Caveolin-1 Mediated Endocytic Pathway is Involved in Encephalomyocarditis Virus Replication in BHK-21 Cells


 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.

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 rst, we con rmed 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.

Methods
Cells, viruses, and antibodies.

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].

qRT-PCR
Total RNA was isolated, followed by qRT-PCR as previously described [22], using primers EMCV-3D qF: GTCATACTATCGTCCAGGGACTCTAT and qR: CATCTGTACTCCACACTCTCGAATG. GAPDH was used as the internal reference and quanti ed using speci c primers; qF: AAGGCCATCACCATCTTCCA and qR: GCCAGTAGACTCCACAACATAC.

Gene overexpression and RNA interference
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 ampli ed by PCR based on the murine caveolin-1 sequence (GenBank accession No. U07645.1). The ampli ed 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 speci c 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 (ThermoFisher, MA, USA) system according to manufacturer's instructions. The silencing e ciencies were measured by qRT-PCR and Western Blotting (WB) analysis. After culturing for 2 days, the cells were infected with EMCV and at 9 hours post-infection (hpi), WB and virus infectivity assays were performed.

Chemical inhibitors and Cell viability Determination
DMEM supplemented with 10% FBS and one of the following chemical inhibitors: Nystatin, pitstop, dynasore, mitmab, cytochalasin D, jasplakinolide, chlorpromazine and 1, 1'-Dithiobis-2-naphthalenol (IPA-3) were purchased from Abcam. Methyl-β-cyclodextrin (MβCD) and NH 4 Cl were purchased from Sigma (Sigma, MO, USA) and ba lomycin A1, EIPA and wortmannin from Solarbio (Solarbio, Beijing, China). Concentration and effects of the inhibitors applied in this study were described in Table 3. Respective cells were treated with inhibitors for one hour before EMCV infection. After RNA interference and chemical inhibitors treatment, cells viability was conducted by the CellTiter 96 ® Aqueous Non-Radioactive Cell Proliferation Assay kit (Promega, WI, USA) according to the manufacturer's instructions. The obtained raw values were converted to percentages in relation to untreated samples and corrected by background absorbance.

Virus infectivity assays, Post-entry inhibitory effects and detection of virus internalization
For virus infectivity assays, cells were incubated with EMCV at 0.1 multiplicity of infection (MOI) for 1 hour 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 rst 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 hours 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].

IFA and Confocal microscopy
Cells were xed 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 hour later, the slides were washed again and 150μl secondary antibody was added and incubated for 1 hour. 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).

Western Blotting
Samples were lysed in NP-40 lysis buffer (Beyotime, Shanghai, China) and concentration was measured using the Pierce BCA Protein Assay Kit (Thermo Fisher Scienti c, 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 hour, the membrane was incubated overnight with the primary antibody at 4°C and then treated with HRP-conjugated secondary antibody for 2 hours at room temperature. The speci c bands of the membrane were analyzed using chemiluminescence (Cowin Bioscience, Beijing, China) and detected using a Electrophoresis gel imaging split system (Gel imaging system GE Healthcare Bio-Sciences AB). Protein ladders (10kDa-180kDa) used in this study was from YEASEN (Yeasen Biotech, Shanghai, China).

Statistical analyses
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 signi cant if P-value was less than 0.05 (*P<0.05; **P<0.01; ***P<0.001).

Results
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 speci c inhibitors were used. The suitable non-toxic concentration of NH 4 Cl and Ba lomycin A1 were measured by the CellTiter 96 ® Aqueous Non-Radioactive Cell Proliferation Assay kit ( Fig. 1d and Fig. 1h).
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 rst 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 uids 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 signi cantly decreased in infected cells compared to control cells in dose dependent manner. This indicated that EMCV infection is sensitive to inhibition of endosomal acidi cation.
Clathrin-mediated endocytosis is not involved in EMCV replication in BHK-21 cells As Clathrin-mediated endocytosis is often related to endosomal acidi cation [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 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.
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 [29]. 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) signi cantly inhibited EMCV proliferation ( Fig. 4; a, b, c, e, f and g). However, their effect on already infected EMCV-cell cultures was not signi cant ( 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 [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 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 signi cantly higher in BHK-Cav1 cells compared to control cells. In order to con rm 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 e cient 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 speci c 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 signi cantly 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 e ciency 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 [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. 7; d and h). Results indicated that these inhibitors signi cantly 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 signi cant 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 signi cantly halted EMCV infection when introduced into cells before infection ( Fig. 8; a, b, c, e, f and g). However, the postinfected treatment was not signi cant ( Fig. 8; i-l).

Discussion
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). For a large number of viruses, they can take adavantage 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 understanded.
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 Ba omycin A1) of endosome acidi cation. 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], MRV [44] and BTV [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 signi cantly decreased by treatment of cells with clathrin speci c inhibitors ( Fig. 2; a, 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 macropinocytosisdependent pathway may be involved in EMCV infection and hence rst, cells were pretreated with macropinocytosis speci c inhibitor 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. 3; a, 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 signi cantly decreased in MβCD and nystatin treated cells compared to control before incubated with EMCV ( Fig. 4; a, b, c, e, f, g), while their effect was limited when added after infection ( Fig. 4; i-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 tra c and other cellular processes, such as endocytosis, signal transductions, protein tra cking 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 pathway and the ndings 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 e ciency compared to the control cells ( Fig. 5; b, c). Conversely, decreased expression of caveolin-1 by siRNA, inhibited the virus replication in BHK-21 cells (Fig. 5; d-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 virla 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. 6; b and c) while EMCV internalization was strongly inhibited when caveolin-1 was downregulated ( Fig. 6; d and e). These ndings, 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 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. 7; a, b, c, e, f and g). However, neither dynasore nor mitmab blocked EMCV infection when added after infection ( Fig. 7; i-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 laments [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. 8; a, b, c, e, f and g) but their effect was limited when added after infection ( Fig. 8; i-l). This revealed that both actin laments and actin reorganization are required for EMCV infection in vitro.

Conclusion
In conclusion, the present study demonstrates for the rst time that caveolin-1, dynamin and actindependent 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.

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Availability of data and materials
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