Duck hepatitis A virus utilizes PCBP2 to facilitate viral translation and replication

Figures

• 

  Figure 1. PCBP2 expression is promoted during DHAV-1 infection. A DEFs were infected with DHAV-1 at an MOI of 0.5 for 12, 24 or 36 h, and the expression of the endogenous PCBP2 protein was measured by western blotting. B The pCAGGS-3 × Flag-PCBP2 plasmid was transfected into DEFs for 12 h, after which the cells were infected with DHAV-1 at an MOI of 0.5 for various durations. The expression of 3 × Flag-PCBP2 was measured by western blotting. C DEFs were infected with DHAV-1 (MOI = 0.1, 0.5, 1 or 2.5) for 24 h, and the expression of the endogenous PCBP2 protein was measured by western blotting. D The pCAGGS-3 × Flag-PCBP2 plasmid was transfected into DEFs for 12 h, after which the cells were infected with DHAV-1 (MOI = 0.1, 0.5, 1 or 2.5) for 24 h. The expression of 3 × Flag-PCBP2 was measured by western blotting. E DEFs were infected with DHAV-1 at an MOI of 0.5 for 12, 24 or 36 h, and the cells were collected to measure the mRNA expression of PCBP2 by quantitative RT‒PCR. Differences between two groups were analysed using Student’s t test and were considered significant at * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

• 

  Figure 2. Overexpression of the PCBP2 protein promotes the replication of DHAV-1. DEFs were transfected with pCAGGS-3 × Flag-PCBP2 or the pCAGGS empty vector. The transfected cells were then infected with DHAV-1 at an MOI of 0.5, and the virions produced were harvested at 12 h, 24 h, 36 h and 48 h. A, B Total viral RNA and negative-strand RNA were quantified via one-step TaqMan fluorescent quantitative RT‒PCR. Virus production in the supernatant was analysed by a TCID50 assay (C), and viral protein expression in the supernatant was analysed by western blotting (D). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

• 

  Figure 3. Knockdown of the PCBP2 protein inhibits replication of DHAV-1. A–C DEFs were transfected with siRNA-NC or siRNAs targeting the PCBP2 gene. After 36 h, cell viability was analysed with a CCK-8 assay, and the mRNA expression and protein expression of PCBP2 were analysed via quantitative RT‒PCR and western blotting, respectively. D, E DEFs were transfected with siRNA-NC or siRNA-PCBP2. After transfection, the cells were infected with DHAV-1 at an MOI of 0.5 and harvested at 12 h, 24 h, 36 h, and 48 h. Viral production in the supernatant was analysed by a TCID50 assay (D), and viral protein expression in the supernatant was analysed by western blotting (E). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

• 

  Figure 4. PCBP2 regulates DHAV-1 translation through interaction with the IRES. A Schematic diagrams of the bicistronic reporter plasmids CMV-RHF and CMV-RHF-IRES. B The effects of PCBP2 on DHAV-1 IRES activity were determined via bicistronic reporter plasmids. DEFs in which PCBP2 was overexpressed or knocked down were transfected with the CMV-RHF or CMV-RHF-IRES plasmid. Twenty-four hours post-transfection, the activities of the FLuc and RLuc reporters were measured. The bars in the histogram indicate FLuc/RLuc activity as percentages. The experiments were performed in triplicate, and the results are presented in the bar graph. The expression levels of PCBP2, 3 × Flag-PCBP2, and actin were measured by western blotting. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. C DEFs were transfected with 3 × Flag-PCBP2 and were then infected with DHAV-1 at 12 h post-transfection. The cell lysates were collected, and a mouse anti-HA antibody or mouse IgG was added. The pulled down RNAs were extracted and amplified via PCR using DHAV-1 IRES primers. D DHAV-1 IRES RNA was pulled down with purified PCBP2 or from cell lysates transfected with the 3 × Flag-PCBP2 plasmid. The 6 × His-tagged PCBP2…

• 

  Figure 5. Identification of interaction regions between the DHAV-1 IRES and PCBP2. A Predicted secondary structure of the DHAV-1 IRES RNA. Four truncations of the IRES were generated: domain Id + Ie, domain Ie, domain II, and domain III. B PCBP2 interaction regions in the DHAV-1 IRES. The truncated sequences of the IRES RNA were transcribed in vitro and biotinylated. Purified PCBP2-6 × His was incubated with these biotinylated RNAs. Nonbiotinylated RNA was used in this assay as a control. The RNA–protein complex-bound beads were pulled down and separated by SDS‒PAGE (12%). An anti-PCBP2 antibody was used to detect PCBP2 in the precipitated complexes. C Schematic diagram of PCBP2 and its truncation mutants. Three truncation mutants of PCBP2 (a), KH1 (b), KH2 (c) and KH3 (d) were generated and fused with 3 × Flag tags at their N termini. D Interaction regions between the PCBP2 protein and the DHAV-1 IRES. Lysates of transfected DEFs were collected 36 h post-transfection and were then incubated with the biotinylated or nonbiotinylated DHAV-1 IRES. Streptavidin beads were used in the pulldown assay, and an anti-PCBP2 antibody was used to detect PCBP2 in the precipitated complexes.

• 

  Figure 6. DHAV-1 3Dpol interacts with the PCBP2 protein and promotes its expression. A, B DEFs were grown in 6‑well plates, and the cells in each well were transfected with 2 µg of pCAGGS‑3D‑HA or 2 µg of pCAGGS‑3 × Flag-PCBP2 alone or co-transfected with 1 µg of pCAGGS‑3D‑HA and 1 µg of pCAGGS‑3 × Flag-PCBP2. The cells were lysed 36 h after transfection, and the lysates were incubated with a mouse anti-Flag or mouse anti-HA antibody and analysed by immunoblotting with an anti-HA or anti-Flag antibody, respectively. C pCAGGS‑3 × Flag‑PCBP2 and pCAGGS‑3D-HA were transfected separately with pCAGGS or pCAGGS‑3 × Flag‑PCBP2 and pCAGGS‑3D-HA were transfected together into cells in 24‑well plates (transfection ratio of 1:1), and cell samples were collected at 24 h after transfection for indirect immunofluorescence experiments to observe the intracellular localization of the 3Dpol and PCBP2 proteins. D DEFs were grown in 12‑well plates and transfected with different doses of 3D‑HA expression plasmids (the amount transfected into each group was 1 µg, and pCAGGS was used to supplement the insufficient group) or with 0.4 µg of pCAGGS‑3 × Flag-PCBP2 plus different doses of pCAGGS‑3D‑HA (the amount…

• 

  Figure 7. Interaction regions between the DHAV-1 3Dpol and PCBP2 proteins. A–D Co-IP assay of exogenous 3 × Flag-tagged truncated PCBP2 and 3Dpol. The cells were lysed 36 h after transfection, and the lysates were incubated with a mouse anti-Flag or mouse anti-HA antibody and analysed by immunoblotting with an anti-HA or anti-Flag antibody, respectively. E, F DEFs were transfected with 3 × Flag-PCBP2, 3 × Flag-PCBP2ΔKH1/3 × Flag-PCBP2ΔKH3, or the control pCAGGS vector and were then infected with DHAV-1 at an MOI of 0.5. The viral copy number was quantified via one-step TaqMan fluorescent quantitative RT–PCR. Western blotting was performed to measure the expression of full-length 3 × Flag-PCBP2, 3 × Flag-PCBP2ΔKH1/3 × Flag-PCBP2ΔKH3, and viral VP3 in DEFs. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.