KIF5B-mediated internalization of FMDV promotes virus infection

Abstract

Foot-and-mouth disease (FMD) is a highly contagious and economically important disease, which is caused by the FMD virus (FMDV). Although the cell receptor for FMDV has been identified, the specific mechanism of FMDV internalization after infection remains unknown. In this study, we found that kinesin family member 5B (KIF5B) plays a vital role during FMDV internalization. Moreover, we confirmed the interaction between KIF5B and FMDV structural protein VP1 by co-immunoprecipitation (Co-IP) and co-localization in FMDV-infected cells. In particular, the stalk [amino acids (aa) 413-678] domain of KIF5B was indispensable for KIF5B-VP1 interaction. Moreover, overexpression of KIF5B dramatically enhanced FMDV replication; consistently, knockdown or knockout of KIF5B suppressed FMDV replication. Furthermore, we also demonstrated that KIF5B promotes the internalization of FMDV via regulating clathrin uncoating. KIF5B also promotes the transmission of viral particles to early and late endosomes during the early stages of infection. In conclusion, our results demonstrate that KIF5B promotes the internalization of FMDV via regulating clathrin uncoating and intracellular transport. This study may provide a new therapeutic target for developing FMDV antiviral drugs.

Keywords: Clathrin; Endosome; FMDV; KIF5B; VP1 protein.

Fig. 1

KIF5B interacted with FMDV VP1 protein. A Exogenous co-immunoprecipitation (Co-IP) assay in HEK-293T cells. HEK-293T cells were transfected with KIF5B-Myc and VP1-HA or vector plasmids, and then cell lysates were immunoprecipitated with HA antibody, followed by immunoblotting with Myc and HA antibodies. B Exogenous co-localization of VP1 and KIF5B. HEK-293T cells were cultured on the bottom of glass cell culture dish for 12 ​h, and then transfected with VP1-HA or KIF5B-Myc plasmids for 24 ​h. Transfected cells were analyzed by immunofluorescence staining with anti-HA (green), anti-Myc (red) and DAPI (blue) under confocal microscopy. C Endogenous interaction between VP1 and KIF5B. FMDV-infected (MOI ​= ​0.5) or mock-infected PK-15 ​cells were used for immunoprecipitation with mouse anti-VP1 antibody and immunoblotted with rabbit anti-KIF5B antibody. D Endogenous co-localization of VP1 and KIF5B. PK-15 ​cells were cultured on the bottom of glass cell culture dish for 24 ​h, and then infected with FMDV (MOI ​= ​0.5) for 8 ​h. Infected cells were analyzed by immunofluorescence staining with anti-KIF5B (green), anti-VP1 (red) and DAPI (blue), and then microscopy.

Fig. 2

FMDV VP1 interacted with the stalk region of KIF5B. A Schematic illustration of KIF5B truncated mutants. B Confocal images of the interaction segments between KIF5B truncated mutants and VP1. HEK-293T cells were cultured on the bottom of glass cell culture dish for 12 ​h and transfected with VP1-HA, KIF5B-1-413-mCherry, KIF5B-413-678-mCherry, or KIF5B-678-963-mCherry plasmids for 24 ​h. Transfected cells were identified by immunofluorescence staining with anti-HA (green), mCherry (red) and DAPI (blue). C The co-localization analysis was expressed as Pearson's correlation coefficient, measured for individual cells. D Co-IP method to validate the interaction segments between KIF5B truncated mutants and VP1. HEK-293T cells were transfected with VP1-HA and KIF5B-Myc or its truncated mutants plasmids. Cell lysates were immunoprecipitated with Myc antibody, followed by immunoblotting with Myc and HA antibodies.

Fig. 3

Overexpression of KIF5B dramatically enhanced FMDV replication. A The effect of KIF5B overexpression on FMDV replication at different time points. BHK-21 ​cells were grown on 6-well plates for 12 ​h, and then transfected with KIF5B-Myc or vector (1 ​μg), respectively. Samples were analyzed by Western blotting. B–D KIF5B promoted FMDV replication in a dose-dependent manner. PK-15 ​cells were grown on 6-well plates for 12 ​h, then transfected with increasing amounts of Myc-KIF5B-expressing plasmids (0, 1, 2 ​μg). Empty vector was used to ensure that each group receives the same amounts of total DNA plasmids. After 24 ​h, cells were infected with equal amounts of FMDV (MOI ​= ​0.5) for 12 ​h. The expression of Myc-KIF5B and FMDV VP1 protein was examined by Western blotting (B). Viral mRNA levels were examined by RT-PCR (C). Cell supernatant and precipitation were mixed, and freeze-thawed for three times, then viral titers were determined by TCID₅₀ assay (D). Data are presented as means ± SDs from three independent experiments. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001; ​∗∗∗∗P < 0.0001. E IFA to detect the impact of KIF5B on FMDV replication. PK-15 ​cells were grown on the bottom of glass cell culture dish for 12 ​h and transfected with KIF5B-Myc or vector plasmids (1 ​μg) for 24 ​h. Cells were infected with FMDV (MOI ​= ​0.5) for 8 ​h, and analyzed by immunofluorescence staining with anti-FMDV (green), anti-Myc (red) and DAPI (blue).

Fig. 4

Down-regulation of KIF5B markedly inhibited FMDV replication. A–B To evaluate the effect of NC siRNA or KIF5B siRNA on KIF5B expression, PK-15 ​cells were transfected with NC siRNA or KIF5B siRNA (siRNA-1642, siRNA-3159, and siRNA-775) for 36 ​h, the knockdown efficiency was determined by RT-PCR (A) and immunoblot analysis (B). C–F The ability of KIF5B-knockdown in FMDV replication. PK-15 ​cells were transfected with NC siRNA or KIF5B-siRNA-1642 for 24 ​h, followed by infection with FMDV at 0, 12, and 24 ​h. The mRNA expression levels of KIF5B and FMDV were measured by RT-PCR (C and D). The protein expression levels of KIF5B and viral VP1 proteins were detected by immunoblot (E). FMDV yields were determined by TCID₅₀ assay (F). Data are presented as means ± SDs from three independent experiments. ∗∗P < 0.01.

Fig. 5

Knockout of KIF5B suppressed FMDV replication. A Schematic chromatogram of gRNA targets at the pKIF5B genomic region. PAM sequences were highlighted in green. sgRNA targeting sites were highlighted in red. B-E PCR amplicon from the KIF5B genome to confirm the genome editing in PK-KIF5B-KO-1 ​cell line (B), IBRS-KIF5B-KO-1 ​cell line (C), PK-KIF5B-KO-2 ​cell line (D), IBRS-KIF5B-KO-2 ​cell line (E). F–G Cell viability of PK-KIF5B-KO cell lines (F), IBRS-KIF5B-KO cell lines (G). H–I Immunoblot confirmed successful knockout of KIF5B in PK-KIF5B-KO cell lines (H), IBRS-KIF5B-KO cell lines (I). J Knockout of pKIF5B inhibits FMDV progeny virion production in PK-15 ​cells. PK-KIF5B-WT and PK-KIF5B-KO cell lines were infected with FMDV (MOI ​= ​0.5) at 0, 6, 12, and 24 ​h. The protein expression levels of KIF5B and viral proteins were detected by immunoblot. K Knockout of pKIF5B decreased FMDV progeny virion production in IBRS cells. IBRS-KIF5B-WT and IBRS-KIF5B-KO cell lines were infected with FMDV (MOI ​= ​0.5) at 0, 6, 12, and 24 ​h. The protein expression levels of KIF5B and viral proteins were detected by immunoblot. L PK-KIF5B-WT and PK-KIF5B-KO cell lines were infected with FMDV (MOI ​= ​0.5) at 0, 6, 12, and 24 ​h. The mRNA expression level of FMDV was measured by RT-PCR. M PK-KIF5B-WT and PK-KIF5B-KO cell lines were infected with FMDV (MOI ​= ​0.5) at 36, and 48 ​h. Supernatant and cell precipitation mixture was freeze-thawed for three times, and FMDV yields were determined by TCID₅₀ assay. Data are presented as means ± SDs from three independent experiments. ∗∗P < 0.01; n.s., not significant.

Fig. 6

KIF5B modulated FMDV internalization. A, B The effect of KIF5B overexpression on FMDV adsorption (A) and internalization (B). PK-15 cells were transfected with KIF5B-Myc or vector plasmids for 24 h, then infected with FMDV at an MOI of 20. The cell-bound and internalized FMDV virions were quantified by RT-PCR. C, D Bound virus (C) and internalized virus (D) were assessed. After adsorption (C) or internalization (D), the cells were fixed at the indicated time points and processed for confocal microscopy with AF594-phalloidin (actin filaments), anti-FMDV (viral particles), and DAPI (cell nuclei). E Endogenous co-localization of FMDV particles and KIF5B in the early stages of FMDV infection (MOI = 10). F, G The effect of KIF5B knockdown on FMDV adsorption (F) and internalization (G). The PK-15 cells were transfected with indicated siRNA (150 nmol/L) for 24 h, and then infected with FMDV at an MOI of 20. H The effect of KIF5B-knockout on FMDV adsorption (H) and internalization (I). PK-KIF5B-KO cells, or KIF5B-KO cells reconstituted with KIF5B, or WT cells were infected with FMDV at an MOI of 20. FMDV virions were quantified by RT-PCR. J Schematic illustration of bicistronic FMDV IRES construct. K The effect of endogenous KIF5B on FMDV IRES-driven translation. PK-15 WT or KIF5B-KO cells were transfected with the bicistronic construct FMDV-IRES or vector plasmids. At 36 h posttransfection, the Rluc and Fluc activities were determined. L The effect of KIF5B on viral RNA synthesis. PK-15 WT or KIF5B-KO cells were transfected with infectious viral RNA (3 μg/well) for 24 h. The viral copy numbers were quantified. M-O Effect of KIF5B on FMDV virion assembly. PK-15 WT or KIF5B-KO cells were transfected with infectious viral RNA (3 μg/well) for 36 h, and the extracellular (M) and intracellular (N) viral RNA levels were measured. The extracellular to intracellular viral RNA levels ratio was calculated to indicate virion assembly/release efficiency (O). Data are presented as means ± SDs from three independent experiments. ∗∗P < 0.01; n.s., not significant.

Fig. 7

KIF5B-mediated FMDV internalization relied on clathrin. A The effect of clathrin-knockdown in PK-15 ​cells. PK-15 ​cells were grown on 6-well plates for 12 ​h, and then transfected with clathrin pX459-gRNA-1 and pX459-gRNA-2 mixture plasmids. Post transfection 24 ​h, cells were selected with puromycin (3 ​μg/mL) for 3 days. The efficacy of polyclonal knockdown cell lines was confirmed by immunoblot assay. B The effect of clathrin-knockdown on KIF5B-regulated FMDV internalization. Clathrin-WT or clathrin-KD cells were transfected with KIF5B-Myc or vector plasmids for 24 ​h, and then infected with FMDV at an MOI of 20 and cultured at 4 ​°C for 1 ​h. The internalized FMDV virions were quantified by RT-PCR. C The effect of KIF5B on clathrin uncoating. Immunoprecipitation of Hsc70 in KIF5B-KO cells or WT cells was performed. The precipitated Hsc70, co-precipitated CHC or input cell lysates were detected by Western blotting analysis. D The effect of KIF5B on FMDV transportation to early endosome or late endosome. PK-KIF5B-KO or WT cells were inoculated with 10 MOI FMDV for 2 ​h at 37 ​°C, and then cells were fixed and stained with guinea pig anti-FMDV (green), mouse anti-Rab5 (red) or mouse anti-Rab7 (red) for confocal microscopy. The co-localization analysis was expressed as Pearson's correlation coefficient, measured for individual cells. Data are presented as means ​± ​SDs from three independent experiments. ∗∗, P ​< ​0.01.

Fig. 8

KIF5B mediated-FMDV internalization model. First, KIF5B directly interacts with VP1 to promote FMDV internalization. Then, KIF5B regulates clathrin uncoating. Finally, KIF5B promotes the transmission of viral particles to early and late endosomes during the early stages of FMDV infection.