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. 2024 Mar 16;39(3):378–389. doi: 10.1016/j.virs.2024.03.005

KIF5B-mediated internalization of FMDV promotes virus infection

Wei Zhang a,b,1, Fan Yang a,b,1, Yang Yang a,b, Weijun Cao a,b, Wenhua Shao a,b, Jiali Wang a,b, Mengyao Huang a,b, Zhitong Chen a,b, Xiaoyi Zhao a,b, Weiwei Li a,b, Zixiang Zhu a,b, Haixue Zheng a,b,⁎
PMCID: PMC11279799  PMID: 38499154

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: FMDV, VP1 protein, KIF5B, Endosome, Clathrin

Highlights

  • •

    KIF5B directly interacts with VP1 to promote FMDV internalization.

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    KIF5B regulates clathrin uncoating.

  • •

    KIF5B promotes the transmission of viral particles to early and late endosomes during the early stages of FMDV infection.

1. Introduction

Foot-and-mouth disease (FMD) is a highly virulent contagious disease for livestock and wild cloven-hoofed animals and is seriously dangerous to animal husbandry production, causing significant economic losses around the world (Mahy, 2005). FMD virus (FMDV) is the pathogen of FMD. The virus invades host cells and relies on their metabolic enzyme system and energy material system to complete its own replication, assembly, and release processes, thus completing its life cycle. Therefore, its life cycle is also influenced by different host cell proteins (Yang et al., 2023). The interaction between host cell proteins and viral proteins determines the viral replication ability, species specificity, and tissue tropism of virus infection (Lawrence et al., 2012). Hence, in-depth research on the interaction between FMDV proteins and host cell proteins and their regulatory mechanisms for FMDV replication is of great significance.

The FMDV genome encodes a polyprotein precursor that is further proteolytically cleaved into four structural proteins (VP4, VP2, VP3, and VP1) and eight nonstructural proteins (Lpro, 2A, 2B, 2C, 3A, 3B, 3Cpro, and 3Dpol) (Feng et al., 2004). The VP1 is exposed on the surface of FMDV capsid and contains the receptor binding site which is a conserved Arg-Gly-Asp (RGD) sequence in the G-H loop of VP1 (Lawrence et al., 2013). The FMDV wild strain can utilize four different RGD-dependent integrin receptors (αVβ1, αVβ3, αVβ6 and αVβ8) to invade cells through a clathrin-dependent endocytosis pathway (Jackson et al., 2000, 2002, 2004; Martin-Acebes et al., 2007; Neff et al., 1998, 2000). In addition, some FMDV strains possess the ability to utilize heparin sulfate (HS) as receptors through a caveola-mediated endocytosis pathway into early endosomes (Bai et al., 2014; O'Donnell et al., 2008).

The VP1 protein exhibits multifunctionality by binding to host cells and eliciting an immune response. The outcome of disease and pathogenesis may be influenced by the interaction between VP1 and host proteins. In order to gain a deeper understanding of the potential pathogenic implications of VP1–host protein interactions, we conducted a yeast two-hybrid assay, as previously documented, and observed that VP1 interacted with host DnaJ heat shock protein family member A3 (DNAJA3). Furthermore, our findings demonstrated that DNAJA3 played a critical role in suppressing the replication of FMDV (Zhang et al., 2019). Cellular kinesin family member 5B (KIF5B) is also identified as a potential target of FMDV VP1 by yeast two-hybrid assay. The kinesin superfamilies (KIFs) are a class of molecular motors and play crucial roles in intracellular transportation, mitosis, cell formation, and cell function (Iworima et al., 2016). They are not only responsible for basic cell activity, like the transport of various membrane organelles, protein complexes, and mRNA, but also can regulate some intracellular molecular signaling pathways (Hirokawa et al., 2009). The mammalian genome contains three KIF5 genes (KIF5A, KIF5B, and KIF5C), and all tissues express KIF5B, while only neuronal tissue expresses KIF5A and KIF5C (DuRaine et al., 2018). KIF5B plays an important role in the replication of viruses such as herpes simplex virus (HSV), vaccinia virus (Vaccinia virus), adenovirus (Adenovirus), human immunodeficiency virus type 1 (HIV-1), and pseudorabies virus (Diwaker et al., 2020; DuRaine et al., 2018; Gao et al., 2017; Malikov et al., 2015; Scherer et al., 2020). Hence, we speculated that KIF5B may also play a crucial role in FMDV replication.

Here, we revealed the critical role of KIF5B in regulating FMDV internalization. Mechanistically, KIF5B interacts with VP1 to promote FMDV internalization via regulating clathrin uncoating, and promotes the transmission of viral particles to early and late endosomes during the early stages of infection. Thus, this study extends the theoretical basis of intracellular transport of FMDV and provides a potential target for the control and treatment of FMDV infection.

2. Materials and methods

2.1. Cells and viruses

Porcine kidney cell line PK-15 (ATCC CCL-33), porcine Instituto Biologico-Rim Suino-2 (IBRS-2) cells (Zhang et al., 2021) and baby hamster kidney-21 (BHK-21) (ATCC CCL-10) cells were cultured in minimum essential medium (MEM, Gibco, USA). Human embryonic kidney 293T (HEK-293T) (ATCC CRL-11268) cells were cultured in Dulbecco modified Eagle medium (DMEM, Gibco, USA). All mediums were supplemented with 10% fetal bovine serum (FBS), 1% streptomycin (0.2 ​mg/mL) and penicillin (200 ​U/mL). All cells were maintained at 37 ​°C with 5% CO2. FMDV type O/BY/CHA/2010 (accession no. JN998085) was isolated from an infected pig in the Baiyun District of Guangzhou city, Guangdong Province in March 2010 and propagated in BHK-21 ​cells (Yang et al., 2020).

2.2. Plasmids and antibodies

The VP1, KIF5B, KIF5B (1–413), KIF5B (413–678) and KIF5B (678–963) genes were cloned into the pcDNA3.1/myc-His A vector (Invitrogen, USA) to obtain HA-VP1, Myc-KIF5B, Myc/mCherry-KIF5B-1-413, Myc/mCherry-KIF5B-413-678 and Myc/mCherry-KIF5B-678-963, respectively. The PCR primer pairs were listed in Supplementary Table S1. All constructed plasmids were analyzed and verified by DNA sequencing. Antibodies against HA (Biolegend, Cat #901513, 1:1000), Flag (Sigma-Aldrich, Cat #F1804, 1:1000), Myc (Sigma-Aldrich, Cat #M5546, 1:1000), β-actin (Sigma-Aldrich, Cat #A5441, 1:1000), KIF5B (Abcam, Cat #ab167429 and ab25715, 1:1000), Clathrin heavy chain/CLTC (Santa cruz, Cat #sc-12734, 1:500), Rab5 (Santa cruz, Cat #sc-46692, 1:200), HSPA8/HSC70 (Santa cruz, Cat #sc-7298, 1:500), Rab7 (Santa cruz, Cat #sc-376362, 1:200), Goat anti-mouse IgG/Alexa Fluor 594 (Invitrogen, Cat # A11005, 1:300), Goat anti-rabbit IgG/Alexa Fluor 488 (Invitrogen, Cat # A11008, 1:300), Goat anti-rabbit IgG/Alexa Fluor 594 (Invitrogen, Cat # A11037, 1:300), and Goat anti-mouse IgG/Alexa Fluor 488 (Invitrogen, Cat # A28175, 1:300) were purchased from the indicated manufacturers. Guinea pig anti-FMDV positive serum (1:1000) and mouse anti-VP1 antibody (1:1000) were obtained from the Lanzhou Veterinary Research Institute (LVRI).

2.3. Coimmunoprecipitation assay (Co-IP) and immunoblot analysis

HEK-293T cells were cultured in 10-cm2 dishes, and the monolayer cells were co-transfected with various indicated plasmids. The transfected cells were cultured and lysed in 1 ​mL of lysis buffer (20 ​mmol/L Tris pH 7.5, 150 ​mmol/L NaCl, 1% Triton X-100, 1 ​mmol/L EDTA, 10 ​mg/mL aprotinin, 10 ​mg/mL leupeptin, and 1 ​mmol/L PMSF). For each sample, 1 ​mL of lysate was incubated with 0.5 ​mg of suitable antibody and 40 ​μL of protein A ​+ ​G Agarose in 20% ethanol (Beyotime) for 12 ​h. The sepharose beads were washed three times with 1 ​mL of lysis buffer containing 500 ​mmol/L NaCl. The precipitates were analyzed by immunoblot assay. For immunoblot, target proteins were resolved by SDS-PAGE and transferred onto a pure nitrocellulose blotting membrane (PALL, Mexico). The membrane was blocked and incubated with appropriate primary antibodies and secondary antibodies. The antibody–antigen complexes were visualized using enhanced chemiluminescence detection reagents (Thermo, USA).

2.4. Immunofluorescence microscopy

HEK-293T, PK-15, or BHK-21 ​cells were seeded on the glass bottom cell culture dish (Nest) for 12 ​h, and then cells were transfected with the indicated plasmids for 24 ​h, followed by infection with or without FMDV. Subsequently, cells were fixed with 4% paraformaldehyde for 30 ​min, and then permeabilized with 0.3% Triton X-100 for another 30 ​min. Fixed cells were incubated with 5% BSA at 4 ​°C for 4 ​h, followed by incubation with primary antibody and Alexa Fluor 488- or 594-conjugated secondary antibody, respectively. The images were acquired with a laser-scanning confocal microscope (LSCM, Leica SP8, Solms, Germany). To quantify the FMDV entry signal, the confocal microscope images were acquired randomly and imported to Image J2 software. Then, the number of virus particles inside the cells was automatically measured with a Macro algorithm in which threshold Intermodes was used to define a single virus particle in the cell and analyzed in ten individual field. Each experiment was performed in triplicate.

2.5. CRISPR/Cas9 knockout

The gRNAs were designed based on recommendation on the Zhang laboratory website (http://crispr.mit.edu/). Generally, to construct gRNA expression plasmid, complementary oligonucleotides encoding gRNA-567 (5′-TGGCGATGTACTTATCGCCG-3′) and gRNA-9913 (5′-AACACCCGGTCAAATGCGTA-3′) were annealed and cloned into BbsI (NEB) sites in pX459 vector (Addgene#62988). And then PK-15 or IBRS-2 ​cells were transfected with pX459-gRNA plasmids using polyplus jetPRIME transfection reagent (jetPRIME®114-15), respectively. After 24 ​h transfection, the cells were selected with puromycin (3 ​μg/mL) for 3 days. The genomic region surrounding the gRNA target site was amplified by PCR using the check primers (F1: 5′-atggctgccatgatggatcggaag-3′; R1: 5′-actccctacttcccaggactccag-3′; F2: 5′-tactgttgcagtacactgaaaggc-3′; R2: 5′-tactgttgggacaacttagcagat-3′), in the end, PCR products were purified and sequenced. The knockout efficacy was confirmed by immunoblot assay with wild-type (WT) PK-15 or IBRS-2 ​cells as a control.

2.6. TCID50 titration

BHK-21 ​cells were used to titrate the released infectious virus. The infected cells were harvested at the indicated time points, and the titers were determined in terms of 50% tissue infection dose (TCID50)/100 ​μL by using the Reed–Muench method (Reed and Muench, 1938).

2.7. RNA extraction and RT-PCR

Total RNAs were extracted with TRIzol Reagent (Invitrogen, USA). Then cDNA was transcribed from the RNA samples with the HiScript II Q RT SuperMix (Vazyme, Nanjing). The generated cDNA was used as the template to detect the expression of FMDV RNA and host cellular mRNA. The QuantStudio system (Thermo Fisher Scientific, USA) and ChamQ Universal SYBR qPCR Master Mix (Vazyme, Nanjing) were used in the real time PCR (RT-PCR) to quantify the abundance of various RNAs. Primers used for RT-PCR assays were listed in Supplementary Table S2. The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used as an internal control. Relative expression of mRNA was calculated using the comparative cycle threshold (2−ΔΔCT) method. All the experiments were repeated three times with duplicates each.

2.8. Adsorption and internalization assay

For FMDV adsorption assay, the KIF5B-transfected cells were infected with FMDV at an MOI of 20 and incubated at 4 ​°C for 1 ​h. After adsorption, the unbound viruses were washed away with ice-cold PBS, and the relative level of cell-bound viral RNA was quantified by RT-qPCR and immunofluorescence microscopy. For FMDV internalization assay, the cells were inoculated with FMDV at an MOI of 20 ​at 4 ​°C for 1 ​h. The unbound viruses were washed away with ice-cold PBS, and then the inoculated cells were transferred at 37 ​°C for 1 ​h to allow viral internalization. The viruses not entering were removed with PBS containing proteinase K, and the entering viral RNA was analyzed by RT-qPCR assay and immunofluorescence microscopy.

2.9. RNA interference (RNAi)

Small interfering RNA (siRNA) used in the RNAi assay was chemically synthesized by GenePharma (Shanghai, China). To knockdown endogenous KIF5B, KIF5B siRNA (KIF5B-Sus-1642: 5′-GCGGUGGAUAAGGAUAUUATT-3′; KIF5B-Sus-3159: 5′-GCAGGAAGUAGAUCGUAUATT-3′; KIF5B-Sus-775: 5′-CCAGAAGGCAUGGGAAUUATT-3′) was transfected into PK-15 ​cells using polyplus jetPRIME transfection reagent (jetPRIME®114-15). Non-targeting siRNA (NC siRNA: 5′-UUCUCCGAACGUGUCACGUTT-3′) was used as a negative control.

2.10. Cell viability assay

Cell Counting Kit-8 (CCK-8) (Yeasen) was used for determining cell viability. Cells were seeded into 96-well plates and cultured for 6–8 ​h, and then 10 ​μL CCK-8 solution were added according to the manufacturer's instructions. After 2 ​h incubation, the absorbance of cells was measured at OD450 nm.

2.11. Statistical analysis

All measured values (following a normal distribution) were represented as the mean with standard error from three independent experiments. Student's t-test was used to analyze the significance [∗P < 0.05 (considered significant); ∗∗P < 0.01 (considered highly significant)].

3. Results

3.1. KIF5B interacted with FMDV VP1 protein

The VP1 protein of FMDV is a multifunctional protein that can bind to host cells, inducing an immune response and cell apoptosis. Yeast two-hybrid system was used to detect host cellular proteins that were potentially associated with VP1 protein. We previously have reported that VP1 interacted with DNAJA3. Cellular KIF5B was another potential combination protein for VP1 by the yeast two-hybrid approach (Zhang et al., 2019; Zhu et al., 2023). To confirm the interaction between VP1 and KIF5B, we carried out co-immunoprecipitation (Co-IP) assays by co-transfecting Myc-KIF5B and HA-VP1 plasmids into HEK-293T cells. After immunoprecipitation with an anti-HA antibody, KIF5B was confirmed to co-precipitate with the VP1 (Fig. 1A). Moreover, confocal microscopy showed co-localization of VP1 and KIF5B in cells (Fig. 1B). Furthermore, the endogenous interactions between VP1 and KIF5B in the process of FMDV infection were detected, and the FMDV-infected cell lysates were immunoprecipitated with anti-VP1 antibodies and probed for the presence of KIF5B. The result showed that KIF5B was pulled down by VP1 in FMDV-infected cells (MOI of 0.5) (Fig. 1C). We also found the co-localization of VP1 and KIF5B in FMDV-infected cells (Fig. 1D). These results suggested that KIF5B directly interacted with FMDV VP1.

Fig. 1.

Fig. 1

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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.

To map KIF5B potential binding site of VP1, we generated KIF5B fragments that fused to mCherry or Myc tag (Fig. 2A). Next, we assessed the co-localization of KIF5B fragments and VP1. We found that VP1 mainly co-localizes with KIF5B stalk domain (413–678 regions), while partially localizes with motor (1–413 regions) and tail (678–963 regions) domains of KIF5B (Fig. 2B). Pearson correlation analysis for the co-localization between VP1 and motor, stalk, or tail domains is 0.45, 0.9, or 0.58, respectively (Fig. 2C). However, Co-IP results show that VP1 only interacts with the stalk domain, but not others (Fig. 2D). These results indicated that stalk domain is the main interaction region with VP1.

Fig. 2.

Fig. 2

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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.

3.2. KIF5B positively modulated FMDV replication

To access the role of KIF5B during FMDV infection, BHK-21 ​cells were transfected with KIF5B-expression plasmids or vectors for 24 ​h, and then transfected cells were infected with FMDV for 0, 2, 4, 8, and 12 ​h. The expression of viral proteins was detected by Western blotting. A considerable enhancement of viral protein expression was observed in KIF5B-overexpression cells compared with cells transfected with vector plasmids (Fig. 3A). In addition, KIF5B enhanced viral replication in a dose-dependent manner (Fig. 3B). We also measured viral RNA in cells and virus titers in the cell supernatant and sediment mixture. As expected, both viral RNA and virus yields were dramatically enhanced in KIF5B-overexpression cells in a dose-dependent manner (Fig. 3C and D). Additionally, confocal images showed that overexpression of KIF5B significantly increased FMDV replication (Fig. 3E).

Fig. 3.

Fig. 3

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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).

Next, we evaluated FMDV replication in KIF5B down-regulated cells. KIF5B was knocked down in PK-15 ​cells with RNAi. Three KIF5B siRNAs were designed and synthesized, and their silencing efficiencies were evaluated by RT-PCR and Western blotting. siRNA-1642 exerted the highest efficiency in decreasing KIF5B expression, both at the mRNA and protein levels (Fig. 4A and B). PK-15 ​cells were transfected with negative control (NC) siRNA or siRNA-1642 and then infected with equal amounts of FMDV for 0, 12, and 24 ​h. RT-PCR indicated that KIF5B-siRNA-1642 substantially suppressed mRNA levels of KIF5B (Fig. 4C). FMDV mRNA levels were extensively diminished in KIF5B knockdown cells compared with the NC siRNA cells (Fig. 4D). Immunoblot assays showed that siRNA-mediated KIF5B knockdown also suppressed FMDV VP1 expression (Fig. 4E). Furthermore, the TCID50 assay showed that KIF5B knockdown also reduced viral titers by ∼1.0665 log10 TCID50/0.1 ​mL (corresponding to ∼11.65-fold) at 24 hpi (Fig. 4F). These results implied that FMDV replication was significantly inhibited in KIF5B knockdown cells.

Fig. 4.

Fig. 4

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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.

3.3. Knockout of KIF5B suppressed FMDV replication

To further verify the influence of KIF5B on FMDV replication, we constructed KIF5B knockout PK-15 and IBRS-2 ​cells with CRISPR/Cas9 genomic editing system. Two guide RNAs (gRNA-567 and gRNA-9913) were designed to target two sites of the exon 1 and exon 2 of KIF5B genome sequences, respectively (Fig. 5A). Genomic sequencing of the gRNA-567 ​cell clone showed that 16 nucleotides were deleted in the PK-15 ​cell line (PK-KIF5B-KO-1) (Fig. 5B), and one nucleotide was inserted in the IBRS-2 (IBRS-KIF5B-KO-1) cell line (Fig. 5C). In the gRNA-9913 ​cell line, 2 nucleotides were deleted in the PK-15 ​cell line (PK-KIF5B-KO-2) (Fig. 5D), and 4 nucleotides were deleted in the IBRS-2 (IBRS-KIF5B-KO-2) cell line (Fig. 5E). The viability of PK-KIF5B-KO-1 and PK-KIF5B-KO-2 ​cell lines was similar to that of wild-type (WT) PK-15 ​cells (Fig. 5F), and the viability of IBRS-KIF5B-KO-1 and IBRS-KIF5B-KO-2 ​cell lines was also similar to that of WT IBRS cells (Fig. 5G). Immunoblot analysis observed that KIF5B expression was deficient in the established cell lines (Fig. 5H and I). These findings demonstrated that KIF5B-KO cell lines were constructed successfully. Likewise, PK-KIF5B-KO cells inhibited FMDV replication compared to WT cells (Fig. 5J). Similarly, FMDV replication was significantly reduced in IBRS-KIF5B-KO cells (Fig. 5K). Meanwhile, the expression of viral RNA as well as the viral titers was also significantly suppressed in KIF5B-KO cells compared with WT cells (Fig. 5L and M). These results suggested that KIF5B knockout significantly decreased FMDV replication.

Fig. 5.

Fig. 5

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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.

3.4. KIF5B modulated FMDV internalization

We next investigated which step in the viral replication cycle is targeted by KIF5B. Firstly, FMDV was adsorbed in KIF5B-Myc-transfected cells at 4 ​°C for 1 ​h, the unbound viruses were extensively washed away with ice-cold PBS. The cell-bound FMDV virions were quantified by RT-PCR. The result showed that KIF5B overexpression did not affect the adsorption of FMDV (Fig. 6A). We subsequently detected whether KIF5B implicates to FMDV internalization. PK-15 ​cells were transfected with KIF5B-Myc plasmids or vector for 24 ​h, and then were inoculated with FMDV at 4 ​°C for 1 ​h and 37 ​°C for another 1 ​h to allow internalization. The RT-PCR showed that KIF5B overexpression significantly promoted FMDV internalization (Fig. 6B). Using confocal microscopy to visualize viral particles to further view bound and internalized virions, we observed that KIF5B had no effect on virus adhesion (Fig. 6C), but facilitated the entry of virions (Fig. 6D). In order to further study the early role of KIF5B in virus entry, cells infected with FMDV (MOI ​= ​10) were incubated at 4 ​°C for 1 ​h and then shifted to 37 ​°C for 1 ​h and 3 ​h. The results showed that there was an obvious co-localization between FMDV particles and KIF5B (Fig. 6E). These results were further validated on knockdown or knockout cell lines. PK-15 ​cells were transfected siRNA-1642 or NC for 24 ​h and then infected with FMDV for adsorption or internalization. The results showed that KIF5B knockdown markedly reduced FMDV internalization but had no effect on FMDV adsorption (Fig. 6F and G). Similarly, we also detected FMDV adsorption and internalization in KIF5B knockout cell lines, and the results were consistent with those in KIF5B knockdown cells (Fig. 6H and I). Furthermore, after supplementing KIF5B plasmids in KIF5B knockdown or knockout cell lines, the inhibitory effect of KIF5B knockout on FMDV internalization was restored (Fig. 6G and I). Taken together, the gain- and loss-of-function experiments indicated that KIF5B positively regulated FMDV internalization.

Fig. 6.

Fig. 6

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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.

Next, we investigated whether KIF5B also affects the translation, RNA synthesis, RNA assembly, or release of FMDV. Firstly, we explored the effect of KIF5B on viral mRNA translation. A bicistronic reporter plasmid was used to evaluate FMDV IRES activity (Fig. 6J), as the translation of the first cistron (Renilla luciferase, Rluc) is cap-dependent, while the translation of the second cistron (Firefly luciferase gene, Fluc) is dependent on FMDV IRES activity. The relative IRES activity was reported as the ratio of Fluc expression to Rluc expression. The bicistronic reporter plasmid was transfected into KIF5B-KO or WT cells. At 36 ​h posttransfection, cell lysates were collected to calculate the ratio of Fluc activity to Rluc activity. The FMDV IRES activity showed no difference between KIF5B-KO cells and WT cells (Fig. 6K). During FMDV infection, genomic RNA acts as the template for translation and RNA replication, closely coupling these processes (Gamarnik and Andino, 1998). To determine the effect of KIF5B on viral RNA synthesis, cells were exposed to infectious viral RNA at 3 ​μg/well in 6-well plates by transfection to bypass viral entry and uncoating steps. Following a 24-h culture, viral copy numbers were quantified by RT-PCR. The viral copy numbers show no significant difference between KIF5B-KO and WT cells (Fig. 6L), suggesting that KIF5B had no effect on viral RNA synthesis. Next, we examined whether KIF5B is involved in FMDV assembly or release. We determined the effect of KIF5B knockout on FMDV intracellular and extracellular viral RNAs. The ratio of extracellular to intracellular RNA levels was calculated to indicate virion assembly and release efficiency. Cells were exposed to infectious viral RNA at 3 ​μg/well in 6-well plates by transfection to bypass viral entry and uncoating steps. Following 24 ​h of culture, viral copy numbers were quantified by RT-PCR. The results showed that KIF5B had no effect on intracellular or extracellular viral RNAs (Fig. 6M and N), leading to no difference in extracellular/intercellular viral RNA ratios (Fig. 6O). These results suggest that KIF5B is only involved in viral internalization.

3.5. KIF5B-mediated FMDV internalization relied on clathrin

The RGD sequence in the G-H loop of VP1 is the binding site of the integrin receptor, which relies on the clathrin-mediated endocytosis to infect the host cell (Martin-Acebes et al., 2007). KIF5B regulates clathrin uncoating, promoting clathrin-mediated endocytosis (Ni et al., 2018). To investigate whether KIF5B-mediated FMDV internalization relies on clathrin, we designed two gRNAs targeting clathrin using the CRISPR/Cas9 genomic editing system and constructed a polyclonal clathrin knockdown (KD) cell line. The knockout effect was detected by Western blotting, indicating that the clathrin was successfully knocked down (Fig. 7A). Clathrin-KD cells were transfected with KIF5B-Myc or empty plasmids for 24 ​h, and then were inoculated with FMDV at 4 ​°C for 1 ​h and 37 ​°C for another 1 ​h to allow internalization. The FMDV internalization level was detected using RT-PCR. The results showed that overexpression of KIF5B substantially promoted FMDV internalization in clathrin-WT cells, while there was no significant change in clathrin-KD cells (Fig. 7B). These data indicated that clathrin is required for KIF5B-mediated FMDV internalization. Previous studies demonstrated that KIF5B regulates clathrin uncoating in mouse cortices (Ni et al., 2018). To investigate whether KIF5B regulates clathrin uncoating in porcine kidney-15 (PK-15), the endogenous Co-IP was performed. Notably, when testing the endogenous clathrin heavy chain (CHC)–Hsc70 interaction that is important for clathrin-coated vesicles (CCV) uncoating by immunoprecipitating PK-KIF5B-KO or WT cell lysate with Hsc70 antibody, we detected less CHC in Hsc70 precipitates in PK-KIF5B-KO cell than WT cell (Fig. 7C). This decreased binding between Hsc70 and CHC in PK-KIF5B-KO cell implied the involvement of endogenous KIF5B in modulating CHC–Hsc70 interaction and possibly CCV uncoating. Our previous studies have confirmed that FMDV utilized the endosomal proteins Rab5 (early endosome) and Rab7 (late endosome) during the processes of entering cells (Chen et al., 2022), and kinesin-1 regulated endocytic trafficking of classical swine fever virus (Lou et al., 2023). To evaluate whether KIF5B promotes FMDV particles for translocating to early endosome or late endosome, PK-KIF5B-KO or WT cells were inoculated with FMDV, and then cells were fixed and stained with indicated antibodies. Confocal microscopy showed that KIF5B functional deficiency blocks FMD virion access to the early endosome or late endosome (Fig. 7D). Overall, these results suggest that KIF5B is involved in CCV uncoating and endocytic trafficking, regulated by early endosomes and late endosomes.

Fig. 7.

Fig. 7

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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.

4. Discussion

FMD is a highly contagious disease caused by FMDV, which is responsible for huge economic losses globally. FMDV parasitizes and proliferates within cells, utilizing various host factors to achieve virus invasion, replication, and the release of infectious viral particles (Liu et al., 2021; Wu et al., 2023). However, the cell proteins involved in FMDV replication are not fully understood. Here, we identified that FMDV structural protein VP1 interacted with the host KIF5B protein, an important molecular motor proteins that directionally transports various cargos, including membranous organelles, protein complexes, and mRNAs.

Among the molecular motors that are involved in intracellular transport, three large superfamilies have been identified: kinesins, dyneins, and myosins (Hirokawa and Takemura, 2005; Vale, 2003). Kinesin superfamily proteins, also known as KIFs, play a vital role in the process of intracellular transport in various cell types. The KIF5 motor complex consists of two kinesin family KIF5 motors, which are also known as kinesin heavy chains (KHCs). Generally, two kinesin light chains (KLCs) associate with the tail domains of the two KIF5 motors. KIF5 motors transport synaptic vesicle precursors (synaptotagmin and synaptobrevin) and membrane organelles such as syntaxin 1 and SNAP25 (Diefenbach et al., 2002; Su et al., 2004). KIF5 motors are thought to be also responsible for transporting mitochondria (Conforti et al., 1999), elongation of neurites (Yamazaki et al., 1995), and polarization of neurons (Nakata and Hirokawa, 2003). KIF5 motors bind to their cargos through at least two different regions. KIF5 stalk domains can bind to KLCs, which in turn associate with certain cargos, or the specific cargo-binding region of the KIF5 tail domain can directly bind to cargos (Diefenbach et al., 1998). Despite the fact that an interaction between KIF5B 1–413 or 678–963 co-localization with VP1 protein was not detected by the Co-IP assay, a co-localization relationship could be observed in co-transfected cells, suggesting that they likely interact within intact cells.

Previous studies have proved that KIF5B plays an essential role in DNA virus replication processes such as HSV, vaccinia virus, adenovirus, HIV-1, and pseudorabies virus (Diwaker et al., 2020; DuRaine et al., 2018; Gao et al., 2017; Malikov et al., 2015; Scherer et al., 2020). Notably, KIF5B is also involved in RNA virus replication. Recently, research showed that KIF5B enhanced CSFV (a positive single-stranded RNA virus) infection and engaged in the transport of multiple vesicles after endocytosis, including late and recycling endosomes and lysosomes (Lou et al., 2023). Consistently, we also demonstrated that KIF5B regulates FMDV (a positive single-stranded RNA virus) replication. To understand the molecular mechanisms of KIF5B-mediated virus replication, we performed several experiments. We observed that KIF5B involved in FMDV internalization, and further study found that KIF5B is an uncoating regulator of CCV in PK-15 ​cells. Upon binding to a specific receptor on the surface of the host cell, FMDV can gain entry into the cell through various pathways. FMDV exploits integrin receptors to infect the cell via the clathrin-mediated endocytosis pathway, subsequently trafficking through early endosomes. On the other hand, FMDV that binds to heparin sulfate (HS) receptors enters the cell through a caveola-mediated endocytosis pathway, also leading to early endosomes. Within the acidic environment of the endosomes, FMDV undergoes viral uncoating and releases RNA. Rab proteins, known as small GTPases, exert regulatory control over the progression from early to late endosomes. The early (Rab5) and late (Rab7) endosomal proteins significantly contribute to the formation of early endosomes and lysosomes (Jordens et al., 2005). The internalization process encompasses the entry of the virus into the cell via either the clathrin- or caveola-mediated endocytosis pathway, subsequently, the virus is transported to the endosome, where it undergoes uncoating and releases its RNA in the acidic environment of the endosomes. KIF5B facilitates viral entry into cells via clathrin-mediated endocytosis pathway by inducing the clathrin uncoating.

Additionally, numerous motor proteins have been identified on endosomes. Motors drive cargos in opposite directions, and this determines the localization of endosomes (Bananis et al., 2000). In the early phase, endosomes that contain RAB5 or RAB4 are driven by cytoplasmic dynein, KIF5, KIF3 and KIFC2 motors. When transferred to late endosomes, these vesicles are labeled with RAB7-bound to cytoplasmic dynein, KIF3, and sometimes KIFC2. Here, we found that KIF5B elevates viral particles converting to early and late endosomes during the early stages of replication. Therefore, KIF5B enhances viral internalization by promoting the transport of viruses to endosomes. Consequently, a reduction in the internalization of viral particles is observed in KIF5B-KO cells.

5. Conclusions

In summary, our study demonstrated that KIF5B is an essential factor for FMDV infection. Furthermore, KIF5B can regulate FMDV internalization. Mechanistically, KIF5B directly interacts with VP1 to promote FMDV internalization via regulating clathrin uncoating, and promotes the transmission of viral particles to early and late endosomes during the early stages of replication (Fig. 8). Hence, our findings provide a new therapeutic target for FMDV prevention.

Fig. 8.

Fig. 8

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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.

Data availability

The authors confirm that the data supporting the findings of this study are available within the article.

Ethics statement

This article does not contain any studies with human or animal subjects performed by any of the authors.

Author contributions

Wei Zhang: conceptualization, data curation, investigation, writing & original draft, writing-review & editing. Fan Yang: data curation, formal analysis, methodology, resources, funding acquisition. Yang Yang: data curation, investigation, methodology. Weijun Cao: investigation, supervision. Wenhua Shao: data curation, validation. Jiali Wang: data curation, methodology, resources. Mengyao Huang: validation, resources. Zhitong Chen: formal analysis, methodology. Xiaoyi Zhao: formal analysis, resources. Weiwei Li and Zixiang Zhu: conceptualization, data curation, formal analysis, project administration. Haixue Zheng: data curation, methodology, project administration, resources, supervision.

Conflict of interest

The authors declare that they have no conflict of interest.

Acknowledgements

This work was supported by the National Natural Sciences Foundation of China (No. 32102639 and 32072831), the National Key Research and Development Program of China (No. 2021YFD1800300), the Gansu Science Foundation for Distinguished Young Scholars (No. 21JR7RA026), the Earmarked Fund for CARS-35, the Strategic Priority Research Program of the National Center of Technology Innovation for Pigs (No. NCTIP-XD/C03), the Science and Technology Major Project of Gansu Province (No. 22ZD6NA001), the Natural Science Foundation of Gansu Province (No. 22JR5RA034 and 23JRRA549), and the open competition program of top ten critical priorities of Agricultural Science and Technology Innovation for the 14th Five-Year Plan of Guangdong Province (No. 2023SDZG02), the Fundamental Research Funds for the Central Universities (No. lzujbky-2022-ey20).

Footnotes

Appendix A

Supplementary data to this article can be found online at https://doi.org/10.1016/j.virs.2024.03.005.

Appendix A. Supplementary data

The following is the Supplementary data to this article:

Multimedia component 1
mmc1.docx (21.5KB, docx)

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Data Availability Statement

The authors confirm that the data supporting the findings of this study are available within the article.

Articles from Virologica Sinica are provided here courtesy of Wuhan Institute of Virology, Chinese Academy of Sciences

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