IGF2BP1 Significantly Enhances Translation Efficiency of Duck Hepatitis A Virus Type 1 without Affecting Viral Replication

Figures

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  Figure 1. Construction schematic of the plasmids used in this study: (A) Construction of monocistronic reporter plasmids; (B) construction of plasmid pRluc; (C) construction of DHAV-1 RNA-launched infectious clones; (D) construction of plasmid pBlue-Ribo-DHAV-1; (E) construction of plasmid pBluescript-polyprotein; (E) construction of plasmid pBlue-Ribo-DHAV-1; and (F) construction of plasmid pcDNA-IGF2BP1.

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  Figure 2. Insulin-like growth factor-2 mRNA-binding protein-1 (IGF2BP1) specifically binds to the 3′ untranslated region (UTR) of duck hepatitis A virus type 1 (DHAV-1). (A) Silver-staining electrophoresis of the proteins that specifically bind to the four labeled RNA probes: the entire 3′ UTR of DHAV-1 with poly(A)25 tail (probe 1), the reverse complementary sequence of the 3′ UTR of DHAV-1 with poly(A)25 tail (probe 2), the 3′ UTR of DHAV-1 alone (probe 3), and the poly(A)25 tail alone (probe 4). The red arrows showed the proteins interacted with probes 1 and 3 rather than the probes 2 and 4. (B) The Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) results of the proteins marked with red arrows in Figure 2A. IGF2BP1 was a common protein that specifically interacted with the probes 1 and 3 (marked with red frames). (C) The purified IGF2BP1-His fusion proteins and in vitro pulling-down proteins with the four RNA probes were conducted by western blot assay using anti-IGF2BP1 or anti-His antibody (1:3000).

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  Figure 3. IGF2BP1 strongly enhances DHAV-1 internal ribosome entry site (IRES)-mediated translation efficiency via binding to 3′ UTR. (A) The IGF2BP1 expression level in ExIGF2BP1 duck embryo epithelial (DEE) and SiIGF2BP1 DEE cells was tested by western blot assay with anti-IGF2BP1 antibody. (B) The same quantity (0.8 µg) of the in vitro transcribed products of linearized plasmids pDHAV-3′UTR-A25 and pDHAV-Δ3′UTR-ΔA25 was transfected into the ExIGF2BP1 DEE, SiIGF2BP1 DEE, and normal DEE cells, and the luciferase activities were detected at 4 hpt. Reporter RNA of pRluc was transfected into the DEE cell line as the control. The FLuc activity of pDHAV-3′UTR-A25 in the normal DEE cell line was set as 100%. (C) The RNA-launched infectious clone pR-DHAV-1 was transfected into the ExIGF2BP1 DEE, SiIGF2BP1 DEE, and normal DEE cells, and the cell lysates were collected from 24 hpt to 60 hpt for western blot analysis with anti-DHAV-1 mAb 4F8. The grayscale value of the normal DEE cells was set as 100%. Columns and bars represent means of three independent transfections and standard deviations. (D) The same quantity (0.8 µg) of the in vitro transcribed products of linearized plasmids…

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  Figure 4. Viral propagation significantly enhanced IGF2BP1 expression level. (A) The DEE cells were transfected with 0.8 µg of the in vitro transcribed RNA of RNA-launched infectious clone pR-DHAV-1 and were collected from 12 hpt to 48 hpt for western blot assay with anti-DHAV-1 mAb 4F8 and anti-IGF2BP1 antibody, respectively (Left). The variation tendency of the VP1 of DHAV-1 and IGF2BP1 in the transfected DEE cells was measured from 12 to 48 hpt (Right). (B) The IGF2BP1 expression level in normal DEE cells (Mock group) and DEE cells transfected with 0.8 µg of pR-DHAV-1 (pR-DHAV-1 group) was measured by immunofluorescence assays (IFA) at 24 hpt with anti-IGF2BP1 antibody. The mean fluorescence intensity was measured by Image J software (National Institutes of Health, Maryland, USA). (C) The DEE cells were transfected with equal copies of the recombinant plasmid pBluescript-polyprotein and pBluescript II KS (+) vector, and the VP1 of DHAV-1 and IGF2BP1 expression levels were detected by western blot from 12 to 48 hpt (Left). The relationship between VP1 of DHAV-1 increment level and IGF2BP1 expression level in the transfected DEE cells was compared (Right). (D) The DEE cells were…

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  Figure 5. IGF2BP1 had no impact on viral replication. Equal amounts of the in vitro transcribed RNA of RNA-launched infectious clone pR-DHAV-1 were transfected into ExIGF2BP1 DEE, SiIGF2BP1 DEE, and normal DEE cells. Total RNA was extracted from the supernatant (A) and DEE cells (B), and the extracted RNA was immediately used for RT-qPCR measurement. Bars represent the mean ± standard deviation of three replicate experiments for each group.