key: cord-0810371-pz5sh1vc
authors: Li, Qiongyi; Liu, Yang; Xu, Shujuan; Zhao, Kexue; Ling, Ying; Liu, Rongxiu; Ali, Amjad; Bai, Jialin
title: Caveolin-1 is involved in encephalomyocarditis virus replication in BHK-21 cells
date: 2021-03-24
journal: Virol J
DOI: 10.1186/s12985-021-01521-3
sha: 908fa7a27346595b95b44deb79a22cb9ec864ac2
doc_id: 810371
cord_uid: pz5sh1vc

BACKGROUND: Encephalomyocarditis virus, member of Cardiovirus genus within Picornaviridae family, is an important pathogen that infects different domestic and wild animals. However, the molecular mechanism of its entry remains unclear. In this study, we investigated the mechanism of EMCV infectivity in relation to endocytic pathway using BHK-21 cells. METHODS: The function of numerous cellular key factors implicated in the various endocytic mechanisms were systematically explored using chemical inhibitors. Furthermore, RNA interference (RNAi) as well as the overexpression of dominant protein combined to virus infectivity assays, and confocal microscopy was used to examine EMCV infection in details. RESULTS: The results indicated that the EMCV entry into BHK-21 cells depends on caveolin, dynamin, and actin but not clathrin nor macropinocytosis pathways. The effects of overexpression and knockdown of caveolin-1, one components of the caveolae, was examined on EMCV infection. The results showed that EMCV infection was positive correlation with caveolin-1 expression. Confocal microscopy analysis and internalization assay showed that caveolin-1 is required at the early stage of EMCV infection. CONCLUSIONS: Caveolin-1, dynamin, and actin-dependent endocytosis pathways are necessary for EMCV infection in vitro.

in their study indicated that EV1 entry to CV-1 cells is dynamin-dependent [13] . Poliovirus infects different host cells using different endocytic pathways, for example, its entery into Hela cells is clathrin-and caveolin-independent while infects brain microvascular endothelial cells, it utilizes caveolin-and dynamin-dependent routes [14] [15] [16] . CBV3 enters into HeLa cells using clathrinmediated endocytosis pathway [17] and coxsackie virus A9 entry into A549 cells is mediated by dynamin, β2-microglobulin, and Arf6 [12] while FMDV internalization is by clathrin-dependent pathway [18] [19] [20] . However, as one member of this family, little is known about the entry mechanism of EMCV.

Based on the above mentioned studies and being a member of the Picornaviridae family, we hypothesized that EMCV may also use the endocytic mechanisms for causing infection. Therefore, we designed this study to investigate EMCV infection with relation to endocytic pathway using BHK-21 cells. At first, we confirmed that the EMCV replication was related to endocytosis. Subsequently, we demonstrated that neither clathrin nor macropinocytosis pathway was involved in virus infection. We have shown that EMCV replication into the BHK-21 cells via caveolin-mediated and dynamin, actindependent pathway.

BHK-21 cells were provided by the Gansu Tech Innovation Center of Animal Cell (Northwest Minzu University, Lanzhou, China) and were maintained in DMEM (Minhai Bio-engineering, Lanzhou, China) supplemented with 10% FBS (Minhai Bio-engineering) in a 5% CO 2 incubator at 37 °C.

The EMCV strain used in the current study was the BHK-21 cells adapted EMCV (GenBank: X74312) and its titer was 10 6.0 TCID 50 ml −1 . The plaque-forming unit (PFU) was calculated as previously described [21] .

Mouse monoclonal antibody (mAb) against Caveolin-1 and rabbit polyclonal antibody (pAb) anti-Caveolin-1 were bought from Beyotime (Beyotime Biotechnology, Shanghai, China) and MAb against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was purchased from Abcam (Abcam, Cambridge, UK). MAb against EMCV-VP1 was kindly donated by Dr. Juan Bai (College of Veterinary Medicine, Nanjing Agricultural University, China). Anti-GFP mAb were purchased from TransGen (TransGen Biotech, Beijing, China).The HRP-labeled secondary antibody, Alexa fluor-488-conjugated anti-mouse and Cy TM 3-conjugated anti-rabbit IgG (H + L) were from Jackson ImmunoResearch Laboratories (Jackson Immu-noResearch Laboratories, PA, USA).

Total RNA was isolated, followed by qRT-PCR as previously described [22] , using primers EMCV-3D qF: GTC ATA CTA TCG TCC AGG GAC TCT AT and qR: CAT CTG TAC TCC ACA CTC TCG AAT G. GAPDH was used as the internal reference and quantified using specific primers; qF: AAG GCC ATC ACC ATC TTC CA and qR: GCC AGT AGA CTC CAC AAC ATAC.

To evaluate the effect of Caveolin-1 in the infection as well as invasion of EMCV into BHK-21 cells, the replication-defective lentivirus system provided by Dr. Enqi Du (Northwest A&F University, China), was used to construct a recombinant plasmid to overexpress caveolin-1. Total RNA was extracted from BHK-21 cells and reverse transcribed into cDNA. The caveolin-1 gene was amplified by PCR based on the murine caveolin-1 sequence (GenBank accession No. U07645.1). The amplified PCR product was digested by restriction endonuclease XbaI and BamH I (NEB, MA, USA) and inserted into pTRIP-CMV-IRES-Puro to construct recombinant plasmid, pTRIP-CAV1, using specific primers ( Table 1) . As a control, a EGFP recombinant plasmid, pTRIP-EGFP, was also constructed. Lentivirus was produced with recombinant lentivirus vector pTRIP-CAV1 and pTRIP-EGFP as described [23] and the transfected cells were named as BHK-CAV1 and BHK-EGFP, respectively.

Moreover, three individual small interfering RNAs (siRNA) against caveolin-1 (Rebobio, Guangdong, China) were designed (Table 2 ) and were employed to transfect the cells using Invitrogen Lipofectamine ™ 2000 (Ther-moFisher, MA, USA) system according to manufacturer's instructions. The silencing efficiencies were measured 

For virus infectivity assays, cells were incubated with EMCV at 0.1 multiplicity of infection (MOI) for 1 h at 37 °C in serum-free medium and then washed three times with pre-warmed phosphate-buffered saline (PBS) and maintained in DMEM with 3% FBS. At the given time points post infection, the virus replication assay was examined by virus yield titration [24] , qRT-PCR and western blotting.

For post-entry inhibitory effects determination, cells were first incubated with EMCV at 0.1 MOI. Then after two hoursrs, cells were washed with PBS and culture medium containing chemical inhibitors was added to the cells accordingly. At 9 h post infection, cells were harvested for further analysis [25] .

EMCV internalizing ability into BHK-21, BHK-Cav1 and BHK-EGFP cells was determined by measuring the quantity of infectious viruses in these cells according to previous report [26] .

Cells were fixed using ice cold 75% ethanol at 4 °C for 30 min. For co-localization studies, cells were permeabilized with 0.1% Triton X-100 when needed. After washing cells on slides with PBS, the suitable primary antibody was added and incubated at 37 °C. 1 h later, the slides were washed again and 150 μl secondary antibody was added and incubated for 1 h. Finally, the samples were counterstained with 4, 6-diamidino-2-phenylindole (DAPI) for 5 min at room temperature and were analyzed under Confocal microscope ZEISS LSM 900 (Zeiss, Oberkochen, Germany).

Samples were lysed in NP-40 lysis buffer (Beyotime, Shanghai, China) and concentration was measured using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, A, USA). Samples were heated at 95 °C 5 min and run on 10% SDS-PAGE gel and were transferred to PVDF membrane (Millipore, MA, USA). After treatment with 5% milk for 1 h, the membrane was incubated overnight with the primary antibody at 4 °C and then treated with HRP-conjugated secondary antibody for 2 h at room temperature. The specific bands of the membrane were 

Results are from three independent experiments and were analyzed with one-way ANOVA using Graphpad PRISM Version 5.0. Data was shown as the means ± standard deviations (SD). Differences were considered statically significant if P-value was less than 0.05 (*P < 0.05; **P < 0.01; ***P < 0.001).

To elucidate whether the endocytic pathway correlated with EMCV replication in BHK-21 cells, endocytosis specific inhibitors were used. The suitable non-toxic concentration of NH 4 Cl and Bafilomycin A1 were measured by the CellTiter 96 ® Aqueous Non-Radioactive Cell Proliferation Assay kit (Fig. 1d, h) . Previous research communicates that NH 4 Cl can hamper the endosomal entry of viruses by preventing pH-dependent activation of the fusion protein and by blocking membrane fusion [27] . In this experiment, BHK-21 cells were first treated with NH 4 Cl and then incubated with the virus. Afterwards; the media was changed to remove unbound viruses. EMCV-infected cells or culture fluids were harvested at 9 h post infection. As shown in Fig. 1a -c, we found that expression of VP1, EMCV-3D and virus titer were significantly decreased in infected cells compared to control cells in dose dependent manner. This indicated that EMCV infection is sensitive to inhibition of endosomal acidification.

Another inhibitor of endocytic compartment acidification, bafilomycin A1, was also tested [28] . Our results showed that 10 nM of bafilomycin A1 cannot block virus replication, while 20 nM of bafilomycin A1 can inhibit the proliferation of EMCV ( Fig. 1e-g) .

Next, we examined which endocytic pathway, including the clathrin-dependent pathway, macropinocytosis and caveolea-dependent pathway [12, 29] , was utilized by EMCV to infect BHK-21 cells.

As Clathrin-mediated endocytosis is often related to endosomal acidification [30] and is a classical pathway for most viruses to enter into host cells [11] . Therefore, we next detected whether EMCV enters into BHK-21 cells through clathrin-mediated endocytosis by using chlorpromazine and Pitstop-2 inhibitors [31, 32] . The Fig. 1 Inhibition of endosomal acidifcation with NH 4 Cl or Baflomycin A1 restrained EMCV replication in BHK-21 cells. NH 4 Cl treatment inhibited EMCV infection. BHK-21 cells were treated with NH 4 Cl, followed by incubation with EMCV at MOI of 0.1. At 9 h post infection (hpi), cells lysates or culture medium was collected, and virus infectivity assay was measured by WB (a), endpoint titration (b) and qRT-PCR (c). Cell viability assay was performed to evaluate the cytotoxicity of the NH 4 Cl (d). Baflomycin A1 (20 nM) treatment halted EMCV replication. BHK-21 cells were treated by Baflomycin A1 as indicated above and infectivity was measured by WB (e), endpoint titration (f) and qRT-PCR (g). Cell viability assay was performed to evaluate the cytotoxicity of Baflomycin A1 (h). Cultures treated with medium only were used as negative controls desirable non-toxic concentration of inhibitors for cells was achieved as indicated (Fig. 2c, f ) . Analysis indicated that neither the expression of EMCV-VP1 (Fig. 2a, b) nor virus titer assays (Fig. 2d , e) were affected by chlorpromazine or Pitstop-2.

To check whether the virus replication is macropinocytosis-mediated, BHK-21 cells were treated with NA + /H + exchanger inhibitor EIPA, Pak-1 inhibitor (1,1′-Dithiobis-2-naphthalenol, IPA-3), and PI3K inhibitor, wortmannin [33] . Virus infectivity assay showed that none of them affected virus replication (Fig. 3a, b, c, e, f, g, i, j, k).

Next, we investigated whether the caveolae-dependent pathway was involved in EMCV infection. Caveolae is rich in cholesterol and sphingolipids and can be disrupted by nystatin or MβCD [29] . The suitable non-toxic concentration was established (Fig. 4d, h) . Results indicated that non-infected cell cultures when treated with certain concentration of nystatin (12.5 μg/ml, 25 μg/ ml) and MβCD (2.5 mM, 5 mM) significantly inhibited EMCV proliferation (Fig. 4a, b, c, e, f and g). However, their effect on already infected EMCV-cell cultures was not significant (Fig. 4i-l) .

Caveolin-1 is the main structural protein of caveolae and is associated with the internalization of many viruses into their respective hosts [35] . In order to explore whether EMCV exploits caveolin-1 during its infection, the expression of caveolin-1 during EMCV infection was investigated. WB analysis indicated that caveolin-1 expression was increased in infected cells in a timedependent manner, consistent with the expression of the EMCV VP1 protein (Fig. 5a) .

To further investigate the impact of caveolin-1 on EMCV infection, overexpression of caveolin-1 was carried out in relation to EGFP and BHK-21 cells (Fig. 5b) . BHK-Cav1 and BHK-EGFP cells were cultured in medium without puromycin at least for 2 weeks prior to EMCV infection. Then BHK-Cav1, BHK-EGFP and BHK-21 cells were incubated with 0.1 MOI EMCV at 37 °C for Fig. 2 Clathrin-mediated endocytosis was not involved in EMCV infection Chlorpromazine treatment had no effect on EMCV replication. BHK-21 cells were treated with chlorpromazine, followed by incubation with EMCV at 0.1 MOI. At 9 hpi, cellular lysates or culture medium was collected, and virus replication assay was assessed by WB (a) and endpoint titration (b). Cell viability assay was performed to evaluate the cytotoxicity of the chlorpromazine (c). Pitstop-2 had also no effect on EMCV replication. BHK-21 cells were treated by Pitstop-2 as indicated above and infectivity was assessed by WB (d) and endpoint titration (e). Cell viability assay was performed to evaluate the cytotoxicity of the Pitstop-2 (f). Cultures treated with medium were used as negative control 1 h and cells were harvested for WB analyses at 12 h post infection.

As shown in Fig. 5b , the expression of VP1 was significantly higher in BHK-Cav1 cells compared to control cells. In order to confirm that EMCV replication is truly upregulated by the overexpression of caveolin-1, culture supernatants were collected at 3-h interval, and viral titers were determined as described previously [24] . The growth kinetics experiment showed that the overall process of virus replication was more efficient in BHK-Cav1 than in BHK-21 and BHK-EGFP cells (Fig. 5c) .

For a more in depth understanding of the molecular pathogenesis of EMCV infection in vitro, knockdown experiments using specific or control siRNA sequences were conducted. RNA interference silenced caveolin-1 expression in BHK-21 cells, in turn, impacted viral infection process as evident by the expression of VP1 (Fig. 5d) , virus titers (Fig. 5e ) and virus copies number (Fig. 5f ) .

To further elaborate caveolin-1involvement in the infection process, the same siRNA experiment was repeated in BHK-Cav1 cells. Results indicated that downregulation of caveolin-1 significantly inhibited the virus replication in BHK-Cav1 cells (Fig. 5g-i) .

To ensure the effect of caveolin-1, we examined the colocalization of virus with caveolin-1 by confocal imaging. Macropinocytosis had no effect on EMCV replication. IPA-3 treatment did not inhibit EMCV replication. WB (a), endpoint titration (b) and qRT-PCR (c) analysis indicated that EMCV proliferation remained unchanged at 9 hpi when BHK-21 cells were treated with IPA-3. Cell viability assay was performed to evaluate the cytotoxicity of the IPA-3 (d). Wortmanin treatment did not inhibit EMCV replication as revealed by WB (e), endpoint titration (f) and qRT-PCR (g) at 9 h post treatment. Cell viability was measured before infectivity test (h). EIPA treatment had also no effect on EMCV replication as evident by WB (i), endpoint titration (j) and qRT-PCR (k). Cell viability was measured before infectivity test (l). Cultures treated with medium were used as negative control As shown in Fig. 6a , after exposed to the EMCV for 120 min, EMCV-VP1 co-localized with caveolin-1 could be observed in infected BHK-21 cells.

As EMCV-VP1 co-localized with caveolin-1 at 120 min post infection, we next investigated caveolin-1 association with EMCV internalization. Increased EMCV internalization efficiency was noticed in BHK-Cav1 as compared to BHK-EGFP or BHK-21 cells (Fig. 6b, c) . Consistent with the results of caveolin-1 overexpression, siRNAs that effectively restrained caveolin-1 expression and inhibited the EMCV internalization (P < 0.01) as compared to control (Fig. 6d, e) .

Dynamin is a kind of large GTPase that can promote the split of endocytic membranes [12] and is considered to have a role in both clathrin-dependent endocytosis and several other endocytic pathways [34] . Therefore, we investigated its potential role in EMCV replication. Two inhibitors of GTPase activity, dynasore [35, 36] and the lipid-binding mitmab were selected [37] , and their optimal concentrations were obtained by cell viability assay (Fig. 7d, h) . Results indicated that these inhibitors significantly inhibited virus replication in BHK-21 before infection when introduced before infection (Fig. 7a, b, c, e, f and g) but their effect on infected cells was not significant when added after infection (Fig. 7i-l) .

Results of the current study suggested that EMCV infection in BHK-21 cells is mediated by caveolin-and dynamin-dependent endocytosis. Next, the role of the Fig. 4 Caveolae is required for EMCV replication. Nystatin treatment inhibited EMCV replication. BHK-21 cells were treated with nystatin before the addition of the virus and virus infectivity assay was assessed at 9 hpi by WB (a), endpoint titration (b) and qRT-PCR (c). Cell viability assay was performed before experiments (d). MβCD treatment inhibits EMCV infectivity. Similar procedure like that of nystatin was adapted for MβCD. The analysis revealed that MβCD treatment inhibits EMCV infectivity as evident by WB (e), endpoint titration (f) and qRT-PCR (g). Cell viability assay was performed to evaluate the cytotoxicity of MβCD (h). Endpoint titration (i) and qRT-PCR (j) showed EMCV replication was no changed at 9 hpi when BHK-21 cells were treated with nystatin after virus was added. Similar procedure like that of nystatin was adapted for MβCD. The analysis revealed that MβCD has no effect on EMCV infectivity by endpoint titration (k) and qRT-PCR (l). Cultures treated with medium were used as negative control cytoskeleton during virus entry was examined by actin disrupting agent (cytochalasin D) and stabilizing compound (jasplakinolide) [38, 39] . The optimal concentrations of these two inhibitors were obtained by cell viability assay (Fig. 8d, h) . We found that both actinstabilizing jasplakinolide and actin-disrupting agent cytochalasin D significantly halted EMCV infection when introduced into cells before infection (Fig. 8a, b, c, e, f and g). However, the post-infected treatment was not significant (Fig. 8i-l) .

Endocytosis is an important cellular process that mediates nutrient uptake, receptor internalization and the regulation of cell signaling (Endocytosis in proliferating, quiescent and terminally differentiated cells. 2018).

Caveolin-1 is associated with EMCV replication. Caveolin-1 was up-regulated with EMCV infection in time dependent manner (a). Increased viral protein production in BHK-Cav1(b). BHK-Cav1, BHK-EGFP and BHK-21 cells were infected with EMCV at 0.1 MOI and at 9 hpi, cellular lysates were collected and immunoblotted by using EMCV-VP1 specific antibodies. The blot was also stained with anti-caveolin-1, anti-GAPDH antibodies and anti-GFP antibodies. Growth kinetics of EMCV in BHK-Cav1, BHK-EGFP and BHK-21 cells (c). BHK-Cav1, BHK-EGFP and BHK-21 cells were incubated with EMCV at 0.1 MOI for 1 h at 37℃. After adsorption, the inocula were removed and cells were supplied with fresh medium. Cultures were harvested at 3-h intervals and virus titers were determined. The expression of EMCV-VP1 (d), virus titers (e) and copy number (f) were significantly reduced at 9 hpi in specific siRNA-transfected BHK-21 cells. Similarly, BHK-Cav1 cells were transfected with indicated siRNAs with reference to controls (NC). The expression of EMCV-VP1 (g), virus titers (h) and copy number (i) decreased significantly at 9 hpi in siRNA-transfected BHK-Cav1 cells For a large number of viruses, they can take advantage of the endocytosis machinery for infecting humans and animals (Endocytosis of Viruses and Bacteria, Pascale Cossart and Ari Helenius). Previous studies showed that members of Picornaviridae family use different endocytic mechanisms for entry into host cells [12, 15, 17, 19, 20] . However, the mechanisms involved in internalization of EMCV are poorly understand.

In the current study, we investigated the role of endocytosis in EMCV infection in BHK-21 cells. Lysosomotropic agents sensitivity is considered a good evidence of endocytosis [40] ; therefore, we pretreated cells with different inhibitors (NH 4 Cl or Baflomycin A1) of endosome acidification. Both reagents partly inhibited the virus infectivity (Fig. 1) , suggesting that endocytosis has a role in EMCV infection.

Earlier studies on both enveloped and nonenveloped viruses, such as HIV [41] , adenovirus [42] , foot-andmouth disease virus [43] , reovirus [44] and bluetongue virus [45, 46] , document that the virus entry into their respective host cells is by clathrin-mediated pathway. But results of our study indicated that neither viral structural proteins nor virus titers were significantly decreased by treatment of cells with clathrin specific inhibitors (Fig. 2a,  b, d, e) suggesting that clathrin-mediated endocytosis might not be an essential pathway for EMCV infection.

As one of the endocytic mechanisms in mammalian cells, macropinocytosis involves internalization of large number of plasma membrane together with extracellular medium and forms micropinosome [47] . Many intracellular pathogens by host cells via macropinocytosis have been described. Some viruses, such as African swine fever virus (ASFV) [48] , Ebola virus [49] and Human cytomegalovirus (HCMV) [50] , use this pathway to gain access to host cell [47] . Then we supposed whether macropinocytosis-dependent pathway may be involved in EMCV infection and hence first, cells were pretreated with macropinocytosis specific inhibitor -1 (a) . Cells treated with virus at MOI of 3 were synchronized on ice for 60 min and incubated at 37 °C for 0, 30, 60, 90 and 120 min before they were washed and fixed. Localization was analyzed by confocal microscopy (Scale bar = 10 µm) after performing a double immunofuorescence staining to visualize early caveolin-1 (left) and EMCV VP1 (middle). Right panel shows merged images; green indicates the virus and red indicates caveolin-1. Cell nuclei are indicated in blue while mock as a negative control. EMCV internalization was increased when caveolin-1 was overexpressed. BHK-Cav1, BHK-EGFP and BHK-21 cells were incubated with 3 MOI of EMCV at 37 °C for 1 h, harvested and cell lysates were prepared by three cycles of freeze-thawing. Virus titer assay (b) and qRT-PCR (c) analysis showed that EMCV internalization was increased in BHK-Cav1. EMCV internalization was decreased when caveolin-1 was down-regulated. BHK-21 cells transfected with negative-control (NC) siRNA and with two specific caveolin-1 siRNAs. Similar procedure was performed as mentioned above. Virus titer assay (d) and qRT-PCR (e) analysis revealed that virus internalization was decreased significantly in siRNA1 and siRNA2 group such as EIPA, IPA-3, wortmannin and then infected with the EMCV. Our results showed that these inhibitors did not affect EMCV infectivity assay (Fig. 3a, b, c, e, f, g, i, j, k) and thus implies that macropinocytosis is not involved in EMCV infection in vitro.

Apart from clathrin-dependent endocytosis, lipid raft and caveolae-dependent endocytosis are alternative endocytic pathways proposed for viruses intake [51] . It has been reported that MβCD could inhibit the CAV9 infection via lipid microdomains [52] , therefore, we examined the role of caveolar/lipid rafts endocytosis in EMCV infection in BHK-21 cells. We found that EMCV infection significantly decreased in MβCD and nystatin treated cells compared to control before incubated with EMCV ( Fig. 4a, b, c, e, f, g), while their effect was limited when added after infection ( Fig. 4i-l) . These results indicated that caveolae is involved in early stage of EMCV replication.

Caveolin-1 is the main structural protein of caveolae and has various functions in endosomal membrane traffic and other cellular processes, such as endocytosis, signal transductions, protein trafficking and secretion [53] [54] [55] [56] . Additionally, caveolin-1 is also involved in many viruses entry process, such as HIV [57] , aquareoviruses [58] , coronavirus [59] , HCV [60] , RSV [61] and CSFV [62] . The dependence of EMCV infection on caveolae-dependent qRT-PCR (c) showed EMCV replication was decreased at 9 hpi when BHK-21 cells were treated with dynasore before virus was added. Cell viability assay was performed before experiments (d). Mitmab treatment inhibited EMCV replication. WB (e), endpoint titration (f) and qRT-PCR (g) were performed at 9 hpi as indicated above. Cell viability was measured before infectivity test (h). Baflomycin A1 (20 nM) treatment halted EMCV replication. BHK-21 cells were treated by Baflomycin A1 as indicated above and infectivity was measured by WB (e), endpoint titration (f) and qRT-PCR (g). Post-BHK-21 infected cells treatments with dynasore had no effect on EMCV replication. Endpoint titration (i) and qRT-PCR (j) showed EMCV replication was no changed at 9 hpi when BHK-21 cells were treated with dynasore after virus was added. Similar procedure like that of dynasore was adapted for mitmab. The analysis revealed that mitmab has no effect on EMCV infectivity by endpoint titration (k) and qRT-PCR (l). Cultures treated with medium were used as negative control pathway and the findings that EMCV infection corresponds to caveolin-1 expression level (Fig. 5a) , directed us to study whether caveolin-1 is an important element involved in the replication process of the virus. To access the possibility, the lentiviral vector overexpressed caveolin-1 and siRNA targeted caveolin-1 were constructed. It is evident when overexpression of caveolin-1 resulted in a clear increase in the infection efficiency compared to the control cells (Fig. 5b, c) . Conversely, decreased expression of caveolin-1 by siRNA, inhibited the virus replication in BHK-21 cells (Fig. 5d-i) . The results highlight the importance of caveolin-1 for EMCV infection in BHK-21 cells.

Entry of viruses into permissive cells is an important stage in the viral pathogenesis [11, 51] and different viruses exploit various cellular endocytic mechanisms to initiate internalization and infection [63] . Results of the co-localization experiment in the current study at different time intervals suggested that there was co-localization of EMCV-VP1 and caveolin-1 at 120 min post infection (Fig. 6a) which implies that that caveolin-1 is required for early stage of EMCV replication. Further, EMCV internalization was enhanced by overexpression of caveolin-1 (Fig. 6b, c) while EMCV internalization was strongly inhibited when caveolin-1 was downregulated (Fig. 6d, e) . These findings, together with the results of co-localization (Fig. 6a) indicated that caveolin-1 is required for the internalization and infection of EMCV in vitro.

Previous studies have pointed out that either overexpressed dominant-negative mutants of dynamin or and qRT-PCR (c) showed EMCV replication was decreased at 9 hpi when BHK-21 cells were treated with cytochalasin D before virus was added. Cell viability assay was performed before experiments (d). Jasplakinolide treatment inhibited EMCV replication. WB (e), endpoint titration (f) and qRT-PCR (g) at 9 hpi was performed as indicated. Cell viability was measured before infectivity test (h). Post-BHK-21 infected cells treatments with cytochalasin D had no effect on EMCV replication. Endpoint titration (i) and qRT-PCR (j) showed EMCV replication was no changed at 9 hpi when BHK-21 cells were treated with cytochalasin D after virus was added. Similar procedure like that of cytochalasin D was adapted for jasplakinolide. The analysis revealed that jasplakinolide has no effect on EMCV infectivity by endpoint titration (k) and qRT-PCR (l). Cultures treated with medium were used as negative control disrupted actin assembly can block caveolae-mediated endocytosis [29, 64] . Therefore, we studied the potential role of dynamin and actin in EMCV infection in BHK-21 cells using two inhibitors of dynamin (dynasore and mitmab). Both inhibitors affected virus replication when added before infection (Fig. 7a, b, c, e, f and g) . However, neither dynasore nor mitmab blocked EMCV infection when added after infection (Fig. 7i-l) . Together, these results suggest that dynamin plays an exclusive role in virus uptake and thereby mediating infectivity.

It has been known that cytochalasin D inhibits actin subunits polymerization, whereas jasplakinolide inhibits the polymerization to stabilize the filaments [38, 65, 66] and we implemented this observation to determine their role in EMCV infection. We discovered that virus replication was decreased in cells pretreated with cytochalasin D and jasplakinolide (Fig. 8a, b, c, e, f and g) but their effect was limited when added after infection (Fig. 8i-l) . This revealed that both actin filaments and actin reorganization are required for EMCV infection in vitro.

In conclusion, the present study demonstrates for the first time that caveolin-1, dynamin and actin-dependent endocytosis pathways are involved in EMCV uptake, internalization and its subsequent replication in BHK-21 cells in vitro. Remarkably, there is a positive correlation between expression level of caveolin-1 and EMCV replication in vitro. Further work is needed to investigate the role of phosphorylation of caveolin-1, and the related singling pathway in regulating EMCV entry and replication.

Abbreviations EMCV: encephalomyocarditis virus; DMEM: Dulbecco's modified eagle medium; FBS: fetal bovine serum; MEM: minimum essential medium; PFU: plaque-forming unit; WB: western blotting; mAb: monoclonal antibody; pAb: polyclonal antibody; qRT-PCR: quantitative reverse transcription polymerase chain reaction; IFA: indirect immunofluorescence assay; DAPI: 4,6-diamidino-2-phenylindole; MOI: multiplicity of infection; hpi: hour post infection.

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Insider information: what viruses tell us about endocytosis

Stabilization of clathrin coated vesicles by amantadine, tromantadine and other hydrophobic amines

Mis-assembly of clathrin lattices on endosomes reveals a regulatory switch for coated pit formation

Macropinocytosis: an endocytic pathway for internalising large gulps

Mechanisms of endocytosis

Iminochromene inhibitors of dynamins I and II GTPase activity and endocytosis

Dynasore-not just a dynamin inhibitor

Myristyl trimethyl ammonium bromide and octadecyl trimethyl ammonium bromide are surface-active small molecule dynamin inhibitors that block endocytosis mediated by dynamin I or dynamin II

Actin filaments disruption and stabilization affect measles virus maturation by different mechanisms

Nocodazole action on tubulin assembly, axonal ultrastructure and fast axoplasmic transport

Dynamin-and clathrin-dependent endocytosis in African swine fever virus entry

Involvement of clathrinmediated endocytosis in human immunodeficiency virus type 1 entry

Adenovirus internalization and infection require dynamin

Analysis of foot-and-mouth disease virus internalization events in cultured cells

NPXY motifs in the beta1 integrin cytoplasmic tail are required for functional reovirus entry

Entry of bluetongue virus capsid requires the late endosome-specific lipid lysobisphosphatidic acid

Bluetongue virus entry into cells

Macropinocytosis: a pathway to protozoan infection

African swine fever virus gets undressed: new insights on the entry pathway

Ebola virus enters host cells by macropinocytosis and clathrin-mediated endocytosis

Cell line models for human cytomegalovirus latency faithfully mimic viral entry by macropinocytosis and endocytosis

Virus entry by endocytosis

Lipid raft microdomains: key sites for Coxsackievirus A9 infectious cycle

The caveolae dress code: structure and signaling

Caveolin-1-mediated expression and secretion of kallikrein 6 in colon cancer cells

The multiple faces of caveolae

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GSTPi stimulates caveolin-1-regulated polyamine uptake via actin remodeling

The role of Caveolin 1 in HIV infection and pathogenesis

Identification of the caveolae/ raft-mediated endocytosis as the primary entry pathway for aquareovirus. Virology

Early events during human coronavirus OC43 entry to the cell

Hepatitis C virus induces the localization of lipid rafts to autophagosomes for its RNA replication

Caveolae provide a specialized membrane environment for respiratory syncytial virus assembly

Caveolin-1-mediated endocytic pathway is involved in classical swine fever virus Shimen infection of porcine alveolar macrophages

Targeting viral entry as a strategy for broad-spectrum antivirals

Regulated portals of entry into the cell

Roles of small GTPase Rac1 in the regulation of actin cytoskeleton during dengue virus infection

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We thank Dr. Enqi Du (Northwest A&F University, China) for providing the replication-defective lentivirus system; Dr. Juan Bai (Nanjing Agricultural University, China) for his kindly supplied the EMCV-VP1 mAb, Prof. Zulqarnain Baloch for critical reading of our manuscript. 1 

Jialin Bai were the project leader, Qiongyi Li were responsible for experimental and project design. Qiongyi Li and Yang Liu performed most of the experiments. Shujuan Xu and Kexue Zhao performed some of the experiments. Ying Ling and Rongxiu Liu prepared the samples. Data analysis was performed by Qiongyi Li and Yang Liu. The paper was written by Qiongyi Li, revised and proofread by Amjad Ali. 

All data included in the manuscript are available.Ethics approval and consent to participate Not applicable.

Not applicable.

The authors declare that there is no conflict of interest.