FMDV 2A), the last 30 amino acids of JEV E protein (E_2388-2477), NS1-NS5 of JEV nonstructural proteins, and the JEV 3′ UTR. To ensure RNA stability and processing, a hepatitis delta virus ribozyme was placed immediately adjacent to the 3′ end of the JEV cDNA, followed by an SV40 poly(A) sequence. (B) Human neuronal NT2 cells transduced with lentivirus carrying shRNA-targeting FBP1 (shFBP1; TRCN0000013293) or LacZ (shLacZ) were selected by puromycin (5 μg/ml). To verify the knockdown effect, cell lysates were harvested for immunoblotting with anti-FBP1 and anti-actin antibodies. NT2-shFBP1 or NT2-shLacZ cells were cotransfected with a Renilla luciferase JEV replicon J-R2A (C), a replication-dead replicon J-R2A-NS5mt (D), or a Renilla luciferase reporter flanked by GAPDH 5′ and 3′ UTRs (E) plus firefly luciferase RNA as a control. At various times posttransfection, cell lysates were collected for dual-luciferase assays. Renilla luciferase activity was normalized to that of firefly luciferase. The results are expressed as averages and standard deviations from two independent samples. The data of shFBP1 and shLacZ at the same time points were compared by two-tailed Student's t test. *, P < 0.05.