Fig 1.
The RHDV genome is associated with the fully processed viral protein genome (VPg) at its terminus.
(A) A schematic diagram of the RHDV genome showing the genes encoding non-structural (NSP1-NSP7) and structural (VP60 and VP10) proteins. (B) The VPg protein interacted with RHDV 5′ -Extreme RNA, as determined by the RNA EMSA assay. Lane 1: negative control: VPg protein (2 μg) + labeled RHDV-NSP2 (6 nM); Lane 2: blank control: VPg protein (2 μg); Lane 3: negative control: labeled 5′ -Extreme RNA (6 nM); Lane 4: VPg protein (1 μg) + labeled 5′ -Extreme RNA (6 nM); Lane 5: VPg protein (2 μg) + labeled 5′ -Extreme RNA (6 nM); Lane 6: competition group: VPg protein (2 μg) + 5′ -Extreme RNA (6 nM) + labeled 5′ -Extreme RNA (6 nM).
Fig 2.
RHDV VPg protein is essential for RHDV translation.
(A) Schematic diagram of the pRHDV-luc, pRHDV-luc/ΔVPg, and pRHDV plasmids [16]. The coding regions of viral structural proteins were replaced with Fluc using fusion PCR. In the fusion PCR results, a gray box denotes the coding region of the viral structural proteins, and lines indicate the 5′ and 3′ UTRs. (B) Thirty-two hours after transfection, the luciferase mRNA levels in cells transfected with pRHDV-luc, pRHDV-luc/ΔVPg + pVPg, or pRHDV-luc/ΔVPg were evaluated by qRT-PCR. The Student’s t test and ANOVA were applied for the statistical analyses, P <0.05 was considered as significantly different (*), and P <0.01 was considered as extremely significant different (**). (C) The expression level of trans-supplemented VPg was evaluated by immunoblotting with VPg polyclonal antibodies. Line 1: pVPg; line 2: pcDNA3.1 vector; line 3: blank control. (D) Relative luciferase activity in RK13 cells carrying pRHDV-luc/ΔVPg, trans-supplemented pVPg, and the parental genotype pRHDV-luc at 12 h, 24 h, 36 h, 48 h, 60 h, and 72 h post-transfection. The luciferase activity in RK13 cells was evaluated by measuring the firefly luciferase activity at different time points after transfection. Renilla luciferase activity measured at the same time points was used to normalize the transfection efficiency. The differences in luciferase activities associated with pRHDV-luc/ΔVPg + pVPg and pRHDV-luc/ΔVPg were compared using SAS 9.1 software. The Student’s t test and ANOVA were used for the statistical analyses. P <0.05 was considered as significantly different (*), and P <0.01 was considered as extremely significant different (**).The experiments were conducted in triplicate, and similar results were obtained from three independent experiments.
Fig 3.
The interaction between VPg and eIF4E in RK13 cells based on the mammalian two-hybrid assay.
RK13 cells (2×105 cells per well in a 6-well plate) were co-transfected with the pACT (1 μg) and pBIND (1 μg) plasmids, pACT-VPg (1 μg) and pBIND (1 μg) plasmids, and pACT-VPg (1 μg) and pBIND-eIF4E (1 μg) plasmids, respectively. RK13 cells co-transfected with the pACT-MyoD (1 μg) and pBIND-ID (1 μg) plasmids served as positive controls [25]. A blank control was also used. In addition, all of the groups were transfected with pGL4.75 (0.2 μg). The cells were lysed at 48 h post-transfection, and Fluc activity was evaluated based on RLUs and normalized according to the results obtained for a co-transfected plasmid encoding the Renilla luciferase. The experiments were conducted in triplicate, and similar results were obtained from three independent experiments. The Student’s t test and ANOVA were applied for the statistical analyses, P <0.01 was considered as extremely significant different (**).
Fig 4.
The interaction between RHDV VPg protein and eIF4E.
(A) Co-IP of VPg and eIF4E protein. RK13 cells were co-transfected with the indicated plasmids (+) or empty vectors (-). Next, whole-cell lysates obtained at 48 h post-transfection were immunoprecipitated (IP) using anti-V5 MAb. The proteins were separated by SDS-PAGE and detected by immunoblotting with specific antibodies. The protein identities are shown below the panel. (B) BiFC of VPg and eIF4E protein. RK-13 cells grown in a 35-mm plate were transiently co-transfected with vectors pVN155(I152L) (1 μg) and pVC155 (1 μg) (B-1), negative control vectors pbJunVN155 (1 μg) and pbFos(ΔZIP)VC155 (1 μg) (B-2), a combination of plasmids pVPg-VN (1 μg) and peIF4E-VC (1 μg) (B-3), the positive control pbJunVN155 and pbFosVC155 (Jun-Fos is a heterodimer[26]) (B-4), the VPg and eIF4E subcellular localization interaction (B-5), and the blank control (B-6). At 24 h post-transfection, the BiFC complexes were visualized under a fluorescence microscope with an appropriate objective. The position of the nuclei was determined using DAPI (blue). (C) BiFC of VPg and eIF4E proteins by flow cytometry. During the BiFC experiments, the green fluorescence observed at 48 h post-transfection was quantified by flow cytometry. C-1: pVN155(I152L) and pVC155 vectors; C-2: negative control pbJunVN155 and pbFos(ΔZIP)VC155; C-3: positive control pbJunVN155 and pbFosVC155; C-4: combination of plasmids pVPg-VN and peIF4E-VC. The gray cloud means negative cells which have no green fluorescence; the green cloud means positive cells which have green fluorescence; X axes represents the fluorescence intensity; Y axes represents cell size; 'E' represents the ratio of the positive cells to the negative cells.
Fig 5.
Knockdown of VPg and eIF4E protein interactions decreased the levels of RHDV translation.
RK13 cells were co-transfected with the p4EBP1 (0 μg, 0.3 μg, 0.6 μg, or 1.0 μg), pRHDV-luc (1 μg), peGFP (0.5μg) and pIRES-Rluc (0.5 μg) plasmids. (A) RK13 cells were lysed at 48 h post-transfection, and the levels of 4EBP1, eGFP and Rluc were determined by immunoblotting with antibodies against the indicated proteins. (B) RK13 cells were lysed at 48 h post-transfection, and Fluc activity was measured based on RLUs and normalized according to the results obtained for a co-transfected plasmid encoding Renilla luciferase. The experiments were conducted in duplicate, and similar results were obtained in two independent experiments.
Fig 6.
Silencing of eIF4E inhibited RHDV translation.
(A) The expression of eIF4E was silenced by siRNA. The 293T cells transfected without siRNA (Mock), eIF4E siRNA (si-eIF4E), untreated (No treat), or treated with different concentrations (nM) of si-eIF4E (as indicated at the top of each lane) were harvested at 48 h post-transfection. The expression of eIF4E was measured by immunoblotting with antibodies against the indicated proteins. (B) The translation of RHDV in eIF4E-silenced siRNA, pRHDV-luc and peGFP plasmids co-transfected cells were conducted as described in panel A. The cells were lysed at 48 h post-transfection, and Fluc activity was measured based on RLUs and normalized according to the results obtained for a co-transfected pIRES-Rluc plasmid encoding Renilla luciferase. The experiments were conducted in duplicate, and similar results were obtained from two independent experiments. The Student’s t test and ANOVA were applied for the statistical analyses, P <0.01 was considered as extremely significant different (**).