- Email: [email protected]
Cholesterol-rich lipid rafts play a critical role in bovine parainfluenza virus type 3 (BPIV3) infection
Research in Veterinary Science 114 (2017) 341–347
Contents lists available at ScienceDirect
Research in Veterinary Science journal homepage: www.elsevier.com/locate/rvsc
Cholesterol-rich lipid rafts play a critical role in bovine parainfluenza virus type 3 (BPIV3) infection
MARK
Liyang Lia,b, Liyun Yub, Xilin Houa,⁎ a b
College of Animal Science and Veterinary Medicine, HeiLongJiang BaYi Agricultural University, Daqing 163319, China College of Life Science and Technology, HeiLongJiang BaYi Agricultural University, Daqing 163319, China
A R T I C L E I N F O
A B S T R A C T
Keywords: BPIV3 Lipid raft Virus entry Virus infection
Lipid rafts are specialized lipid domains enriched in cholesterol and sphingolipid, which can be utilized in the lifecycle of numerous enveloped viruses. Bovine parainfluenza virustype3 (BPIV3) entry to cell is mediated by receptor binding and membrane fusion, but how lipid rafts in host cell membrane and BPIV3 envelope affect virus infection remains unclear. In this study, we investigated the role of lipid rafts in the different stages of BPIV3 infection. The MDBK cells were treated by methyl-β-cyclodextrin (MβCD) to disrupt cellular lipid raft, and the virus infection was determined. The results showed that MβCD significantly inhibited BPIV3 infection in a dose-dependent manner, but didn't block the binding of virus to the cell membrane. Whereas, the MDBK cells treated by MβCD after virus-entry had no effects on the virus infection, to suggest that BPIV3 infection was associated with lipid rafts in cell membrane during viral entry stage. To further confirm lipid rafts in viral envelope also affected BPIV3 infection, we treated BPIV3 with MβCD to determine the virus titer. We found that disruption of the viral lipid raft caused a significant reduction of viral yield. Cholesterol reconstitution experiment showed that BPIV3 infection was successfully restored by cholesterol supplementation both in cellular membrane and viral envelope, which demonstrated that cholesterol-rich lipid rafts played a critical role in BPIV3 infection. These findings provide insights on our understanding of the mechanism of BPIV3 infection and imply that lipid raft might be a good potential therapeutic target to prevent virus infection.
1. Introduction BPIV3, the member of family Paramyxoviridae, Respirovirus genera, is an enveloped, single-strand negative-sense RNA virus. BPIV3 is widely spread in many countries according to the results of serological survey (Kale et al., 2013; Solis-Calderon et al., 2007). BPIV3 normally infected adult and young cattle with other viral and bacterial pathogens, which can result of bovine respiratory disease complex (BRDC) (Kirchhoff et al., 2014). BRDC caused large economic losses for cattle industry in the world every year due to high morbidity-mortality rates (Grissett et al., 2015). The genome length of BPIV3 is approximately 15 k nt, and the genome encodes six structural proteins and three nonstructural proteins (Ellis, 2010). Six structural proteins includes nucleoprotein (N), phosphoprotein (P), large proteins (L), matrix protein (M), hemagglutinin-neuraminidase (HN) and the homotrimeric fusion (F). The P gene also encodes three accessory non-structural proteins, the C, V and D proteins. As membrane glycoprotein, the HN protein is the chief structural protein in the interaction between BPIV3 and host. The HN protein binds to the receptor protein in host cell surface to
⁎
cause a conformational change in the fusion protein (Jardetzky and Lamb, 2014). When bound, the HN protein interacted with the F protein, which resulting in fusion between the BPIV3 envelope and host cell membrane (Ellis, 2010). Once fusion occurs, there is a refolding of the proteins in the cell membrane that allows the viral nucleocapsid to enter the cytoplasm. Following the membrane fusion, the host cells are infected by BPIV3 (Jardetzky and Lamb, 2014). Lipid raft distributed on the cell membrane, which mainly composed of cholesterol, sphingomyelin, and lecithin (Munro, 2003), performs special functions in many biological processes, such as the separation of proteins, cell adhesion, migration, apoptosis, and synaptic transmission (Brown and London, 2000). Lipid raft in Influenza A virus has been proved to be related to the virus entry process, viral protein translation and targeted transport, virus assembly and budding process (Barman and Nayak, 2007; Bhattacharya et al., 2006; Ma et al., 2015; Ono and Freed, 2001), which indicated that lipid rafts play a remarkable role in the process of enveloped-virus infection. It is well known that membrane fusion and entering into target cells are critical steps for enveloped-virus infection. When enveloped-virus invaded host cells,
Corresponding author. E-mail address: [email protected] (X. Hou).
http://dx.doi.org/10.1016/j.rvsc.2017.04.009 Received 8 December 2016; Received in revised form 14 March 2017; Accepted 18 April 2017 0034-5288/ © 2017 Published by Elsevier Ltd.
Research in Veterinary Science 114 (2017) 341–347
L. Li et al.
10,0000 × g for 2 h. The TCID50 were measured by Reed-Muench method after resuspending the virus pellet in PBS and discarding the supernatant or the virus was finally stored at − 80 °C until be used.
lipid rafts may provide a platform for the interaction between the virus and receptor protein (Rawat et al., 2004). Hepatitis C virus entering the cell from the tight junction via endocytosis and fusion is mediated by envelope glycoproteins (Burlone and Budkowska, 2009). The clathrinmediated endocytosis mechanism is pH-dependent (Blanchard et al., 2006) but the cell entry is also dependent on an intact microtubule network firstly for virus transport between its cell attachment site and the site where fusion takes place and also later for the nucleocapsid release into the cytoplasm (Roohvand et al., 2009). Some reports suggest that lipid rafts promote fusion of the viral and target membranes and infection, which were proved by some experiments, for example, insertion of 25-hydroxycholesterol into the cellular membranes blocks virus-cell fusion and protects cells from infection by a number of enveloped viruses, including vesicular stomatitis virus, herpes simplex virus, HIV, Ebola virus, Rift Valley fever virus, Russian spring-summer encephalitis virus, and Nipah virus. The most likely and general mechanism of fusion regulation by cholesterol appears to be concentration of virus-specific protein molecules in one location, in rafts (Sviridov and Bukrinsky, 2014). Lipid rafts have been found to take part in the infection process of some paramyxoviruses. When MβCD deletes cholesterol and disrupts lipid rafts, NDV infectivity and the binding ability of NDV-HN to the receptor obviously decreased (Martin et al., 2012). Rafts are required for release of infectious RSV virion particles, since intact raft domains are necessary for loading cholesterol into the RSV virion particle (Chang et al., 2012). When PIV-5 was grown in negative Cav1MDCK cells, caveolae were mainly concerned in lipid rafts, the infectivity of virus was reduced (Ravid et al., 2010). In addition, it has been reported that some paramyxoviruses for infection require lipid raft in viral envelop rather than in target cell membrane, such as canine distemper virus and genus Morbillivirus (Imhoff et al., 2007). These data implied that lipid rafts both in cellular membrane and viral envelope were critical for different enveloped-virus efficient infection. Recently, it was reported that cholesterol-depletion drugs MβCD could inhibit Paramyxoviruses infection and influence virus entry process. The process of virus entry into cells is significant for elucidating the mechanism of pathogenesis and developing effective antiviral therapy. However, there is a lack of detailed and direct evidence to prove what function lipid rafts have in BPIV3 entry process and virus replication. Furthermore, we still have little information about the process of BPIV3 entering into host cells and how BPIV3 utilize lipid rafts. Thus, in this study, we investigated the role of lipid rafts in the different stages of BPIV3 infection, and provide solid evidence to verify that BPIV3 entry is associated with lipid rafts. We also showed that lipid rafts both in cell membrane and viral envelope played an important role in BPIV3 infection. These results not only provided new insights on our understanding of the mechanism of BPIV3 infection, but also demonstrated that lipid rafts might be a good potential therapeutic target in virus infection.
2.2. Antibodies and reagents Methyl-β-cyclodextrin (MβCD), MTT, and cholesterol were bought from Sigma-Aldrich and DMEM, FBS were purchased from Gibco Technology. The secondary antibodies, Goat anti-rabbit IgG-FITC, were bought from Boosen Biological Technology. We purchased the Amplex® red cholesterol assay kit from Life Technology and the BCA protein concentration assay kit from Beyotime Technology. The rabbit antiBPIV3 serum was prepared by our laboratory. 2.3. Cholesterol measurement and cell viability assay Cholesterol located in MDBK cells membrane and viral envelop were measured according to the protocol of Amplex® red cholesterol assay kit. Cytotoxic effects of MβCD were evaluated by the MTT (3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazolimromide) assay. MDBK cells in a 96-well plate were cultured in 100 μl DMEM containing 2.5, 5, 10, 15, 20 mM MβCD for 1 h at 37 °C. Culture medium was replaced with fresh medium and cells were continued culturing for 48 h. Subsequently, the culture medium was discarded and fresh medium containing 30 μl of 2 mg/ml MTT were added. After incubation with MTT at 37 °C for 4 h, the supernatant was discarded and 150 μl of DMSO was added per well to dissolve the crystal for 10 min. The optical density value (OD) of each well was recorded at 490 nm using a plate reader. 2.4. Indirect immunofluorescent assay (IFA) The MDBK cells fixed with 4% paraformaldehyde for 10 min were permeabilized with 0.1% Trinton X-100 for 20 min. And then the plate was blocked by 1% BSA for 2 h. The cells were washed three times with PBS and incubated with 200 μl rabbit polyclonal anti-BPIV3 serum (1:100) for 1 h, followed by incubation with 200 μl FITC-conjugated Goat anti-rabbit IgG (1:100) for 1 h at room temperature. The cells were washed extensively with PBS, and 90% glycerol was added to mount cells. The cells were examined and images were captured by using fluorescence microscope. 2.5. Flow cytometer The MDBK cells were washed once with PBS and fixed in 4% paraformaldehyde for 10 min. The cells were incubated with 200 μl rabbit polyclonal anti-BPIV3 serum (1:100) for 1 h, and FITC-conjugated Goat anti-rabbit IgG (1:100) for 1 h at room temperature at darkness. Then the cells were washed with PBS to remove the unbound antibodies. The cells were resuspended in PBS and analyzed with FACS Calibur cytometer.
2. Materials and methods 2.1. Cells and virus purification
2.6. Statistical analysis MDBK cells were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) with 100 μg/ml penicillin and 100 μg/ml streptomycin. All cells were cultured at 37 °C in a humidified atmosphere with 5% CO2. BPIV3 virus (DQ strain) was isolated and identified in Prevention Veterinary Laboratory at Heilongjiang Bayi Agricultural University. In brief, BPIV3 was propagated in MDBK monolayer cells for 1 h at 37 °C with occasional shaking. The cells were cultured in DMEM supplemented with 2% FBS for 24 h, and virus were harvested until appeared 80% cytopathic effect (CPE) for 48 h after infection. The collected virus was added into the gradient sucrose solution (20%, 40% and 60%) and ultra-centrifuged at 10, 0000 × g for 2 h. The virus layer between 40% and 60% sucrose solution was suspended in PBS, followed by ultra-centrifugation at
All experiments were performed with at least three independent replicates. All data were analyzed by SPSS 19.0 software using Student's t-test. P < 0.05 and P < 0.01 were considered significant difference. 3. Results 3.1. Depletion of cellular cholesterol inhibits infection of BPIV3 To investigate whether intact lipid rafts are associated with BPIV3 infection, we used MβCD, a drug widely used to sequester cholesterol, to remove MDBK cellular membrane cholesterol, and tested the effect of lipid rafts on the infection of BPIV3. The results showed that the MDBK 342
Research in Veterinary Science 114 (2017) 341–347
L. Li et al.
Fig. 1. Removal of cell membrane cholesterol affected infectivity of BPIV3. (A) The MβCD- dependent changes of cells membrane cholesterol concentration after MDBK cells were treated by MβCD in different concentration. (B) No toxicity effect of MβCD on MDBK cells by MTT assay. (C and D) The MDBK cells were treated by MβCD different concentration for 1 h at 37 °C, then were infected by BPIV3 (MOI = 1). After 24 h infection, the infected cells measured by IFA were showed that MβCD significantly inhibited the BPIV3 infection in a dose-dependent manner (C) and the TCID50 in the supernatants were significantly decreased (D). (E) The cholesterol replenished experiment. After treatment of 10 mM MβCD for 1 h at 37 °C, the 400 μg/ ml exogenous cholesterol in DMEM was replenished into MDBK cells for 1 h, then BPIV3 infected (MOI = 1). After 24 h infection, TCID50 of the supernatants were gradually restored (E). The data statistic was based on three independent experiments. The significant difference were marked by *(P < 0.05), **(P < 0.01), ***(P < 0.001).
specific effects of MβCD. After the cells were treated with 10 mM MβCD for 1 h at 37 °C, the 400 μg/ml exogenous cholesterol was added into culture medium to replenished cellular cholesterol. MDBK cells were infected with BPIV3 (MOI = 1) for 24 h, and then virus titers were determined. As expected, infectivity of BPIV3 was successfully restored after the supplement of exogenous cholesterol, which proved that the inhibition of MβCD on BPIV3 was indeed due to its specific sequestering of cholesterol (Fig. 1E).
cells treatment with 10 mM MβCD for 1 h effectively reduced the membrane cholesterol level approximately 50% compared to that in mock-treated cells (Fig. 1A). Moreover, the MβCD-treated cells exhibited the same relative viability in mock-treated cells, excluding the non-specific effects of cytotoxicity caused by MβCD (Fig. 1B). Subsequently, MβCD-treated cells were infected with BPIV3 at the MOI of 1 for 24 h, the culture supernatants were collected and the virus titers were measured. The results indicated that MβCD significantly inhibited the BPIV3 infection in a dose-dependent manner (Fig. 1C). The virus titer reduced > 103.5-fold when treated with 20 mM MβCD compared with the mock-treated cells (P < 0.01). Next, we used IFA to further confirm the inhibition of MβCD on BPIV3 infection. We found that disruption of lipid rafts by MβCD obviously decreased BPIV3 infection, which was in accordance with the virus titer measurement (Fig. 1D). Moreover, we performed a cholesterol reconstitution experiment to confirm whether inhibition of BPIV3 infection was caused by the non-
3.2. Depletion of cellular cholesterol didn't block the binding of BPIV3 The entry process of BPIV3 was initiated once the virus bound and fused with cell membrane. To elucidate the underlying mechanism how lipid rafts influence BPIV3 infection, we determined whether depletion of membrane cholesterol blocked BPIV3 binding to cell membrane. BPIV3 virions could bind to the MDBK cell surface, but couldn't 343
Research in Veterinary Science 114 (2017) 341–347
L. Li et al.
Fig. 2. Removal of cell membrane cholesterol did not block the binding of BPIV3. (A) The TCID50 results of BPIV3 binding on target cells. The MDBK cells were treated with 0, 10 and 20 mM MβCD for 1 h, then BPIV3 infected (MOI = 1) at 4 °C for 1 h. The TCID50 in cell lysates made no difference were measured in different groups. (B) The FACS results of BPIV3 binding on target cells were consistent with that of A. The MDBK cells were treated with 0, 10 and 20 mM MβCD for 1 h, then BPIV3 infected (MOI = 1) at 4 °C for 1 h, the uninfected cells were negative control. The MDBK cells were fixed in 4% paraformaldehyde, then the cells were incubated with 200 μl rabbit polyclonal anti-serum against BPIV3 (1:100) at 4 °C for 1 h. FITC-conjugated Goat anti-rabbit IgG was added and incubated for 30 min at 37 °C avoid light. Each sample was analyzed with FACS Calibur cytometer. The data statistic was based on three independent experiments.
penetrate cell membrane at 4 °C. The cells were lysed and virus titers were measured in mock-treated and MβCD-treated cells. At the same time, the amount of cell-bound viral virions was analyzed by Flow cytometer. The most interesting finding was that the TCID50 were not affected when cells were treated with 10 and 20 mM MβCD, which indicated cellular cholesterol had no effect on the BPIV3 binding to cell surface (Fig. 2A). The FACS results further confirmed that depletion of cellular cholesterol didn't block the BPIV3 binding, which was suggesting that lipid rafts might affect the entry process (Fig. 2B).
3.3. Depletion of cellular cholesterol post virus-entry not effect on viral infection The process of BPIV3 entry includes early binding and subsequent internalization. In order to investigate effects of cell membrane cholesterol depletion on BPIV3 infection at post-entry stage, As shown in Fig. 3, comparing the mock group with the post-entry group, TCID50 had not significant difference (P > 0.05). But, TCID50 significantly decreased in pre-entry group (P < 0.001). These results indicated that lipid raft were mainly effect on BPIV3 infection in process of the virus entry.
Fig. 3. Depletion of cellular cholesterol at post virus-entry did not effect on viral infection, however, TCID50 was significantly decreased in pre-60 min group. The MDBK cells grown in 24-well plates were treated with 10 mM MβCD at 60 min before virus infection and 30, 60, 90 min after virus infection for 1 h at 37 °C. In the mock group, the MDBK cells were treated by DMEM for 1 h at 37 °C, then the virus infection. In pre-60 min group, the MDBK cells were treated by 10 mM MβCD for 1 h at 37 °C, then the virus infection. After 24 h infection, we collected the culture supernatant to measure viral titers by TCID50. The data statistic was based on three independent experiments. The significant difference were marked by *(P < 0.05), **(P < 0.01), ***(P < 0.001).
3.4. Removal of viral envelope cholesterol affects virus infection To investigate that the viral envelope cholesterol effected on BPIV3 infection, we used MβCD to remove cholesterol from the viral envelope. After drug treatment, the level of viral envelope residual cholesterol was detected. We used the treated virus to infect cells. TCID50 were used to detect the infected cells after 24 h infection. Fig. 4A showed the cholesterol in virus envelope was reduced along with increasing MβCD concentrations. MβCD treatment caused a significant decrease in virus titers. In the 20 mM treated group, virus titer decreased > 102.4-fold compared with the untreated group (Fig. 4B). To demonstrate decline of BPIV3 infection was caused by treatment of MβCD, we replenished the exogenous cholesterol. As results in Fig. 4C, the infectivity of BPIV3 was restored after the supplement of exogenous cholesterol. These results indicate that the removal of viral envelope cholesterol can inhibit the infection of BPIV3.
4. Discussion Lipid rafts effect many biological processes, including membrane transport, signal transduction, separation of proteins, cell adhesion and migration, viral infection, and synaptic transmission (Rietveld and Simons, 1998; Simons and Ikonen, 1997). A large amount of evidences suggest that lipid raft is essential in the process of virus entering into target cells, especially enveloped virus (Chan et al., 2010). It is reported that the decreased infectivity of porcine reproductive and respiratory syndrome virus (PRRSV) could cause to decrease the expression of receptor on the cell membrane surface after removing lipid rafts (Yang et al., 2015). Recently, the functions of lipid rafts in the process of 344
Research in Veterinary Science 114 (2017) 341–347
L. Li et al.
Fig. 4. Removal of viral envelope cholesterol affects virus infection. (A) The level of viral envelope residual cholesterol after MβCD treatment. The purified virions were treated with 5, 10, 20 mM MβCD at 37 °C for 1 h, the mock group was treated with DMEM, then the MβCD was removed by ultracentrifugation. The cholesterol in virus envelope was reduced along with increasing MβCD concentrations. (B) The TCID50 significantly went down when some of viral envelope cholesterol were removed. The virions treated by MβCD were used to infect the MDBK cells. After 24 h infection, the TCID50 in supernatants were determined. (C) The cholesterol replenished experiment. The TCID50 went up again after the supplement of exogenous cholesterol in growth medium. The purified virions were treated with 10 mM MβCD at 37° for 1 h, the 400 μg/ml exogenous cholesterol in DMEM was replenished into the MDBK cells for 1 h, then the drugs were removed by ultracentrifugation. The treated virus infected the MDBK cells. The TCID50 were measured after 24 h infection. The data statistic was based on three independent experiments. The significant difference were marked by *(P < 0.05), **(P < 0.01), ***(P < 0.001).
dependently destroyed by MβCD. Secondly, when exogenous cholesterol was replenished to target cells membrane after treatment of MβCD, the virus infectivity was surprisingly restored. Then we investigated the effect of cholesterol in cells membrane on the viral binding and post-infection. The results of viral binding experiment showed that cholesterol did not affect BPIV3 binding to the MDBK cells. Whether the cholesterol affect the biochemical events in post-infection or not, we conducted the experiment that MβCD treated the post-infected cells at different times. The removal of cholesterol in post-infected stage could not inhibit viral infection, however, the changes were slight compare with the treatment in pre-infection. These results revealed that infectiousness of BPIV3 particles in raft (and cholesterol) deficient MDBK cells is severely compromised but BPIV3 binding to the cells was not blocked after removing the cholesterol in the MDBK cell membrane, suggesting that BPIV3 fusion may be influenced by lipid rafts. For paramyxoviruses entry, Fusion of enveloped viruses with the plasma membrane of a receptive host cell is a prerequisite for viral entry and infection (Bossart and Broder, 2013). It's widely assumed that enveloped viruses enter the cell through two main mechanisms: by direct fusion of the viral envelope with the plasma membrane or by endocytosis. However, the details of viral entry mechanism remain further study for clarifying the decreased BPIV3 virus infectivity relating to the virus entry cell ways via membrane fusion way or endocytosis way or both ways. All the findings indicated cholesterol played a critical role in process of BPIV3 entry to the target cells, which
Caprine herpesvirus type 1 (CpHV-1) and Cyprinus herpesvirus 3 (CyHV-3) infection are also proved. Most interestingly, the cell membrane cholesterol was required for CpHV-1 replication at the virus entry phase. Furthermore, a dose-dependent reduction of the virus yield was observed after MβCD treatment. Their data also demonstrated that cholesterol is mainly required during virus entry rather than during the post-entry stage (Pratelli and Colao, 2016). Cholesterol-rich lipid rafts are important for the CyHV-3 replication cycle especially during entry and egress (Brogden et al., 2015). Although some studies have reported that rafts play an critical role during the paramyxovirus infection such as NDV (Dolganiuc et al., 2003; Laliberte et al., 2006, 2007), RSV (Chang et al., 2012), no study were performed for an essential function of rafts during BPIV3 infection. In this study, we explored the role of lipid rafts on the target cell membrane and the viral envelope in BPIV3 infection for the first time. The drugs of interfering with the cell membrane lipid raft structure are commonly used to investigate lipid rafts, MβCD is a common drug to remove cholesterol (Simons and Toomre, 2000). Entrying, binding and post-entry are key steps for process of virus infection. In order to study the role of raft in cell membrane during BPIV3 infection, MDBK cells were treated with MβCD for disrupting lipid rafts in the plasma membrane. Our results indicated lipid rafts was required in BPIV3 entry to cells. Firstly, when the MDBK cells with MβCD were incubated together before BPIV3 infected, the infectivity of BPIV3 was significantly decreased along with the cholesterol residues of target cells dose345
Research in Veterinary Science 114 (2017) 341–347
L. Li et al.
envelope was required for BPIV3 infectivity. These results can help us understand the mechanism of BPIV3 infection. In addition, lipid rafts could be a good potential therapeutic target to prevent BPIV3 infection. To achieving this goal, many detail complex mechanisms of BPIV3 fusion, endocytosis, binding, replication and budding need study in the future.
was similar to other enveloped viruses. In process of CpHV-1 infection, an obvious reduction in virus infectivity was detected after MβCDtreated virus envelope (Pratelli and Colao, 2016). Similar results were reported in CyHV-3 infection which suggested that lipid rafts were important during virus entering and egress (Brogden et al., 2015). These views were also supported by our research results that the relationship between the infectivity of BPIV3 and the cholesterol of cells membrane were partially correlated. BPIV3 entered into the target cell requiring fusion of target cells and viral membrane. The receptor binding protein HN could bind to the surface receptor of target cells to induce conformational change. Then, the fusion protein F was activated before membrane fusion, following nucleoprotein enter cytolymph to replicate (Jardetzky and Lamb, 2014). The content of cholesterol in cell membrane can maintaining the fluidity of cell membrane (Burger et al., 2000). Removed cholesterol can reduce the radial diffusion of the membrane. The ability of invading the cell membrane for BPIV3 may be affected by decreased diffusion ability of the membrane. In addition, lipid environment in the cell membrane, including cholesterol, has influence on ion channels (Chang et al., 1995). Paramyxoviruses enter the target cell by binding to a cell surface receptor and then fusing the viral envelope with the target cell membrane, allowing the release of the viral genome into the cytoplasm (Palgen et al., 2015). Lipid rafts in the cell membrane was identified by viral specific protein to regulate the process of virus invading cells (Teissier and Pecheur, 2007). Taken together, viruses achieve host cell entry in two principal mechanism: direct fusion pathway and endocytic pathway including clathrin-mediated, clathrin-independent pathway, raft-dependent pathways, and macropinocytosis (Huang et al., 2017). Our findings imply that as an enveloped virus, BPIV3 infection may associate with the rafts-related membrane fusion mechanism. Although the HN protein carried by the Respirovirus, Rubulavirus, and Avulavirus genera possesses both sialic acid-binding (hemagglutinating) and sialic acid-cleaving (neuraminidase) activities (Palgen et al., 2015), but sialic acid receptor that BPIV3 employed for HN binding and whether the HN-receptor binding affects lipid raft-dependence vrial entry or not are still unknown, which remains further studies. Cholesterol in viral envelope can sustain integrity of virus particles (Fujita et al., 2011). In this study, after the purified BPIV3 virions were treated by MβCD in different concentration, the virus infectivity decreased in dose-dependent manner. The lessened infectivity may be caused by destroyed viral envelope integrity owing to depletion of cholesterol. In the process of Bovine herpesvirus 1 (BoHV-1) infection, the virus infectivity was reduced by removing viral envelop cholesterol with MβCD, exogenous cholesterol can partially reverse the effect (Zhu et al., 2010). In our experiments, cholesterol removed from BPIV3 envelope might destroyed the virus particles, which resulted in the ability of BPIV3 infection decrease. The previous researches have been shown that infectivity of virus particles was decreased by treatment high concentration MβCD because the loss of N protein in PRRSV infection process (Yang et al., 2015). Fujita et al. (2011) also demonstrated infectivity of MβCD treated hemagglutinating virus of Japan (HVJ) was lower than wild-type HVJ. In addition, lipid raft play a role as chemically entities by modulating the curvature of the membranes, and further affected fusion process (Teissier and Pecheur, 2007). Cholesterol depletion resulted in viral particles structure change to decline BPIV3 particles infectivity. It have been shown that intact lipid rafts of viral envelope is required for the viral invasion in many Paramyxoviridae members, such as respiratory syncytial virus (RSV) (Chang et al., 2012), newcastle disease virus (NDV) (Martin et al., 2012), measles virus (MeV) (Avota and Schneider-Schaulies, 2014), sendai virus (SeV) (Fujita et al., 2011). Our researches suggest it may be a typical feature of Paramyxoviridae viruses that lipid rafts in viral envelope effected virus entry into host cells. The virus entry into target cells is a complex process, including many factors. In this study, we first demonstrated lipid raft was essential to BPIV3 entry into MDBK cells. Furthermore, lipid raft in viral
Conflicts of interest None. Acknowledgements This project was supported by the fund of Heilongjiang Education Department Research Program (12521372), National Key Technology R & D Program (2012BAD12B05-2), China. References Avota, E., Schneider-Schaulies, S., 2014. The role of sphingomyelin breakdown in measles virus immunmodulation. Cell. Physiol. Biochem. 34, 20–26. Barman, S., Nayak, D.P., 2007. Lipid raft disruption by cholesterol depletion enhances influenza a virus budding from MDCK cells. J. Virol. 81, 12169–12178. Bhattacharya, J., Repik, A., Clapham, P.R., 2006. Gag regulates association of human immunodeficiency virus type 1 envelope with detergent-resistant membranes. J. Virol. 80, 5292–5300. Blanchard, E., Belouzard, S., Lucie Goueslain, L., Wakita, T., Dubuisson, J., Wychowski, C., Rouille, Y., 2006. Hepatitis C virus entry depends on clathrin-mediated endocytosis. J. Virol. 80, 6964–6972. Bossart, K.N., Broder, C.C., 2013. Paramyxovirus entry. In: Pöhlmann, S. (Ed.), Viral Entry Into Host Cell, Spring Street, New York, USA. Brogden, G., Adamek, M., Proepsting, M.J., Ulrich, R., Naim, H.Y., Steinhagen, D., 2015. Cholesterol-rich lipid rafts play an important role in the Cyprinid herpesvirus 3 replication cycle. Vet. Microbiol. 179, 204–212. Brown, D.A., London, E., 2000. Structure and function of sphingolipid- and cholesterolrich membrane rafts. J. Biol. Chem. 275, 17221–17224. Burger, K., Gimpl, G., Fahrenholz, F., 2000. Regulation of receptor function by cholesterol. Cell. Mol. Life Sci. 57, 1577–1592. Burlone, M.E., Budkowska, A., 2009. Hepatitis C virus cell entry: role of lipoproteins and cellular receptors. J. Gen. Virol. 90, 1055–1070. Chan, R.B., Tanner, L., Wenk, M.R., 2010. Implications for lipids during replication of enveloped viruses. Chem. Phys. Lipids 163, 449–459. Chang, H.M., Reitstetter, R., Mason, R.P., Gruener, R., 1995. Attenuation of channel kinetics and conductance by cholesterol - an interpretation using structural stress as a unifying concept. J. Membr. Biol. 143, 51–63. Chang, T.H., Segovia, J., Sabbah, A., Mgbemena, V., Bose, S., 2012. Cholesterol-rich lipid rafts are required for release of infectious human respiratory syncytial virus particles. Virology 422, 205–213. Dolganiuc, V., McGinnes, L., Luna, E.J., Morrison, T.G., 2003. Role of the cytoplasmic domain of the Newcastle disease virus fusion protein in association with lipid rafts. J. Virol. 77, 12968–12979. Ellis, J.A., 2010. Bovine parainfluenza-3 virus. In: Veterinary Clinics of North AmericaFood Animal Practice. vol. 26. pp. 575–57 +. Fujita, H., Tamai, K., Kawachi, M., Saga, K., Shimbo, T., Yamazaki, T., Kaneda, Y., 2011. Methyl-beta cyclodextrin alters the production and infectivity of Sendai virus. Arch. Virol. 156, 995–1005. Grissett, G.P., White, B.J., Larson, R.L., 2015. Structured literature review of responses of cattle to viral and bacterial pathogens causing bovine respiratory disease complex. J. Vet. Intern. Med. 29, 770–780. Huang, R., Zhu, G., Zhang, J., Lai, Y., Xu, Y., He, J., Xie, J., 2017. Betanodavirus-like particles enter host cells via clathrin-mediated endocytosis in a cholesterol-, pH- and cytoskeleton-dependent manner. Vet. Res. 48. Imhoff, H., von Messling, V., Herrler, G., Haas, L., 2007. Canine distemper virus infection requires cholesterol in the viral envelope. J. Virol. 81, 4158–4165. Jardetzky, T.S., Lamb, R.A., 2014. Activation of paramyxovirus membrane fusion and virus entry. Curr. Opin. Virol. 5, 24–33. Kale, M., Dylek, O., Sybel, H., Pehlivanoglu, F., Turutoglu, H., 2013. Some viral and bacterial respiratory tract infections of dairy cattle during the summer season. Acta Vet-Beogr. 63, 227–236. Kirchhoff, J., Uhlenbruck, S., Goris, K., Keil, G.M., Herrler, G., 2014. Three viruses of the bovine respiratory disease complex apply different strategies to initiate infection. Vet. Res. 45. Laliberte, J.P., McGinnes, L.W., Peeples, M.E., Morrison, T.G., 2006. Integrity of membrane lipid rafts is necessary for the ordered assembly and release of infectious Newcastle disease virus particles. J. Virol. 80, 10652–10662. Laliberte, J.P., McGinnes, L.W., Morrison, T.G., 2007. Incorporation of functional HN-F glycoprotein-containing complexes into Newcastle disease virus is dependent on cholesterol and membrane lipid raft integrity. J. Virol. 81, 10636–10648. Ma, K., Wang, Y.J., Wang, J.F., 2015. Influenza virus assembly and budding in lipid rafts. Prog. Biochem. Biophys. 42, 495–500.
346
Research in Veterinary Science 114 (2017) 341–347
L. Li et al.
Roohvand, F., Maillard, P., Lavergne, J.P., Boulant, S., Walic, M., Andreo, U., Goueslain, L., Helle, F., Mallet, A., McLauchlan, J., Budkowska, A., 2009. Initiation of hepatitis C virus infection requires the dynamic microtubule network. J. Biol. Chem. 284, 13778–13791. Simons, K., Ikonen, E., 1997. Functional rafts in cell membranes. Nature 387, 569–572. Simons, K., Toomre, D., 2000. Lipid rafts and signal transduction. Nat. Rev. Mol. Cell Biol. 1, 31–39. Solis-Calderon, J.J., Segura-Correa, J.C., Aguilar-Romero, F., Segura-Correa, V.M., 2007. Detection of antibodies and risk factors for infection with bovine respiratory syncytial virus and parainfluenza virus-3 in beef cattle of Yucatan, Mexico. Prev. Vet. Med. 82, 102–110. Sviridov, D., Bukrinsky, M., 2014. Interaction of pathogens with host cholesterol metabolism. Curr. Opin. Lipidol. 25, 333–338. Teissier, E., Pecheur, E.I., 2007. Lipids as modulators of membrane fusion mediated by viral fusion proteins. Eur. Biophys. J. Biophys. Lett. 36, 887–899. Yang, Q., Zhang, Q., Tang, J., Feng, W.H., 2015. Lipid rafts both in cellular membrane and viral envelope are critical for PRRSV efficient infection. Virology 484, 170–180. Zhu, L.Q., Ding, X.Y., Tao, J., Wang, J.Y., Zhao, X., Zhu, G.Q., 2010. Critical role of cholesterol in bovine herpesvirus type 1 infection of MDBK cells. Vet. Microbiol. 144, 51–57.
Martin, J.J., Holguera, J., Sanchez-Felipe, L., Villar, E., Munoz-Barroso, I., 2012. Cholesterol dependence of Newcastle disease virus entry. Biochim. Biophys. Acta Biomembr. 1818, 753–761. Munro, S., 2003. Lipid rafts: elusive or illusive? Cell 115, 377–388. Ono, A., Freed, E.O., 2001. Plasma membrane rafts play a critical role in HIV-1 assembly and release. Proc. Natl. Acad. Sci. U. S. A. 98, 13925–13930. Palgen, J.L., Eric, M., Jurgens, E.M., Moscona, A., Porotto, M., Palermo, L.M., 2015. Unity in diversity: shared mechanism of entry among paramyxoviruses. Prog. Mol. Biol. Transl. Sci. 129, 1–32. Pratelli, A., Colao, V., 2016. Critical role of the lipid rafts in caprine herpesvirus type 1 infection in vitro. Virus Res. 211, 186–193. Ravid, D., Leser, G.P., Lamb, R.A., 2010. A role for caveolin 1 in assembly and budding of the paramyxovirus parainfluenza virus 5. J. Virol. 84, 9749–9759. Rawat, S.S., Gallo, S.A., Eaton, J., Martin, T.D., Ablan, S., KewalRamani, V.N., Wang, J.M., Blumenthal, R., Puri, A., 2004. Elevated expression of GM3 in receptor-bearing targets confers resistance to human immunodeficiency virus type 1 fusion. J. Virol. 78, 7360–7368. Rietveld, A., Simons, K., 1998. The differential miscibility of lipids as the basis for the formation of functional membrane rafts. Biochim. Biophys. Acta Rev. Biomembr. 1376, 467–479.
347
Contents lists available at ScienceDirect
Research in Veterinary Science journal homepage: www.elsevier.com/locate/rvsc
Cholesterol-rich lipid rafts play a critical role in bovine parainfluenza virus type 3 (BPIV3) infection
MARK
Liyang Lia,b, Liyun Yub, Xilin Houa,⁎ a b
College of Animal Science and Veterinary Medicine, HeiLongJiang BaYi Agricultural University, Daqing 163319, China College of Life Science and Technology, HeiLongJiang BaYi Agricultural University, Daqing 163319, China
A R T I C L E I N F O
A B S T R A C T
Keywords: BPIV3 Lipid raft Virus entry Virus infection
Lipid rafts are specialized lipid domains enriched in cholesterol and sphingolipid, which can be utilized in the lifecycle of numerous enveloped viruses. Bovine parainfluenza virustype3 (BPIV3) entry to cell is mediated by receptor binding and membrane fusion, but how lipid rafts in host cell membrane and BPIV3 envelope affect virus infection remains unclear. In this study, we investigated the role of lipid rafts in the different stages of BPIV3 infection. The MDBK cells were treated by methyl-β-cyclodextrin (MβCD) to disrupt cellular lipid raft, and the virus infection was determined. The results showed that MβCD significantly inhibited BPIV3 infection in a dose-dependent manner, but didn't block the binding of virus to the cell membrane. Whereas, the MDBK cells treated by MβCD after virus-entry had no effects on the virus infection, to suggest that BPIV3 infection was associated with lipid rafts in cell membrane during viral entry stage. To further confirm lipid rafts in viral envelope also affected BPIV3 infection, we treated BPIV3 with MβCD to determine the virus titer. We found that disruption of the viral lipid raft caused a significant reduction of viral yield. Cholesterol reconstitution experiment showed that BPIV3 infection was successfully restored by cholesterol supplementation both in cellular membrane and viral envelope, which demonstrated that cholesterol-rich lipid rafts played a critical role in BPIV3 infection. These findings provide insights on our understanding of the mechanism of BPIV3 infection and imply that lipid raft might be a good potential therapeutic target to prevent virus infection.
1. Introduction BPIV3, the member of family Paramyxoviridae, Respirovirus genera, is an enveloped, single-strand negative-sense RNA virus. BPIV3 is widely spread in many countries according to the results of serological survey (Kale et al., 2013; Solis-Calderon et al., 2007). BPIV3 normally infected adult and young cattle with other viral and bacterial pathogens, which can result of bovine respiratory disease complex (BRDC) (Kirchhoff et al., 2014). BRDC caused large economic losses for cattle industry in the world every year due to high morbidity-mortality rates (Grissett et al., 2015). The genome length of BPIV3 is approximately 15 k nt, and the genome encodes six structural proteins and three nonstructural proteins (Ellis, 2010). Six structural proteins includes nucleoprotein (N), phosphoprotein (P), large proteins (L), matrix protein (M), hemagglutinin-neuraminidase (HN) and the homotrimeric fusion (F). The P gene also encodes three accessory non-structural proteins, the C, V and D proteins. As membrane glycoprotein, the HN protein is the chief structural protein in the interaction between BPIV3 and host. The HN protein binds to the receptor protein in host cell surface to
⁎
cause a conformational change in the fusion protein (Jardetzky and Lamb, 2014). When bound, the HN protein interacted with the F protein, which resulting in fusion between the BPIV3 envelope and host cell membrane (Ellis, 2010). Once fusion occurs, there is a refolding of the proteins in the cell membrane that allows the viral nucleocapsid to enter the cytoplasm. Following the membrane fusion, the host cells are infected by BPIV3 (Jardetzky and Lamb, 2014). Lipid raft distributed on the cell membrane, which mainly composed of cholesterol, sphingomyelin, and lecithin (Munro, 2003), performs special functions in many biological processes, such as the separation of proteins, cell adhesion, migration, apoptosis, and synaptic transmission (Brown and London, 2000). Lipid raft in Influenza A virus has been proved to be related to the virus entry process, viral protein translation and targeted transport, virus assembly and budding process (Barman and Nayak, 2007; Bhattacharya et al., 2006; Ma et al., 2015; Ono and Freed, 2001), which indicated that lipid rafts play a remarkable role in the process of enveloped-virus infection. It is well known that membrane fusion and entering into target cells are critical steps for enveloped-virus infection. When enveloped-virus invaded host cells,
Corresponding author. E-mail address: [email protected] (X. Hou).
http://dx.doi.org/10.1016/j.rvsc.2017.04.009 Received 8 December 2016; Received in revised form 14 March 2017; Accepted 18 April 2017 0034-5288/ © 2017 Published by Elsevier Ltd.
Research in Veterinary Science 114 (2017) 341–347
L. Li et al.
10,0000 × g for 2 h. The TCID50 were measured by Reed-Muench method after resuspending the virus pellet in PBS and discarding the supernatant or the virus was finally stored at − 80 °C until be used.
lipid rafts may provide a platform for the interaction between the virus and receptor protein (Rawat et al., 2004). Hepatitis C virus entering the cell from the tight junction via endocytosis and fusion is mediated by envelope glycoproteins (Burlone and Budkowska, 2009). The clathrinmediated endocytosis mechanism is pH-dependent (Blanchard et al., 2006) but the cell entry is also dependent on an intact microtubule network firstly for virus transport between its cell attachment site and the site where fusion takes place and also later for the nucleocapsid release into the cytoplasm (Roohvand et al., 2009). Some reports suggest that lipid rafts promote fusion of the viral and target membranes and infection, which were proved by some experiments, for example, insertion of 25-hydroxycholesterol into the cellular membranes blocks virus-cell fusion and protects cells from infection by a number of enveloped viruses, including vesicular stomatitis virus, herpes simplex virus, HIV, Ebola virus, Rift Valley fever virus, Russian spring-summer encephalitis virus, and Nipah virus. The most likely and general mechanism of fusion regulation by cholesterol appears to be concentration of virus-specific protein molecules in one location, in rafts (Sviridov and Bukrinsky, 2014). Lipid rafts have been found to take part in the infection process of some paramyxoviruses. When MβCD deletes cholesterol and disrupts lipid rafts, NDV infectivity and the binding ability of NDV-HN to the receptor obviously decreased (Martin et al., 2012). Rafts are required for release of infectious RSV virion particles, since intact raft domains are necessary for loading cholesterol into the RSV virion particle (Chang et al., 2012). When PIV-5 was grown in negative Cav1MDCK cells, caveolae were mainly concerned in lipid rafts, the infectivity of virus was reduced (Ravid et al., 2010). In addition, it has been reported that some paramyxoviruses for infection require lipid raft in viral envelop rather than in target cell membrane, such as canine distemper virus and genus Morbillivirus (Imhoff et al., 2007). These data implied that lipid rafts both in cellular membrane and viral envelope were critical for different enveloped-virus efficient infection. Recently, it was reported that cholesterol-depletion drugs MβCD could inhibit Paramyxoviruses infection and influence virus entry process. The process of virus entry into cells is significant for elucidating the mechanism of pathogenesis and developing effective antiviral therapy. However, there is a lack of detailed and direct evidence to prove what function lipid rafts have in BPIV3 entry process and virus replication. Furthermore, we still have little information about the process of BPIV3 entering into host cells and how BPIV3 utilize lipid rafts. Thus, in this study, we investigated the role of lipid rafts in the different stages of BPIV3 infection, and provide solid evidence to verify that BPIV3 entry is associated with lipid rafts. We also showed that lipid rafts both in cell membrane and viral envelope played an important role in BPIV3 infection. These results not only provided new insights on our understanding of the mechanism of BPIV3 infection, but also demonstrated that lipid rafts might be a good potential therapeutic target in virus infection.
2.2. Antibodies and reagents Methyl-β-cyclodextrin (MβCD), MTT, and cholesterol were bought from Sigma-Aldrich and DMEM, FBS were purchased from Gibco Technology. The secondary antibodies, Goat anti-rabbit IgG-FITC, were bought from Boosen Biological Technology. We purchased the Amplex® red cholesterol assay kit from Life Technology and the BCA protein concentration assay kit from Beyotime Technology. The rabbit antiBPIV3 serum was prepared by our laboratory. 2.3. Cholesterol measurement and cell viability assay Cholesterol located in MDBK cells membrane and viral envelop were measured according to the protocol of Amplex® red cholesterol assay kit. Cytotoxic effects of MβCD were evaluated by the MTT (3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazolimromide) assay. MDBK cells in a 96-well plate were cultured in 100 μl DMEM containing 2.5, 5, 10, 15, 20 mM MβCD for 1 h at 37 °C. Culture medium was replaced with fresh medium and cells were continued culturing for 48 h. Subsequently, the culture medium was discarded and fresh medium containing 30 μl of 2 mg/ml MTT were added. After incubation with MTT at 37 °C for 4 h, the supernatant was discarded and 150 μl of DMSO was added per well to dissolve the crystal for 10 min. The optical density value (OD) of each well was recorded at 490 nm using a plate reader. 2.4. Indirect immunofluorescent assay (IFA) The MDBK cells fixed with 4% paraformaldehyde for 10 min were permeabilized with 0.1% Trinton X-100 for 20 min. And then the plate was blocked by 1% BSA for 2 h. The cells were washed three times with PBS and incubated with 200 μl rabbit polyclonal anti-BPIV3 serum (1:100) for 1 h, followed by incubation with 200 μl FITC-conjugated Goat anti-rabbit IgG (1:100) for 1 h at room temperature. The cells were washed extensively with PBS, and 90% glycerol was added to mount cells. The cells were examined and images were captured by using fluorescence microscope. 2.5. Flow cytometer The MDBK cells were washed once with PBS and fixed in 4% paraformaldehyde for 10 min. The cells were incubated with 200 μl rabbit polyclonal anti-BPIV3 serum (1:100) for 1 h, and FITC-conjugated Goat anti-rabbit IgG (1:100) for 1 h at room temperature at darkness. Then the cells were washed with PBS to remove the unbound antibodies. The cells were resuspended in PBS and analyzed with FACS Calibur cytometer.
2. Materials and methods 2.1. Cells and virus purification
2.6. Statistical analysis MDBK cells were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) with 100 μg/ml penicillin and 100 μg/ml streptomycin. All cells were cultured at 37 °C in a humidified atmosphere with 5% CO2. BPIV3 virus (DQ strain) was isolated and identified in Prevention Veterinary Laboratory at Heilongjiang Bayi Agricultural University. In brief, BPIV3 was propagated in MDBK monolayer cells for 1 h at 37 °C with occasional shaking. The cells were cultured in DMEM supplemented with 2% FBS for 24 h, and virus were harvested until appeared 80% cytopathic effect (CPE) for 48 h after infection. The collected virus was added into the gradient sucrose solution (20%, 40% and 60%) and ultra-centrifuged at 10, 0000 × g for 2 h. The virus layer between 40% and 60% sucrose solution was suspended in PBS, followed by ultra-centrifugation at
All experiments were performed with at least three independent replicates. All data were analyzed by SPSS 19.0 software using Student's t-test. P < 0.05 and P < 0.01 were considered significant difference. 3. Results 3.1. Depletion of cellular cholesterol inhibits infection of BPIV3 To investigate whether intact lipid rafts are associated with BPIV3 infection, we used MβCD, a drug widely used to sequester cholesterol, to remove MDBK cellular membrane cholesterol, and tested the effect of lipid rafts on the infection of BPIV3. The results showed that the MDBK 342
Research in Veterinary Science 114 (2017) 341–347
L. Li et al.
Fig. 1. Removal of cell membrane cholesterol affected infectivity of BPIV3. (A) The MβCD- dependent changes of cells membrane cholesterol concentration after MDBK cells were treated by MβCD in different concentration. (B) No toxicity effect of MβCD on MDBK cells by MTT assay. (C and D) The MDBK cells were treated by MβCD different concentration for 1 h at 37 °C, then were infected by BPIV3 (MOI = 1). After 24 h infection, the infected cells measured by IFA were showed that MβCD significantly inhibited the BPIV3 infection in a dose-dependent manner (C) and the TCID50 in the supernatants were significantly decreased (D). (E) The cholesterol replenished experiment. After treatment of 10 mM MβCD for 1 h at 37 °C, the 400 μg/ ml exogenous cholesterol in DMEM was replenished into MDBK cells for 1 h, then BPIV3 infected (MOI = 1). After 24 h infection, TCID50 of the supernatants were gradually restored (E). The data statistic was based on three independent experiments. The significant difference were marked by *(P < 0.05), **(P < 0.01), ***(P < 0.001).
specific effects of MβCD. After the cells were treated with 10 mM MβCD for 1 h at 37 °C, the 400 μg/ml exogenous cholesterol was added into culture medium to replenished cellular cholesterol. MDBK cells were infected with BPIV3 (MOI = 1) for 24 h, and then virus titers were determined. As expected, infectivity of BPIV3 was successfully restored after the supplement of exogenous cholesterol, which proved that the inhibition of MβCD on BPIV3 was indeed due to its specific sequestering of cholesterol (Fig. 1E).
cells treatment with 10 mM MβCD for 1 h effectively reduced the membrane cholesterol level approximately 50% compared to that in mock-treated cells (Fig. 1A). Moreover, the MβCD-treated cells exhibited the same relative viability in mock-treated cells, excluding the non-specific effects of cytotoxicity caused by MβCD (Fig. 1B). Subsequently, MβCD-treated cells were infected with BPIV3 at the MOI of 1 for 24 h, the culture supernatants were collected and the virus titers were measured. The results indicated that MβCD significantly inhibited the BPIV3 infection in a dose-dependent manner (Fig. 1C). The virus titer reduced > 103.5-fold when treated with 20 mM MβCD compared with the mock-treated cells (P < 0.01). Next, we used IFA to further confirm the inhibition of MβCD on BPIV3 infection. We found that disruption of lipid rafts by MβCD obviously decreased BPIV3 infection, which was in accordance with the virus titer measurement (Fig. 1D). Moreover, we performed a cholesterol reconstitution experiment to confirm whether inhibition of BPIV3 infection was caused by the non-
3.2. Depletion of cellular cholesterol didn't block the binding of BPIV3 The entry process of BPIV3 was initiated once the virus bound and fused with cell membrane. To elucidate the underlying mechanism how lipid rafts influence BPIV3 infection, we determined whether depletion of membrane cholesterol blocked BPIV3 binding to cell membrane. BPIV3 virions could bind to the MDBK cell surface, but couldn't 343
Research in Veterinary Science 114 (2017) 341–347
L. Li et al.
Fig. 2. Removal of cell membrane cholesterol did not block the binding of BPIV3. (A) The TCID50 results of BPIV3 binding on target cells. The MDBK cells were treated with 0, 10 and 20 mM MβCD for 1 h, then BPIV3 infected (MOI = 1) at 4 °C for 1 h. The TCID50 in cell lysates made no difference were measured in different groups. (B) The FACS results of BPIV3 binding on target cells were consistent with that of A. The MDBK cells were treated with 0, 10 and 20 mM MβCD for 1 h, then BPIV3 infected (MOI = 1) at 4 °C for 1 h, the uninfected cells were negative control. The MDBK cells were fixed in 4% paraformaldehyde, then the cells were incubated with 200 μl rabbit polyclonal anti-serum against BPIV3 (1:100) at 4 °C for 1 h. FITC-conjugated Goat anti-rabbit IgG was added and incubated for 30 min at 37 °C avoid light. Each sample was analyzed with FACS Calibur cytometer. The data statistic was based on three independent experiments.
penetrate cell membrane at 4 °C. The cells were lysed and virus titers were measured in mock-treated and MβCD-treated cells. At the same time, the amount of cell-bound viral virions was analyzed by Flow cytometer. The most interesting finding was that the TCID50 were not affected when cells were treated with 10 and 20 mM MβCD, which indicated cellular cholesterol had no effect on the BPIV3 binding to cell surface (Fig. 2A). The FACS results further confirmed that depletion of cellular cholesterol didn't block the BPIV3 binding, which was suggesting that lipid rafts might affect the entry process (Fig. 2B).
3.3. Depletion of cellular cholesterol post virus-entry not effect on viral infection The process of BPIV3 entry includes early binding and subsequent internalization. In order to investigate effects of cell membrane cholesterol depletion on BPIV3 infection at post-entry stage, As shown in Fig. 3, comparing the mock group with the post-entry group, TCID50 had not significant difference (P > 0.05). But, TCID50 significantly decreased in pre-entry group (P < 0.001). These results indicated that lipid raft were mainly effect on BPIV3 infection in process of the virus entry.
Fig. 3. Depletion of cellular cholesterol at post virus-entry did not effect on viral infection, however, TCID50 was significantly decreased in pre-60 min group. The MDBK cells grown in 24-well plates were treated with 10 mM MβCD at 60 min before virus infection and 30, 60, 90 min after virus infection for 1 h at 37 °C. In the mock group, the MDBK cells were treated by DMEM for 1 h at 37 °C, then the virus infection. In pre-60 min group, the MDBK cells were treated by 10 mM MβCD for 1 h at 37 °C, then the virus infection. After 24 h infection, we collected the culture supernatant to measure viral titers by TCID50. The data statistic was based on three independent experiments. The significant difference were marked by *(P < 0.05), **(P < 0.01), ***(P < 0.001).
3.4. Removal of viral envelope cholesterol affects virus infection To investigate that the viral envelope cholesterol effected on BPIV3 infection, we used MβCD to remove cholesterol from the viral envelope. After drug treatment, the level of viral envelope residual cholesterol was detected. We used the treated virus to infect cells. TCID50 were used to detect the infected cells after 24 h infection. Fig. 4A showed the cholesterol in virus envelope was reduced along with increasing MβCD concentrations. MβCD treatment caused a significant decrease in virus titers. In the 20 mM treated group, virus titer decreased > 102.4-fold compared with the untreated group (Fig. 4B). To demonstrate decline of BPIV3 infection was caused by treatment of MβCD, we replenished the exogenous cholesterol. As results in Fig. 4C, the infectivity of BPIV3 was restored after the supplement of exogenous cholesterol. These results indicate that the removal of viral envelope cholesterol can inhibit the infection of BPIV3.
4. Discussion Lipid rafts effect many biological processes, including membrane transport, signal transduction, separation of proteins, cell adhesion and migration, viral infection, and synaptic transmission (Rietveld and Simons, 1998; Simons and Ikonen, 1997). A large amount of evidences suggest that lipid raft is essential in the process of virus entering into target cells, especially enveloped virus (Chan et al., 2010). It is reported that the decreased infectivity of porcine reproductive and respiratory syndrome virus (PRRSV) could cause to decrease the expression of receptor on the cell membrane surface after removing lipid rafts (Yang et al., 2015). Recently, the functions of lipid rafts in the process of 344
Research in Veterinary Science 114 (2017) 341–347
L. Li et al.
Fig. 4. Removal of viral envelope cholesterol affects virus infection. (A) The level of viral envelope residual cholesterol after MβCD treatment. The purified virions were treated with 5, 10, 20 mM MβCD at 37 °C for 1 h, the mock group was treated with DMEM, then the MβCD was removed by ultracentrifugation. The cholesterol in virus envelope was reduced along with increasing MβCD concentrations. (B) The TCID50 significantly went down when some of viral envelope cholesterol were removed. The virions treated by MβCD were used to infect the MDBK cells. After 24 h infection, the TCID50 in supernatants were determined. (C) The cholesterol replenished experiment. The TCID50 went up again after the supplement of exogenous cholesterol in growth medium. The purified virions were treated with 10 mM MβCD at 37° for 1 h, the 400 μg/ml exogenous cholesterol in DMEM was replenished into the MDBK cells for 1 h, then the drugs were removed by ultracentrifugation. The treated virus infected the MDBK cells. The TCID50 were measured after 24 h infection. The data statistic was based on three independent experiments. The significant difference were marked by *(P < 0.05), **(P < 0.01), ***(P < 0.001).
dependently destroyed by MβCD. Secondly, when exogenous cholesterol was replenished to target cells membrane after treatment of MβCD, the virus infectivity was surprisingly restored. Then we investigated the effect of cholesterol in cells membrane on the viral binding and post-infection. The results of viral binding experiment showed that cholesterol did not affect BPIV3 binding to the MDBK cells. Whether the cholesterol affect the biochemical events in post-infection or not, we conducted the experiment that MβCD treated the post-infected cells at different times. The removal of cholesterol in post-infected stage could not inhibit viral infection, however, the changes were slight compare with the treatment in pre-infection. These results revealed that infectiousness of BPIV3 particles in raft (and cholesterol) deficient MDBK cells is severely compromised but BPIV3 binding to the cells was not blocked after removing the cholesterol in the MDBK cell membrane, suggesting that BPIV3 fusion may be influenced by lipid rafts. For paramyxoviruses entry, Fusion of enveloped viruses with the plasma membrane of a receptive host cell is a prerequisite for viral entry and infection (Bossart and Broder, 2013). It's widely assumed that enveloped viruses enter the cell through two main mechanisms: by direct fusion of the viral envelope with the plasma membrane or by endocytosis. However, the details of viral entry mechanism remain further study for clarifying the decreased BPIV3 virus infectivity relating to the virus entry cell ways via membrane fusion way or endocytosis way or both ways. All the findings indicated cholesterol played a critical role in process of BPIV3 entry to the target cells, which
Caprine herpesvirus type 1 (CpHV-1) and Cyprinus herpesvirus 3 (CyHV-3) infection are also proved. Most interestingly, the cell membrane cholesterol was required for CpHV-1 replication at the virus entry phase. Furthermore, a dose-dependent reduction of the virus yield was observed after MβCD treatment. Their data also demonstrated that cholesterol is mainly required during virus entry rather than during the post-entry stage (Pratelli and Colao, 2016). Cholesterol-rich lipid rafts are important for the CyHV-3 replication cycle especially during entry and egress (Brogden et al., 2015). Although some studies have reported that rafts play an critical role during the paramyxovirus infection such as NDV (Dolganiuc et al., 2003; Laliberte et al., 2006, 2007), RSV (Chang et al., 2012), no study were performed for an essential function of rafts during BPIV3 infection. In this study, we explored the role of lipid rafts on the target cell membrane and the viral envelope in BPIV3 infection for the first time. The drugs of interfering with the cell membrane lipid raft structure are commonly used to investigate lipid rafts, MβCD is a common drug to remove cholesterol (Simons and Toomre, 2000). Entrying, binding and post-entry are key steps for process of virus infection. In order to study the role of raft in cell membrane during BPIV3 infection, MDBK cells were treated with MβCD for disrupting lipid rafts in the plasma membrane. Our results indicated lipid rafts was required in BPIV3 entry to cells. Firstly, when the MDBK cells with MβCD were incubated together before BPIV3 infected, the infectivity of BPIV3 was significantly decreased along with the cholesterol residues of target cells dose345
Research in Veterinary Science 114 (2017) 341–347
L. Li et al.
envelope was required for BPIV3 infectivity. These results can help us understand the mechanism of BPIV3 infection. In addition, lipid rafts could be a good potential therapeutic target to prevent BPIV3 infection. To achieving this goal, many detail complex mechanisms of BPIV3 fusion, endocytosis, binding, replication and budding need study in the future.
was similar to other enveloped viruses. In process of CpHV-1 infection, an obvious reduction in virus infectivity was detected after MβCDtreated virus envelope (Pratelli and Colao, 2016). Similar results were reported in CyHV-3 infection which suggested that lipid rafts were important during virus entering and egress (Brogden et al., 2015). These views were also supported by our research results that the relationship between the infectivity of BPIV3 and the cholesterol of cells membrane were partially correlated. BPIV3 entered into the target cell requiring fusion of target cells and viral membrane. The receptor binding protein HN could bind to the surface receptor of target cells to induce conformational change. Then, the fusion protein F was activated before membrane fusion, following nucleoprotein enter cytolymph to replicate (Jardetzky and Lamb, 2014). The content of cholesterol in cell membrane can maintaining the fluidity of cell membrane (Burger et al., 2000). Removed cholesterol can reduce the radial diffusion of the membrane. The ability of invading the cell membrane for BPIV3 may be affected by decreased diffusion ability of the membrane. In addition, lipid environment in the cell membrane, including cholesterol, has influence on ion channels (Chang et al., 1995). Paramyxoviruses enter the target cell by binding to a cell surface receptor and then fusing the viral envelope with the target cell membrane, allowing the release of the viral genome into the cytoplasm (Palgen et al., 2015). Lipid rafts in the cell membrane was identified by viral specific protein to regulate the process of virus invading cells (Teissier and Pecheur, 2007). Taken together, viruses achieve host cell entry in two principal mechanism: direct fusion pathway and endocytic pathway including clathrin-mediated, clathrin-independent pathway, raft-dependent pathways, and macropinocytosis (Huang et al., 2017). Our findings imply that as an enveloped virus, BPIV3 infection may associate with the rafts-related membrane fusion mechanism. Although the HN protein carried by the Respirovirus, Rubulavirus, and Avulavirus genera possesses both sialic acid-binding (hemagglutinating) and sialic acid-cleaving (neuraminidase) activities (Palgen et al., 2015), but sialic acid receptor that BPIV3 employed for HN binding and whether the HN-receptor binding affects lipid raft-dependence vrial entry or not are still unknown, which remains further studies. Cholesterol in viral envelope can sustain integrity of virus particles (Fujita et al., 2011). In this study, after the purified BPIV3 virions were treated by MβCD in different concentration, the virus infectivity decreased in dose-dependent manner. The lessened infectivity may be caused by destroyed viral envelope integrity owing to depletion of cholesterol. In the process of Bovine herpesvirus 1 (BoHV-1) infection, the virus infectivity was reduced by removing viral envelop cholesterol with MβCD, exogenous cholesterol can partially reverse the effect (Zhu et al., 2010). In our experiments, cholesterol removed from BPIV3 envelope might destroyed the virus particles, which resulted in the ability of BPIV3 infection decrease. The previous researches have been shown that infectivity of virus particles was decreased by treatment high concentration MβCD because the loss of N protein in PRRSV infection process (Yang et al., 2015). Fujita et al. (2011) also demonstrated infectivity of MβCD treated hemagglutinating virus of Japan (HVJ) was lower than wild-type HVJ. In addition, lipid raft play a role as chemically entities by modulating the curvature of the membranes, and further affected fusion process (Teissier and Pecheur, 2007). Cholesterol depletion resulted in viral particles structure change to decline BPIV3 particles infectivity. It have been shown that intact lipid rafts of viral envelope is required for the viral invasion in many Paramyxoviridae members, such as respiratory syncytial virus (RSV) (Chang et al., 2012), newcastle disease virus (NDV) (Martin et al., 2012), measles virus (MeV) (Avota and Schneider-Schaulies, 2014), sendai virus (SeV) (Fujita et al., 2011). Our researches suggest it may be a typical feature of Paramyxoviridae viruses that lipid rafts in viral envelope effected virus entry into host cells. The virus entry into target cells is a complex process, including many factors. In this study, we first demonstrated lipid raft was essential to BPIV3 entry into MDBK cells. Furthermore, lipid raft in viral
Conflicts of interest None. Acknowledgements This project was supported by the fund of Heilongjiang Education Department Research Program (12521372), National Key Technology R & D Program (2012BAD12B05-2), China. References Avota, E., Schneider-Schaulies, S., 2014. The role of sphingomyelin breakdown in measles virus immunmodulation. Cell. Physiol. Biochem. 34, 20–26. Barman, S., Nayak, D.P., 2007. Lipid raft disruption by cholesterol depletion enhances influenza a virus budding from MDCK cells. J. Virol. 81, 12169–12178. Bhattacharya, J., Repik, A., Clapham, P.R., 2006. Gag regulates association of human immunodeficiency virus type 1 envelope with detergent-resistant membranes. J. Virol. 80, 5292–5300. Blanchard, E., Belouzard, S., Lucie Goueslain, L., Wakita, T., Dubuisson, J., Wychowski, C., Rouille, Y., 2006. Hepatitis C virus entry depends on clathrin-mediated endocytosis. J. Virol. 80, 6964–6972. Bossart, K.N., Broder, C.C., 2013. Paramyxovirus entry. In: Pöhlmann, S. (Ed.), Viral Entry Into Host Cell, Spring Street, New York, USA. Brogden, G., Adamek, M., Proepsting, M.J., Ulrich, R., Naim, H.Y., Steinhagen, D., 2015. Cholesterol-rich lipid rafts play an important role in the Cyprinid herpesvirus 3 replication cycle. Vet. Microbiol. 179, 204–212. Brown, D.A., London, E., 2000. Structure and function of sphingolipid- and cholesterolrich membrane rafts. J. Biol. Chem. 275, 17221–17224. Burger, K., Gimpl, G., Fahrenholz, F., 2000. Regulation of receptor function by cholesterol. Cell. Mol. Life Sci. 57, 1577–1592. Burlone, M.E., Budkowska, A., 2009. Hepatitis C virus cell entry: role of lipoproteins and cellular receptors. J. Gen. Virol. 90, 1055–1070. Chan, R.B., Tanner, L., Wenk, M.R., 2010. Implications for lipids during replication of enveloped viruses. Chem. Phys. Lipids 163, 449–459. Chang, H.M., Reitstetter, R., Mason, R.P., Gruener, R., 1995. Attenuation of channel kinetics and conductance by cholesterol - an interpretation using structural stress as a unifying concept. J. Membr. Biol. 143, 51–63. Chang, T.H., Segovia, J., Sabbah, A., Mgbemena, V., Bose, S., 2012. Cholesterol-rich lipid rafts are required for release of infectious human respiratory syncytial virus particles. Virology 422, 205–213. Dolganiuc, V., McGinnes, L., Luna, E.J., Morrison, T.G., 2003. Role of the cytoplasmic domain of the Newcastle disease virus fusion protein in association with lipid rafts. J. Virol. 77, 12968–12979. Ellis, J.A., 2010. Bovine parainfluenza-3 virus. In: Veterinary Clinics of North AmericaFood Animal Practice. vol. 26. pp. 575–57 +. Fujita, H., Tamai, K., Kawachi, M., Saga, K., Shimbo, T., Yamazaki, T., Kaneda, Y., 2011. Methyl-beta cyclodextrin alters the production and infectivity of Sendai virus. Arch. Virol. 156, 995–1005. Grissett, G.P., White, B.J., Larson, R.L., 2015. Structured literature review of responses of cattle to viral and bacterial pathogens causing bovine respiratory disease complex. J. Vet. Intern. Med. 29, 770–780. Huang, R., Zhu, G., Zhang, J., Lai, Y., Xu, Y., He, J., Xie, J., 2017. Betanodavirus-like particles enter host cells via clathrin-mediated endocytosis in a cholesterol-, pH- and cytoskeleton-dependent manner. Vet. Res. 48. Imhoff, H., von Messling, V., Herrler, G., Haas, L., 2007. Canine distemper virus infection requires cholesterol in the viral envelope. J. Virol. 81, 4158–4165. Jardetzky, T.S., Lamb, R.A., 2014. Activation of paramyxovirus membrane fusion and virus entry. Curr. Opin. Virol. 5, 24–33. Kale, M., Dylek, O., Sybel, H., Pehlivanoglu, F., Turutoglu, H., 2013. Some viral and bacterial respiratory tract infections of dairy cattle during the summer season. Acta Vet-Beogr. 63, 227–236. Kirchhoff, J., Uhlenbruck, S., Goris, K., Keil, G.M., Herrler, G., 2014. Three viruses of the bovine respiratory disease complex apply different strategies to initiate infection. Vet. Res. 45. Laliberte, J.P., McGinnes, L.W., Peeples, M.E., Morrison, T.G., 2006. Integrity of membrane lipid rafts is necessary for the ordered assembly and release of infectious Newcastle disease virus particles. J. Virol. 80, 10652–10662. Laliberte, J.P., McGinnes, L.W., Morrison, T.G., 2007. Incorporation of functional HN-F glycoprotein-containing complexes into Newcastle disease virus is dependent on cholesterol and membrane lipid raft integrity. J. Virol. 81, 10636–10648. Ma, K., Wang, Y.J., Wang, J.F., 2015. Influenza virus assembly and budding in lipid rafts. Prog. Biochem. Biophys. 42, 495–500.
346
Research in Veterinary Science 114 (2017) 341–347
L. Li et al.
Roohvand, F., Maillard, P., Lavergne, J.P., Boulant, S., Walic, M., Andreo, U., Goueslain, L., Helle, F., Mallet, A., McLauchlan, J., Budkowska, A., 2009. Initiation of hepatitis C virus infection requires the dynamic microtubule network. J. Biol. Chem. 284, 13778–13791. Simons, K., Ikonen, E., 1997. Functional rafts in cell membranes. Nature 387, 569–572. Simons, K., Toomre, D., 2000. Lipid rafts and signal transduction. Nat. Rev. Mol. Cell Biol. 1, 31–39. Solis-Calderon, J.J., Segura-Correa, J.C., Aguilar-Romero, F., Segura-Correa, V.M., 2007. Detection of antibodies and risk factors for infection with bovine respiratory syncytial virus and parainfluenza virus-3 in beef cattle of Yucatan, Mexico. Prev. Vet. Med. 82, 102–110. Sviridov, D., Bukrinsky, M., 2014. Interaction of pathogens with host cholesterol metabolism. Curr. Opin. Lipidol. 25, 333–338. Teissier, E., Pecheur, E.I., 2007. Lipids as modulators of membrane fusion mediated by viral fusion proteins. Eur. Biophys. J. Biophys. Lett. 36, 887–899. Yang, Q., Zhang, Q., Tang, J., Feng, W.H., 2015. Lipid rafts both in cellular membrane and viral envelope are critical for PRRSV efficient infection. Virology 484, 170–180. Zhu, L.Q., Ding, X.Y., Tao, J., Wang, J.Y., Zhao, X., Zhu, G.Q., 2010. Critical role of cholesterol in bovine herpesvirus type 1 infection of MDBK cells. Vet. Microbiol. 144, 51–57.
Martin, J.J., Holguera, J., Sanchez-Felipe, L., Villar, E., Munoz-Barroso, I., 2012. Cholesterol dependence of Newcastle disease virus entry. Biochim. Biophys. Acta Biomembr. 1818, 753–761. Munro, S., 2003. Lipid rafts: elusive or illusive? Cell 115, 377–388. Ono, A., Freed, E.O., 2001. Plasma membrane rafts play a critical role in HIV-1 assembly and release. Proc. Natl. Acad. Sci. U. S. A. 98, 13925–13930. Palgen, J.L., Eric, M., Jurgens, E.M., Moscona, A., Porotto, M., Palermo, L.M., 2015. Unity in diversity: shared mechanism of entry among paramyxoviruses. Prog. Mol. Biol. Transl. Sci. 129, 1–32. Pratelli, A., Colao, V., 2016. Critical role of the lipid rafts in caprine herpesvirus type 1 infection in vitro. Virus Res. 211, 186–193. Ravid, D., Leser, G.P., Lamb, R.A., 2010. A role for caveolin 1 in assembly and budding of the paramyxovirus parainfluenza virus 5. J. Virol. 84, 9749–9759. Rawat, S.S., Gallo, S.A., Eaton, J., Martin, T.D., Ablan, S., KewalRamani, V.N., Wang, J.M., Blumenthal, R., Puri, A., 2004. Elevated expression of GM3 in receptor-bearing targets confers resistance to human immunodeficiency virus type 1 fusion. J. Virol. 78, 7360–7368. Rietveld, A., Simons, K., 1998. The differential miscibility of lipids as the basis for the formation of functional membrane rafts. Biochim. Biophys. Acta Rev. Biomembr. 1376, 467–479.
347