The role of endocytosis in EMCV replication in BHK-21 cells
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 NH4Cl and Bafilomycin A1 were measured by the CellTiter 96® Aqueous Non-Radioactive Cell Proliferation Assay kit (Fig. 1d and Fig. 1h).
Previous research communicates that NH4Cl can hamper the endosomal entry of viruses by preventing pH-dependent activation of the fusion protein and by blocking membrane fusion . In this experiment, BHK-21 cells were first treated with NH4Cl 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 hours post infection. As shown in Fig. 1a, 1b and 1c, 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 . Our results showed that 10nM of bafilomycin A1 cannot block virus replication, while 20nM of bafilomycin A1 can inhibit the proliferation of EMCV (Fig. 1e, 1f and 1g).
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.
Clathrin-mediated endocytosis is not involved in EMCV replication in BHK-21 cells
As Clathrin-mediated endocytosis is often related to endosomal acidification  and is a classical pathway for most viruses to enter into host cells . 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 desirable non-toxic concentration of inhibitors for cells was achieved as indicated (Fig. 2; c, f). Analysis indicated that neither the expression of EMCV-VP1 (Fig. 2; a, b) nor virus titer assays (Fig. 2d, 2e) were affected by chlorpromazine or Pitstop-2.
EMCV replication in BHK-21 cells is independent of macropinocytosis
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 . Virus infectivity assay showed that none of them affected virus replication (Fig. 3; a, b, c, e, f, g, i, j, k).
Caveolae is required for EMCV replication in BHK-21 cells.
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 . The suitable non-toxic concentration was established (Fig. 4d and 4h). Results indicated that non-infected cell cultures when treated with certain concentration of nystatin (12.5μg/ml, 25μg/ml) and MβCD (2.5mM, 5mM) significantly inhibited EMCV proliferation (Fig. 4; a, b, c, e, f and g). However, their effect on already infected EMCV-cell cultures was not significant (Fig. 4; i-l).
Caveolin-1 facilitates EMCV infection
Caveolin-1 is the main structural protein of caveolae and is associated with the internalization of many viruses into their respective hosts . 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 time-dependent 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 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 . 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 down-regulation of caveolin-1 significantly inhibited the virus replication in BHK-Cav1 cells (Fig. 5; g, h, i).
Caveolin-1 is essential for EMCV infection by involving in internalization
Co-localization of virus particles with caveolin-1 was investigated using confocal imaging. As shown in Fig. 6a, after 120 min of exposer to EMCV at MOI of 3, EMCV-VP1 co-localized with caveolin-1 in infected BHK-21 cells.
As EMCV-VP1 co-localized with caveolin-1 at 120min 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. 6; b and 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. 6; d and e).
Dynamin is needed for EMCV replication in BHK-21 cells
Dynamin is a kind of large GTPase that can promote the split of endocytic membranes  and is considered to have a role in both clathrin-dependent endocytosis and several other endocytic pathways . Therefore, we investigated its potential role in EMCV replication. Two inhibitors of GTPase activity, dynasore [35, 36] and the lipid-binding mitmab were selected , and their optimal concentrations were obtained by cell viability assay (Fig. 7; d and h). Results indicated that these inhibitors significantly inhibited virus replication in BHK-21 before infection when introduced before infection (Fig. 7; a, b, c, e, f and g) but their effect on infected cells was not significant when added after infection (Fig. 7; i-l).
Role of actin in EMCV infection in BHK-21 cells
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 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. 8; d and h). We found that both actin-stabilizing jasplakinolide and actin-disrupting agent cytochalasin D significantly halted EMCV infection when introduced into cells before infection (Fig. 8; a, b, c, e, f and g). However, the post-infected treatment was not significant (Fig. 8; i-l).