Duck hepatitis A virus 3CD and 3D proteins are key facilitators of hnRNP K expression and cytoplasmic entry, and hijack hnRNP K to facilitate viral translation and replication

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  Fig. 1. hnRNP K expression and nucleocytoplasmic shuttling are promoted during DHAV-1 infection. (A) DEF cells were infected with 0.5 MOI DHAV-1 for 6, 12, 24, and 48 h, and the expression of endogenous hnRNP K protein was detected by Western blotting. (B) Plasmid pCAGGS-hnRNP K-His were transfected into DEF cells for 12h , and then cells were infected with 0.5 MOI DHAV-1 for a gradient time, hnRNP K-His was detected by Western blotting. (C) Plasmid pCAGGS-hnRNP K-His were transfected into DEF cells for 12h , and then cells were mock infected or infected with 0.5 MOI DHAV-1. After 24 h, the cells were stained with antibodies against dsRNA (green), His (red), and DAPI (blue). The localization of hnRNP K was observed and imaged using a microscope. (D) After infecting DEF cells with 1.0 MOI DHAV-1 for 24 h, the cytoplasm and nucleus were extracted separately, and the expression of endogenous hnRNP K in the cytoplasm and nucleus was detected by Western blotting. (E) Plasmid pCAGGS-hnRNP K-His were transfected into DEF cells for 12h , and then cells were infected with 0.5 MOI DHAV-1 for 24 h. The cell samples were subjected to immunoprecipitation with an anti-His antibody against hnRNP K-His.…

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  Fig. 2. hnRNP K promotes DHAV-1 IRES-mediated translation and DHAV-1 replication. (A) Schematic diagram of the bicistronic luciferase plasmid (CMV-Rluc-IRES-Fluc). (B) The CMV-Rluc-IRES-Fluc was transfected into hnRNP K-overexpressing DEF cells. At 24 h post-transfection, the RLuc and FLuc activities were analyzed. The level of hnRNP K overexpression was detected by Western blotting. (C) The CMV-Rluc-IRES-Fluc was transfected into hnRNP K-knockdown DEF cells. At 24 h post-transfection, the RLuc and FLuc activities were analyzed. The knockdown effect of hnRNP K was detected at the protein level by Western blotting. (D) The effect of hnRNP K-knockdown on DEF cell activity was detected by CCK8 assay at different time points. (E) hnRNP K-overexpressing DEF cells were infected with 0.01 MOI DHAV-1, and the RNA samples were harvested at the indicated time points. The viral copy number was detected by RT-qPCR. (F) hnRNP K-overexpressing DEF cells were infected with 0.5 MOI DHAV-1, and the protein samples were harvested at the indicated time points. The hnRNP K overexpression and viral VP3 levels in DEF cells were detected by Western blotting. (G) hnRNP K-knockdown DEF cells were infected with…

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  Fig. 3. Interaction of hnRNP K with the DHAV-1 IRES and 3′UTR. (A) Purified hnRNP K-6 × His was incubated with biotinylated DHAV-1 IRES, nonbiotinylated DHAV-1 IRES, biotin-16-UTP, or no RNA. The RNA-protein complexes were precipitated with streptavidin magnetic beads and analyzed by Western blotting with an anti-hnRNP K antibody. (B) Purified hnRNP K-6 × His was incubated with biotinylated DHAV-1 3′UTR, nonbiotinylated DHAV-1 3′UTR, biotin-16-UTP, or no RNA. The RNA-protein complexes were analyzed by Western blotting. (C) Schematic diagram of hnRNP K and its truncated mutants. (D) Extracts of 293T cells overexpressing 3 × Flag-tagged hnRNP K truncated mutants were incubated with biotinylated or unlabeled DHAV-1 IRES. The bound proteins were resolved by SDS-PAGE and detected by Western blotting using an anti-Flag antibody. (E) Extracts of 293T cells overexpressing 3 × Flag-tagged hnRNP K truncated mutants were incubated with biotinylated or unlabeled DHAV-1 3′UTR. hnRNP K truncated mutants were visualized by Western blotting with an anti-Flag antibody. Each independent experiment was repeated in triplicate (n = 3).

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  Fig. 4. Screening for viral proteins that activate the hnRNP K protein. (A to E) Expression plasmids of viral proteins VP0 (A), 2BC (B), 2B (C), 3CD (D), and 3D (E) were transfected into DEF cells, and hnRNP K protein expression was detected by Western blotting after 24 h. (F) DEF cells were transfected with different doses of 3CD expression plasmids (the total transfection amount for each group was 1 µg, the amount of pCAGGS-3CD-HA was 0 / 0.25 / 0.5 / 0.75 / 1 µg, and the insufficient amount was supplemented with pCAGGS plasmid). The endogenous hnRNP K protein was detected by Western blotting after 24 h. (G) DEF cells were transfected with different doses of 3C expression plasmids (the total transfection amount for each group was 1 µg, the amount of pCMV-3C-HA was 0 / 0.25 / 0.5 / 0.75 / 1 µg, and the insufficient amount was supplemented with pCMV plasmid). The endogenous hnRNP K protein was detected by Western blotting after 24 h. (H) DEF cells were transfected with different doses of 3D expression plasmids (the total transfection amount for each group was 1 µg, the amount of pCAGGS-3D-HA was 0 / 0.25 / 0.5 / 0.75 / 1 µg, and the insufficient amount was supplemented with pCAGGS…

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  Fig. 5. The 3CD and 3D proteins interact with the hnRNP K protein and affect its localization in cells. (A to B) pCAGGS-3CD-HA (A) or pCAGGS-3D-HA (B) and pCAGGS-hnRNP K-His were co-transfected into 293T cells. The cell samples were harvested 24 h after transfection and subjected to immunoprecipitation with an anti-HA antibody or a mouse IgG antibody. The immunoprecipitate was analyzed for Western blotting. (C) pCAGGS-3CD-HA and pCAGGS-hnRNP K-His were co-transfected, or both were separately co-transfected with pCAGGS into DEF cells. After 24 h, cells were fixed, and the localization of 3CD-HA and hnRNP K-His were detected by indirect immunofluorescence. (D) pCAGGS-3D-HA and pCAGGS-hnRNP K-His were co-transfected, or both were separately co-transfected with pCAGGS into DEF cells. After 24 h, cells were fixed for indirect immunofluorescence to observe the protein localization. Each independent experiment was repeated in triplicate (n = 3).

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  Fig. 6. hnRNP K interacts with 3CD and 3D via the KH1 and KI domains. (A to D) Expression plasmids of 3CD and hnRNP K truncated mutant were co-transfected into 293T cells. The cell samples were harvested 24 h after transfection and subjected to immunoprecipitation with an anti-HA antibody or a mouse IgG antibody. The immunoprecipitate was analyzed for Western blotting. (E to H) Expression plasmids of 3D and hnRNP K truncated mutant were co-transfected into 293T cells. The cell samples were harvested 24 h after transfection and subjected to immunoprecipitation with an anti-HA antibody or a mouse IgG antibody. The immunoprecipitate was analyzed for Western blotting. Each independent experiment was repeated in triplicate (n = 3).

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  Fig. 7. Interactions of the hnRNP KΔ1-110 and hnRNP KΔ191-328 mutants with 3CD and 3D. (A) pCAGGS-hnRNP K-His or pCAGGS-hnRNP KΔ1-110-His or pCAGGS-hnRNP KΔ191-328-His and pCAGGS-3CD-HA were co-transfected into 293T cells. The cell samples were harvested 24 h after transfection and were incubated with an anti-His antibody or a mouse IgG antibody for immunoprecipitation. The protein-protein complexes were analyzed by Western blotting. (B) pCAGGS-hnRNP K-His or pCAGGS-hnRNP KΔ1-110-His or pCAGGS-hnRNP KΔ191-328-His and pCAGGS-3D-HA were co-transfected into 293T cells. The cell samples were harvested 24 h after transfection and were incubated with an anti-His antibody or a mouse IgG antibody for immunoprecipitation. The protein-protein complexes were analyzed by Western blotting. (C) pCAGGS-3CD-HA or pCAGGS-3D-HA and pCAGGS-hnRNP KΔ1-110-His or pCAGGS-hnRNP KΔ191-328-His were co-transfected into DEF cells. After 24 h, cells were fixed, and indirect immunofluorescence detected the protein localization. Each independent experiment was repeated in triplicate (n = 3).

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  Fig. 8. 3CD and 3D interact with the DHAV-1 UTRs. (A to D) The interaction between 3CD/3D and DHAV-1 IRES/3′UTR. Cell lysates prepared from 293T cells were subjected to a biotin-RNA pulldown assay. The RNA-protein complexes were precipitated with streptavidin magnetic beads and analyzed by Western blotting. (A) Extracts of 293T cells overexpressing 3CD-HA were incubated with biotinylated or unlabeled DHAV-1 IRES. (B) Extracts of 293T cells overexpressing 3CD-HA were incubated with biotinylated or unlabeled DHAV-1 3′UTR. (C) Extracts of 293T cells overexpressing 3D-HA were incubated with biotinylated or unlabeled DHAV-1 IRES. (D) Extracts of 293T cells overexpressing 3D-HA were incubated with biotinylated or unlabeled DHAV-1 3′UTR. Each independent experiment was repeated in triplicate (n = 3).

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  Fig. 9. Effect of the interaction between hnRNP K and 3CD/3D on DHAV-1 translation and replication. (A) DEF cells were first transfected with hnRNP K-His, hnRNP KΔ1-110-His, hnRNP KΔ191-328-His, or pCAGGS vector control. After 12 h, the CMV-Rluc-IRES-Fluc was transfected into DEF cells. At 24 h post-transfection, the RLuc and FLuc activities were analyzed. Expression levels of hnRNP K and its mutants were detected by Western blotting. (B to C) Extracts of 293T cells overexpressing hnRNP KΔ1-110-His (B) or hnRNP KΔ191-328-His (C) were incubated with biotinylated DHAV-1 IRES, nonbiotinylated DHAV-1 IRES, biotin-16-UTP, or no RNA. The bound proteins were resolved by SDS-PAGE and detected by Western blotting using an anti-His antibody. (D) DEF cells were transfected with hnRNP K-His, hnRNP KΔ1-110-His, or pCAGGS vector control and then infected with DHAV-1. At the indicated time points, the RNA samples were harvested to detect the viral copy number. The protein samples were harvested to detect full-length hnRNP K and hnRNP KΔ1-110 and viral VP3 levels in the cells. (E) DEF cells were transfected with hnRNP K-His, hnRNP KΔ191-328-His, or pCAGGS vector control and then infected with DHAV-1.…

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  Fig. 10. Schematic representation of the mechanism delineated in this study. DHAV-1 binds to a cellular receptor, and its genome is released into the cytoplasm. The viral proteins 3CD and 3D interact with hnRNP K and assist in the nucleocytoplasmic shuttling of hnRNP K. 3CD, 3D, and hnRNP K all bind to the DHAV-1 IRES and 3′UTR. These three proteins may form complexes, and then regulate viral translation and replication.