Entry of sapelovirus into IPEC-J2 cells is dependent on caveolae-mediated endocytosis

IPEC-J2 cells were cultured in RPMI 1640 medium (Gibco, USA), supplemented with 10% fetal bovine serum (FBS, Gibco) in 5% CO₂ at 37 °C. The PSV (csh) strain was isolated and preserved in our laboratory [13]. Mouse polyclonal anti-PSV VP1 antibody was generated by our laboratory.

Inhibitors chlorpromazine (CPZ), ammonium chloride (NH₄Cl), chloroquine (CQ), methyl-β-cyclodextrin (MβCD), nystatin, dynasore, 5-(N-ethyl-N-isopropyl) amiloride (EIPA), and wortmannin were purchased from Sigma, dissolved in water or DMSO and preserved in − 80 °C. Alexa Fluor 594-conjugated cholera toxin B (CTB) and Alexa Fluor 568-conjugated transferrin (Tfn) were purchased from Invitrogen (Carlsbad, CA, United States). Cell viability upon inhibitor treatment was assessed by employing the cell counting kit-8 (CCK-8, Beyotime Biotechnology, Shanghai, China). Briefly, cells were seeded in 96-well cell culture plates and subsequently treated with drugs at different concentrations for 24 h. After incubation with 10 μl CCK-8 for 2 h at 37 °C, the data of absorbance at a wavelength of 450 nm were collected.

IPEC-J2 cell monolayers were grown in 24-well plates. After washing with DPBS for three times, the cells were pre-treated with inhibitors at the indicated concentrations for 1 h at 37 °C. For virus entry and replication assay, PSV was then added (MOI = 0.5). After incubation for 1 h, the unbound virus was removed by three washes with DPBS, and fresh medium was added to the cells. At 24 hpi, cells were lysed for virus titers determined by TCID₅₀ or VP1 protein expression levels assays detected by western blot.

Plasmid expressing GFP-tagged Eps15 (WT), Eps15 (EpsΔ95/295), caveolin 1 wild type (Cav WT), caveolin 1 DN mutant (Y14F), dynamin-2 (WT) and dynamin-2 (K44A) were constructed by our laboratory and sequenced by Sangon (Shanghai, China). To determinate the infectivity of PSV in cells transfected with WT or DN mutant, IPEC-J2 cells grown on 24-well plates were fist transfected with 0.5 μg of plasmids for 24 h. Cells were then infected with PSV (MOI = 0.5), and virus replication was detected with western blot.

After siRNA transfection for 30 h, cells were lysed and total RNA was extracted using TRIzol (Invitrogen). The mRNA levels of clathrin heavy chains (CHC) and caveolin-1 were checked by RT-qPCR. RT-qPCR was conducted with SYBR green master mix on an ABI 7500 Real-Time PCR System and 7500 System Software (Applied Biosystems, Alameda, CA, USA). For western blot analysis, cells were lysed in RIPA lysis buffer. After being separated by SDS-PAGE, the proteins were electrotransferred onto PVDF membranes and then immunoblotted with mouse anti-PSV VP1 antibody (1:1000) and anti-mouse secondary antibodies conjugated to HRP (1:10,000). α-tubulin was used as a loading control. Finally, bands were developed with ECL prime western blot detection reagent (GE Healthcare), and then quantified with Image Pro-Plus software.

PK-15 cells seeded in 12-well plates with coverslips were left untreated or pretreated with indicated inhibitors for 1 h, and incubated with 50 μg/ml Alexa Fluor 568-conjugated Tfn or 10 μg/ml Alexa Fluor 594 conjugated-CTB at 37 °C for 60 min. Then, cells were washed with cold PBS for three times, fixed with cold 0.4% paraformaldehyde. Cell nuclei were stained with DAPI and cells were observed by confocal microscopy.

Inhibitor treated and mock-treated IPEC-J2 cells infected with PSV were harvested at 24 h post-infection through freezing and thawing for three times, and then centrifuged to remove cell debris. Confluent cell monolayers in 96-well cell culture plates were incubated at 37 °C for 1 h with 10-fold serial dilutions of collected virus (100 μl/well). About 4–5 days later, cytopathic effect was recorded and virus titers were calculated using the Reed-Muench method and recorded as TCID₅₀/100 μl.

To determine the effect of endosomal acidification on PSV infectivity, we exposed IPEC-J2 cells to increasing concentrations of CQ and NH₄Cl for 60 min. The cells were then exposed to PSV at an MOI of 0.5 for 24 h for titer measurement and VP1 protein synthesis detection. NH₄Cl and CQ, both lysosomotropic weak bases, inhibit endosomal acidification. Data from cell viability assays determined the subtoxic concentrations of the drugs (Fig. 1a and b). Compared to mock-treated cells (6.38 log₁₀TCID₅₀/100 μl or 6.22 log₁₀TCID₅₀/100 μl), the virus titer from cells treated with 5 mM NH₄Cl or 10 μM CQ was 5.57 log₁₀TCID₅₀/100 μl or 5.38 log₁₀TCID₅₀/100 μl, respectively (Fig. 1a and b). Likewise, viral VP1 protein synthesis was also diminished by NH₄Cl and CQ in a concentration-dependent manner (Fig. 1c). Together, these findings suggest that PSV enters IPEC-J2 cells uses a pH-dependent and, therefore, most likely an endosomal cell entry pathway.

CPZ is a commonly used chemical inhibitor for interfering with CME through preventing the assembly of clathrin lattices on endosomal membranes and the assembly of clathrin coated pits at the cell surface [25]. To examine the role of clathrin-dependent endocytic pathways in PSV entry and infection of IPEC-J2 cells, we first tested the effect of CPZ on transferrin uptake, which is a model for the CME pathway. As shown in Fig. 2a, when treated with10 μM CPZ, the signal intensity of fluorescently labeled transferrin was obviously reduced. Then we treated IPEC-J2 cells with increasing concentrations of CPZ at subtoxic concentrations (Fig. 2b). However, PSV virus titers or VP1 protein synthesis did not appear to be reduced in the presence of CPZ (Fig. 2b, c). We next tested the effect of knocking down CHC on PSV infection. The siRNA efficiency against CHC was examined by RT-qPCR (Fig. 2d). IPEC-J2 cells were then transfected with siRNA negative control, a non-targeting CHC, or a siRNA targeting against CHC. At 30 h after transfection, cells were infected with PSV (MOI = 0.5), and at 24 h post infection cell lysates were collected, and detected by western blot. The siRNA targeting CHC also did not reduce viral infection (Fig. 2e). To further confirm that PSV enters IPEC-J2 cells does not require CME, we transfected Eps15, which is crucial component of clathrin coated pits and interacts with adaptor protein 2 [26]. Similarly, no significant reduction in VP1 protein synthesis was observed with transfecting wild-type or DN mutant Eps15 (Fig. 2f). Collectively, these data indicate that CME may be not an essential pathway for PSV infection.

Caveolae are plasma membrane invaginations which are involved in numerous cellular processes including transport, signaling, and tumor suppression. The formation of caveolae depends on the expression of caveolin-1 (Cav1). Cav1 directly interacts with cholesterol of the plasma membrane [27] and depletion of the cholesterol makes caveolae flatten [28]. To investigate the possible involvement of caveolae/lipid rafts in PSV entry, we first tested the effect of cholesterol sequestrator nystatin and the caveolae-dependent endocytic inhibitor MβCD on CTB uptake assays. A significant reduction in the signal intensity of fluorescently labeled CTB was observed upon either 5 μM nystatin or 2 mM MβCD pretreatment, indicating a block in CTB uptake (Fig. 3a). Therefore, prior to PSV infection, we treated IPEC-J2 cells with increasing concentrations of nystatin and MβCD at subtoxic concentrations (Fig. 3b). We observed that 10 μM nystatin and 3 mM MβCD reduced virus titers by about 1 log₁₀ and 2 log₁₀, respectively, compared with the untreated controls (Fig. 3b). Similarly, at an MOI of 0.5, VP1 protein levels decreased by 53 and 62% in 5 μM nystatin and 10 μM nystatin pretreatment, respectively, and by 77 and 96% in 2 mM MβCD and 3 mM MβCD pretreatment (Fig. 3c).

Furthermore, cell perturbation assays using siCav were carried out to evaluate the role of caveolin in virus infection. The siRNA efficiency was first detected using RT-qPCR, and siCav could specifically decrease caveolin expression (Fig. 3d). Then, siNC or siCav was transfected into the IPEC-J2 cells. At 30 h after transfection, we performed PSV entry assays, and found that siCav specifically decreased viral VP1 protein expression approximately 45% (Fig. 3e). We also transfected IPEC-J2 cells with a dominant-negative form of the protein with a Y14F mutation, which has been reported to inhibit caveolae dependent endocytosis [29]. IPEC-J2 cells were transfected with either caveolin-1 WT or DN plasmid caveolin-1 Y14F as well as control vector before virus infection. We found that PSV VP1 protein synthesis was significantly decreased by approximately 80% in cells expressing caveolin-1 DN compared to cells transfected with empty vector (Fig. 3f). Together, the data suggest that PSV enters IPEC-J2 cells depend on caveolae and lipid raft.

Dynamin-2, a regulatory GTPase, is essential for the fission of caveolae from the plasma membrane [30]. Dynasore, a cell-permeable dynamin GTPase activity inhibitor, was used to detect the role of dynamin during PSV infection in this study. We observed about 2 log₁₀ decrease in virus titer in cells pretreated with 10 μM dynasore compared with the untreated cells (Fig. 4a). Furthermore, dynasore reduced PSV VP1 protein synthesis by 60% at 5 μM, whereas at a concentration of 10 μM, dynasore reduced PSV VP1 protein synthesis by 70% (Fig. 4b). To further verify the possible involvement of dynamin in PSV entry, we transfected IPEC-J2 cells with plasmids encoding wild-type dynamin-GFP or the DN mutant dynamin (K44A) and found that expression of the DN mutant dynamin (K44A) could decrease PSV VP1 protein synthesis by approximately 71% (Fig. 4c), suggesting that PSV entry into IPEC-J2 cells require dynamin.

Macropinosome formation depends on Na⁺/H⁺ exchangers, and EIPA is an Na⁺/H⁺ antiport inhibitor, therefore, inhibits macropinocytosis-mediated endocytosis [12]. Wortmannin is a covalent inhibitor of PI3K, which is involved in multiple stages of macropinocytosis [31]. To investigate whether PSV entry involves macropinocytosis and PI3K, PSV was added to IPEC-J2 cells pretreated with EIPA or wortmannin. EIPA had no effect on PSV titer or VP1 protein synthesis at different concentrations, as indicated by TCID₅₀ assays or western blot (Fig. 5a,b). However, 5 μM wortmannin decreased virus titer by about 1 log₁₀ and decrease PSV VP1 protein synthesis by 21%, respectively (Fig. 5c, d). These findings suggested that PSV entry and replication does not depend on macropinocytosis, but requires P13K.