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Tubulin mediates Portunus trituberculatus reovirus infection
Aquaculture 448 (2015) 196–202
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Tubulin mediates Portunus trituberculatus reovirus infection Jing Fang 1, Dengfeng Li ⁎,1, Ran Xu, Liping Zhang, Lianguo Liu, Annan Guo School of Marine Sciences, Ningbo University, Ningbo 315000, PR China
a r t i c l e
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Article history: Received 14 April 2015 Received in revised form 31 May 2015 Accepted 1 June 2015 Available online 4 June 2015 Keywords: Portunus trituberculatus Reovirus Receptor Tubulin VOPBA Coimmunoprecipitation
a b s t r a c t Reoviruses are widespread and infect a broad range of hosts. To date, no study has been reported on an aquatic reovirus receptor. By using viral overlay protein binding assay (VOPBA), swimming crab Portunus trituberculatus reovirus (SCRV) was found to bind to a protein of approximately 550 kDa. MALDI-TOF MS–MS analysis revealed that the protein shares the closest homology with β-tubulin. Mouse membrane proteins were tested by western blot with antibodies against the SCRV-binding protein and mouse tubulin, and uniform positive bands were obtained. The results indicated that the SCRV-binding protein was tubulin. The interaction between tubulin and SCRV was further confirmed with coimmunoprecipitation. SCRV infection in vitro could be blocked by a tubulin-specific antibody. The role of tubulin as a major cell surface protein has been reported previously. These findings suggest that tubulin mediates SCRV infection and may function as a receptor for SCRV. Statement of relevance: Swimming crab reovirus (SCRV) spread widely, caused losses of millions of dollars in crab cultivation industry in China. Up to now, no studies have been reported on reovirus receptor in aquatic organisms. This paper found that tubulin serves as a receptor for SCRV in swimming crab. It proposes a new drug target for preventing or controlling SCRV. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Various reoviruses have been found to infect diverse host species, including mammals, birds, reptiles, fish, crustaceans, marine protists, insects, ticks, arachnids, plants and fungi (Mohd et al., 2005). Viruses of the family Reoviridae are distributed widely in aquatic environments and have been isolated from a broad range of aquatic organisms (Attoui et al., 2005; He et al., 2011). Reoviruses are causative agents of serious diseases with high mortality in the cultured Chinese mitten crab Eriocheir sinensis, the mud crab Scylla serrata and the swimming crab Portunus trituberculatus (Deng et al., 2012; Guo et al., 2008; Li, 2013; Li et al., 2012; Montanie et al., 1993; Zhang et al., 2004). Viral infection in host cells is mediated by the initial attachment of a virus to a receptor on the cell surface, which is required for successful entry into the cell and the initiation of infection (Marsh and Helenius, 2006). The receptors of mammalian and Muscovy duck reoviruses
have been reported (Barton et al., 2001; Bernardo and Terence, 2011; Co et al., 1985; Konopka-Anstadt et al., 2014; Noseworthy et al., 1983; Paul and Lee, 1987; Sun, 2004). There are some reports on the interaction between reovirus and grass carp (Chen et al., 2013; Heng et al., 2011; Su et al., 2009), although the infectious mechanism of aquatic reovirus is still unclear. To date, no studies have been reported on reovirus receptors in aquatic organisms. Swimming crab reovirus (SCRV) is widespread and has caused the loss of millions of dollars in the crab cultivation industry in China (Li, 2013; Li et al., 2012). The mechanism of viral infection is poorly understood. In this study, a potential binding receptor of SCRV was screened by viral overlay protein binding assay (VOPBA) and identified by coimmunoprecipitation and an infection blocking test. 2. Materials and methods 2.1. SCRV virion purification
Abbreviations: VOPBA, viral overlay protein binding assay; SCRV, swimming crab Portunus trituberculatus reovirus; EDTA, ethylenediaminetetraacetic acid; PMSF, phenylmethylsulfonyl fluoride; min, minutes; h, hours; TM2, Tris-buffered saline with Mg2+ and Na+; TNE2, Tris-EDTA-buffered saline with 400 mM NaCl; PBS, phosphatebuffered saline; TEM, transmission electron microscopy; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; PVDF, polyvinylidene fluoride; PBST, PBS containing 0.1% Tween 20; RT, room temperature; HRP, horseradish peroxidase; OPD, Ophenylenediamine; NP40, Nonidet P-40; NETN, Tris-EDTA-buffered saline with NP40; FITC, fluorescein isothiocyanate. ⁎ Corresponding author at: Department of Marine Sciences, Ningbo University, Fenghua Road No. 818, 315211 Ningbo, PR China. E-mail address: [email protected] (D. Li). 1 J.F. and D.L. contributed equally to this work.
http://dx.doi.org/10.1016/j.aquaculture.2015.06.001 0044-8486/© 2015 Elsevier B.V. All rights reserved.
Intact SCRV virions were purified as described previously with some modifications (Li, 2013; Xie et al., 2005) from the tissues of artificially infected P. trituberculatus. Briefly, tissues were homogenized in precooled TNE2 buffer [50 mM Tris–HCl; 400 mM NaCl; 5 mM ethylenediaminetetraacetic acid (EDTA), pH 8.5; and 1 mM phenylmethylsulfonyl fluoride (PMSF), which was freshly added] at 4 °C and then centrifuged at 8000 × g for 10 minutes (min) at 4 °C. The collected supernatant fluid was centrifuged again as described above. The supernatant was then collected and centrifuged at 38,000 × g for 1.5 hours (h) at 4 °C. The sediment was immersed in TNE2 buffer containing 1 mM PMSF at
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2.4. Mass spectrometry (MS) analysis of the SCRV-binding protein To investigate the elements comprising the identified SCRV-binding protein, a protein band that corresponded to the viral binding band on the VOPBA was excised from the 8% SDS-PAGE gel and sent to Guangzhou Fitgene Biotechnology Company Limited (PR China) for Micromass Maldi Time-of-Flight Mass Spectrometry (MALDI-TOF-MS) analysis. To confirm their validity, assays including gel excision and MALDI-TOF-MS were repeated twice. 2.5. Preparation of polyclonal antibodies against the SCRV-binding protein
Fig. 1. Electron micrograph of purified SCRV virions negatively stained with 2% uranyl acetate.
4 °C overnight. The sediment was gently resuspended and then centrifuged at 3500 ×g for 5 min at 4 °C. The supernatant was centrifuged at 38,000 × g for 1.5 h at 4 °C. Following this, the upper loose pellet was rinsed out carefully in TM2 buffer (50 mM Tris–HCl; 10 mM MgCl2; 1.5% NaCl; and 1 mM PMSF, pH 7.5–7.6) at 4 °C, and the lower white pellet was immersed again in TNE2 buffer at 4 °C overnight as described above. After centrifugation at 5000 × g for 5 min, the virus particles were sedimented by centrifugation at 38,000 × g for 1.5 h at 4 °C and then resuspended in phosphate-buffered saline (PBS, 8 g of NaCl, 0.2 g of KCl, 1.44 g of NaHPO4 and 0.24 g of KH2PO4, dissolved in 1 l distilled water at pH 7.4) and stored at −80 °C until use. The purified virus sample was detected by negative staining under a transmission electron microscope (TEM).
The SCRV-binding protein identified from swimming crab gill was excised from the 8% SDS-PAGE gel. After homogenization and suspension in 0.9% NaCl, the mixture was emulsified in an equal volume of complete Freund's adjuvant (Sigma) and was used to subcutaneously immunize ICR mice. After 2 weeks, the first booster immunization was administered to each mouse, which included the protein suspended in incomplete Freund's adjuvant (Sigma). The other two booster injections were given at 1-week intervals. Mouse sera were obtained 7 days after the last injection and stored at − 80 °C for later use. Non-immunized mouse serum was also prepared as a control. The specificity and presence of murine antibodies against the SCRV-binding protein were identified by western blot. The titers of the mouse sera were determined by ELISA according to a previous method (Yang et al., 2013). 2.6. Identification of the SCRV-binding protein by western blot analysis Mouse membrane proteins were separated on 8% SDS-PAGE gels and electrophoretically transferred to PVDF membranes (Millipore). Mouse anti-SCRV-binding protein polyclonal sera and a commercial mouse anti-β-tubulin antibody (purchased based on the MS results) were used as primary antibodies in western blot analysis. 2.7. Coimmunoprecipitation assays
2.2. Membrane protein preparation Cell membrane preparations were acquired as previously described with minor modifications (Jindadamrongwech et al., 2004). Briefly, healthy swimming crab was dissected, and gill tissue was homogenized in 10 volumes of ice-cold buffer M (100 mM Tris–HCl, pH 8.0; 2 mM MgCl2; 1 mM EDTA; 0.2% Triton X-100; and 1 mM PMSF). Nuclei and cell debris were removed by centrifugation at 600 × g for 3 min at 4 °C. The supernatant was then centrifuged at 6000 × g for 5 min at 4 °C to remove membranous organelles and then again at 35,000 × g for 2 h to pellet membrane proteins. The pellet was resuspended in buffer M and stored at −80 °C for later use. 2.3. Virus overlay protein binding assay (VOPBA) Membrane proteins were separated on an 8% gel using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a 0.45 μm polyvinylidene fluoride (PVDF) membrane (Millipore). The membrane was blocked overnight at 4 °C in 5% lowfat milk in PBST (PBS containing 0.5% Tween-20) and washed three times with PBST. The membrane was then incubated with 12.9 μg purified SCRV in PBS for 1 h at room temperature (RT). Following this, the membrane was washed three times with PBST and incubated with a specific chicken polyclonal anti-SCRV antibody at room temperature for 2 h. After washing three times with PBST, the membrane was incubated with horseradish peroxidase (HRP)-conjugated sheep antichicken IgG (ComWin Biotech, CWBIO) (diluted 1:5000 in 0.5% skim milk in PBST) for 1 h at RT. After three washes with PBST, the viral binding bands on the membrane were developed with O-phenylenediamine (OPD, Invitrogen) and then photographed.
The binding activity of tubulin to SCRV was detected by coimmunoprecipitation analysis. Coimmunoprecipitation assays were carried out according to a previously described method (Li et al., 2007) with some modifications. A 500 μl aliquot of membrane protein from swimming crab gill was incubated with 30 μl purified SCRV on a rocker for 8 h at 4 °C. A 10 μl aliquot of either mouse anti-tubulin polyclonal antibody or IgG against an irrelevant protein (as a negative control) was added to each tube and incubated overnight at 4 °C. Subsequently, 60 μl of agarose beads conjugated with protein A-G (Santa Cruz) was added to the mixture and incubated for 3 h at 4 °C with gentle rocking. Following this, the agarose beads were collected by centrifugation and washed four times with high-salt NETN buffer (20 mM Tris–HCl, 0.5% NP-40, 500 mM NaCl, 1 mM EDTA, 25 μg/ml PMSF, pH 7.6) and once with NETN buffer (20 mM Tris–HCl, 0.5% NP-40, 100 mM NaCl, 1 mM EDTA, 25 μg/ml PMSF, pH 7.6). The bound proteins were dissociated from the antibodies by boiling in loading buffer for 4 min and were then separated on 12% SDS-PAGE gels. SCRV was then detected by standard western blotting techniques with an anti-SCRV polyclonal antibody. Similarly, an immunoprecipitation of tubulin with the anti-SCRV polyclonal antibody was performed. Either 500 μl membrane protein from swimming crab gill or 500 μl PBS (as a negative control) was incubated with 30 μl purified SCRV on a rocker for 8 h at 4 °C. A 10 μl aliquot of mouse anti-SCRV polyclonal antibody was added to each tube and incubated overnight at 4 °C. Subsequently, a process was performed following the steps of the immunoprecipitation assay on SCRV, except that bound proteins were separated on 8% SDS-PAGE gels, and tubulin was detected by standard western blotting techniques with an antitubulin polyclonal antibody.
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2.8. SCRV infection inhibition assay in vitro A virus infection inhibition assay was carried out in vitro with hemocytes from swimming crab. Hemocyte suspensions were placed into coated 96-well cell culture (BIOFIL, Canada) plates with either antitubulin polyclonal antibodies (1/10, 1/20 and 1/40 dilutions) or PBS (virus control) for 1 h at RT with gentle shaking. Simultaneously, SCRV was labeled with fluorescein isothiocyanate (FITC) as previously described (Li et al., 2007). Briefly, purified virions were incubated in 1 mg/ml FITC at room temperature for 1 h, followed by centrifugation at 10,000 × g for 10 min. Pellets of the labeled virions were resuspended in TM2 buffer (50 mM Tris–HCl, 10 mM MgCl2, 1.5% NaCl, pH 7.5) and then sedimented again by centrifugation at 10,000 × g for 10 min to remove free FITC. After washing three times, labeled virions were suspended in PBS. FITC-labeled SCRV (FITC-SCR) was added to each well and incubated for 30 min at RT with gentle shaking. Subsequently, the mixtures were removed, and unbound SCRV was washed off with PBS. The treated cells were observed under a microscope.
3. Results 3.1. Virus purification Fig. 2. VOPBA analysis of SCRV binding to P. Trituberculatus cell membrane proteins. (A) SDS-PAGE profile of P. Trituberculatus cell membrane proteins. (B) Detection of the positions of the SCRV-binding proteins by VOPBA. The prominent binding bands with molecular weights of approximately 550 kDa are indicated with black arrows on the right.
Purified SCRV was negatively stained with 2% uranyl acetate and observed under a transmission electron microscope (TEM). The results indicated that the virions were pure and intact (Fig. 1).
Fig. 3. Mass spectrographic analysis of the SCRV-binding protein for the first time. (A) Fingerprint of the protein; matched peaks are marked by asterisks. (B) Mass spectrographic analysis of the fingerprint of the protein using Mascot; protein scores greater than 41 are significant (p b 0.05). Accession information is as follows. NCBI accession no: gi|338224308, score: 48, description: beta-I tubulin (Scylla paramamosain).
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Fig. 4. Mass spectrographic analysis of the SCRV-binding protein for the second time. (A) Fingerprint of the protein; matched peaks are marked by asterisks. (B) Mass spectrographic analysis of the fingerprint of the protein using Mascot; protein scores greater than 47 are significant (p b 0.05). Accession information is as follows. NCBI accession no: gi|338224308, score: 48, description: beta-I tubulin (Scylla paramamosain).
3.2. Screening of the virus binding protein in swimming crab gill cells by VOPBA
3.5. Co-immunoprecipitation assay confirms that swimming crab tubulin interacts with SCRV proteins
Swimming crab gill cell-membrane proteins were prepared and separated by SDS-PAGE (Fig. 2A, Lane 1) and then transferred onto PVDF membranes, followed by incubation with SCRV. The membranes were subsequently incubated with polyclonal antibodies against SCRV, and HRP-conjugated antibody was added to visualize the reactive bands. The VOPBA results showed that SCRV could specifically bind to a swimming crab cell membrane protein with a molecular mass of approximately 550 kDa (Fig. 2B, Lane 1).
To demonstrate the interaction between tubulin and SCRV protein, a coimmunoprecipitation of SCRV and swimming crab tubulin was performed. The results showed that SCRV was precipitated with an Ab against tubulin as a membrane protein of swimming crab, while SCRV could not be precipitated with an Ab against an irrelevant protein (Fig. 6). In turn, tubulin and SCRV were both immunoprecipitated with an anti-SCRV Ab.
3.6. Neutralization of cell surface tubulin blocked SCRV infection in vitro 3.3. MALDI-TOF MS–MS analysis Based on searching NCBI BLAST with the peptide mass fingerprints, MALDI-TOF analysis of the 550 kDa protein twice exhibited homology to β-tubulin (Figs. 3, 4).
To test if tubulin plays a role in mediating the infection of SCRV, infection-blocking assays were carried out on hemocytes of P. Trituberculatus in vitro. Hemocytes pretreated with different dilutions of anti-tubulin polyclonal antibodies were infected with SCRV (Fig. 7). Hemocytes pretreated with PBS were employed as a control. The results
3.4. Western blot analysis To verify that the 550 kDa SCRV-binding protein was tubulin, mouse membrane proteins were respectively separated on 8% SDSPAGE gels and electrophoretically transferred to 0.45 μm PVDF membranes. Mouse anti-SCRV-binding-protein polyclonal sera and a commercial mouse anti-β-tubulin antibody were used as primary antibodies respectively in western blot analysis. The results showed that the immunized sera against the 550 kDa SCRV-binding protein, coincident with the commercial mouse anti-β-tubulin antibody (Fig. 5A, lane 1), could specifically recognize mouse tubulin (Fig. 5B, lane 1).
Fig. 5. Identification of SCRV-binding protein by western blot analysis. (A) A commercial mouse anti-β-tubulin antibody was used as a primary antibody in western blot analysis. Lane 1: mouse membrane proteins + mouse-anti-β-tubulin antibody. (B) Mouse antiSCRV-binding-protein polyclonal sera were used as primary antibodies in western blot analysis. Lane 1: mouse membrane proteins + immunized sera.
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Fig. 6. Co-immunoprecipitation of SCRV and tubulin. Membrane proteins from swimming crabs were mixed with SCRV and immunoprecipitated with anti-tubulin antibodies (lane 1) or an antibody against an irrelevant protein (negative control, lane 3), in addition to protein A-G agarose. The immunoprecipitates were subjected to western blot analysis using antibodies against SCRV as outlined in Materials and methods section. Lane 1: swimming crab membrane proteins + SCRV + anti-tubulin Ab. Lane 2: SCRV. Lane 3: swimming crab membrane proteins + SCRV + Ab against irrelevant protein.
demonstrated that the neutralization of cell surface tubulin with antitubulin antibodies (1/10 dilution) significantly inhibited the infection of SCRV in vitro. 4. Discussion The swimming crab P. Trituberculatus (Miers, 1876) (Crustacea: Decapoda: Brachyura), also known as gazami crab, horse crab or Japanese blue crab, is the most widely fished species of crab in the world, producing a yield of over 300,000 tons annually. In recent years, P. trituberculatus crab farms in China suffered from an outbreak epidemic that led to mass mortality, which heavily impacted the industry. SCRV is a primary lethal pathogen, which spreads widely and causes losses of millions of dollars in the crab cultivation industry annually. The similarity of nucleic acid sequences between SCRV and mud crab S. Serrata reovirus was 99%, although SCRV cannot infect mud crab (unpublished). No effective strategy for preventing or controlling SCRV is available. Studies on the interactions between SCRV and swimming crab cells, especially identification of the virus receptor, are critical for understanding its infectious mechanism, which has considerable implications for developing strategies to prevent and control infection. The purpose of this study was to screen for cell receptors of SCRV. Viral overlay protein binding assay (VOPBA) has been used
to identify the receptor proteins of various viruses, such as dengue virus (Jindadamrongwech and Smith, 2004), pancreatic necrosis virus (Orpetveit et al., 2008), fowl adenovirus (Taharaguchi et al., 2007), bovine adenovirus (Li et al., 2009), and hepatitis E virus (Zhang et al., 2011). In a VOPBA, membrane proteins are separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto polyvinylidene fluoride (PVDF) membranes; the membranes containing transferred proteins are incubated with viral particles and tested with virus-specific antibodies (Yang et al., 2013). This assay is sensitive enough to detect 5 mg of purified glycophorin or to detect the binding of less than 100 mg of protein from a crude membrane preparation (Gershoni et al., 1986). In this study, by applying VOPBA, an approximately 550 kDa protein in the membrane fraction of swimming crab gill cells was found to interact with SCRV. Twice, the results of MALDI-TOF analysis of the 550 kDa protein exhibited homology to β-tubulin. To determine whether the 550 kDa protein was tubulin, the SCRV-binding protein was identified using western blot analysis (Fig. 5). The results clearly indicated that antibodies against the 550 kDa swimming crab protein could specifically recognize the mouse protein coincident with the ability of a commercial mouse anti-β-tubulin antibody. These results indicated that the SCRV-binding protein is tubulin and suggested that tubulin may exist either as a multimeric form or bound to GTP and nucleotides in
Fig. 7. Infection inhibition assay using anti-tubulin polyclonal antibody in vitro. Hemocytes were incubated with different dilutions of anti-tubulin polyclonal antibodies for 1 h at RT and then FITC-SCRV was added to each well and incubated for 30 min at RT with gentle shaking. Cells were viewed under a microscope. Scale bar = 500 μm.
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swimming crab. Tubulin heterodimers have long been known to be involved in the assembly and transport of various viruses (Hong and Ng, 1987; Ng and Hong, 1989; Ogino et al., 2003; Ruthel et al., 2005; Smith and Enquist, 2002). It has been reported that DENV2 through DENV2E binds directly to tubulin or to a tubulin-like protein in C6/36 mosquito cells (Chee and Sazaly, 2004). Reoviruses have been reported to interact with microtubules (Babiss et al., 1979; Heinemann et al., 2011; Kim et al., 2004; Kobayashi et al., 2009; Parker et al., 2002). Reovirus cell entry requires functional microtubules, which facilitate the targeting of reovirus to acidified endosomes for viral disassembly. Microtubule inhibitors can block various stages of the reovirus life cycle (Huang et al., 2011; Mainou et al., 2013). Generally, tubulin is considered to be an intracellular protein and is a cytoskeletal element that is essential for intracellular transport and cell division in all eukaryotes (Nogales et al., 1998). Earlier, we were confused in the face of our results. Therefore, we consulted a number of relevant studies and eventually obtained references (Por et al., 1991; Quillen et al., 1985; Rubin et al., 1982; Stephens, 1981) which reported that tubulin can also act as a cell surface protein. Stephens (1981) identified that tubulin was a membrane protein. Rubin et al. (1982) reported that tubulin was a major cell surface protein in human lymphoid cells of leukemic origin. Quillen et al. (1985) found that tubulin can exist either in or on the outer surfaces of living cells. Por et al. (1991) provided evidence for the presence of tubulin on the surface of a human monocyte-like cell line, U937. Coimmunoprecipitation of tubulin and SCRV identified the interaction between them. To further confirm the role of tubulin in mediating the infection of SCRV, infection-blocking assays were carried out on hemocytes of P. Trituberculatus in vitro. Antibodies against tubulin were able to inhibit virus entry into cells. All of the above findings suggest that tubulin mediates the infection of SCRV in swimming crab. 5. Conclusion This is the first study to reveal that tubulin serves as a receptor for SCRV in swimming crab using VOPBA, mass spectrometry, western blot, coimmunoprecipitation and infectivity reduction via anti-tubulin antibodies. It proposes a new drug target for preventing or controlling SCRV. This is also the first study to find that tubulin serves as a receptor for viruses and to identify a reovirus receptor in aquatic organisms. Acknowledgments This work was financially supported by the Spark Program of China (no. 2012GA701006), the Zhejiang Provincial Natural Science Foundation (no. LY12C19004), the Science Foundation of the Department of Education of Zhejiang Province (Y201327815), the Agricultural Program of Ningbo, Zhejiang, China (no. 2011C10004), and the Open Foundation of Key Disciplines of Zhejiang Province (E02009124200) and was sponsored by the K.C. Wong Magna Fund of Ningbo University. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. References Attoui, H., Belhouchet, M., Mohd, J.F., Biagini, P., Cantaloube, J.F., de, M.P., de, L.X., 2005. Expansion of family Reoviridae to include nine-segmented dsRNA viruses: isolation and characterization of a new virus designated Aedes pseudoscutellaris reovirus assigned to a proposed genus (Dinovernavirus). Virology 343, 212–223. Babiss, L.E., Luftig, R.B., Weatherbee, J.A., Weihing, R.R., Ray, U.R., Fields, B.N., 1979. Reovirus serotypes 1 and 3 differ in their in vitro association with microtubules. J. Virol. 30, 863–874. Barton, E.S., Forrest, C., Connolly, J.L., 2001. Junction adhesion molecule is a receptor for reovirus. Cell 104, 441–451. Bernardo, A.M., Terence, S.D., 2011. Src kinase mediates productive endocytic sorting of reovirus during cell entry. J. Virol. 85, 3203–3205.
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Tubulin mediates Portunus trituberculatus reovirus infection Jing Fang 1, Dengfeng Li ⁎,1, Ran Xu, Liping Zhang, Lianguo Liu, Annan Guo School of Marine Sciences, Ningbo University, Ningbo 315000, PR China
a r t i c l e
i n f o
Article history: Received 14 April 2015 Received in revised form 31 May 2015 Accepted 1 June 2015 Available online 4 June 2015 Keywords: Portunus trituberculatus Reovirus Receptor Tubulin VOPBA Coimmunoprecipitation
a b s t r a c t Reoviruses are widespread and infect a broad range of hosts. To date, no study has been reported on an aquatic reovirus receptor. By using viral overlay protein binding assay (VOPBA), swimming crab Portunus trituberculatus reovirus (SCRV) was found to bind to a protein of approximately 550 kDa. MALDI-TOF MS–MS analysis revealed that the protein shares the closest homology with β-tubulin. Mouse membrane proteins were tested by western blot with antibodies against the SCRV-binding protein and mouse tubulin, and uniform positive bands were obtained. The results indicated that the SCRV-binding protein was tubulin. The interaction between tubulin and SCRV was further confirmed with coimmunoprecipitation. SCRV infection in vitro could be blocked by a tubulin-specific antibody. The role of tubulin as a major cell surface protein has been reported previously. These findings suggest that tubulin mediates SCRV infection and may function as a receptor for SCRV. Statement of relevance: Swimming crab reovirus (SCRV) spread widely, caused losses of millions of dollars in crab cultivation industry in China. Up to now, no studies have been reported on reovirus receptor in aquatic organisms. This paper found that tubulin serves as a receptor for SCRV in swimming crab. It proposes a new drug target for preventing or controlling SCRV. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Various reoviruses have been found to infect diverse host species, including mammals, birds, reptiles, fish, crustaceans, marine protists, insects, ticks, arachnids, plants and fungi (Mohd et al., 2005). Viruses of the family Reoviridae are distributed widely in aquatic environments and have been isolated from a broad range of aquatic organisms (Attoui et al., 2005; He et al., 2011). Reoviruses are causative agents of serious diseases with high mortality in the cultured Chinese mitten crab Eriocheir sinensis, the mud crab Scylla serrata and the swimming crab Portunus trituberculatus (Deng et al., 2012; Guo et al., 2008; Li, 2013; Li et al., 2012; Montanie et al., 1993; Zhang et al., 2004). Viral infection in host cells is mediated by the initial attachment of a virus to a receptor on the cell surface, which is required for successful entry into the cell and the initiation of infection (Marsh and Helenius, 2006). The receptors of mammalian and Muscovy duck reoviruses
have been reported (Barton et al., 2001; Bernardo and Terence, 2011; Co et al., 1985; Konopka-Anstadt et al., 2014; Noseworthy et al., 1983; Paul and Lee, 1987; Sun, 2004). There are some reports on the interaction between reovirus and grass carp (Chen et al., 2013; Heng et al., 2011; Su et al., 2009), although the infectious mechanism of aquatic reovirus is still unclear. To date, no studies have been reported on reovirus receptors in aquatic organisms. Swimming crab reovirus (SCRV) is widespread and has caused the loss of millions of dollars in the crab cultivation industry in China (Li, 2013; Li et al., 2012). The mechanism of viral infection is poorly understood. In this study, a potential binding receptor of SCRV was screened by viral overlay protein binding assay (VOPBA) and identified by coimmunoprecipitation and an infection blocking test. 2. Materials and methods 2.1. SCRV virion purification
Abbreviations: VOPBA, viral overlay protein binding assay; SCRV, swimming crab Portunus trituberculatus reovirus; EDTA, ethylenediaminetetraacetic acid; PMSF, phenylmethylsulfonyl fluoride; min, minutes; h, hours; TM2, Tris-buffered saline with Mg2+ and Na+; TNE2, Tris-EDTA-buffered saline with 400 mM NaCl; PBS, phosphatebuffered saline; TEM, transmission electron microscopy; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; PVDF, polyvinylidene fluoride; PBST, PBS containing 0.1% Tween 20; RT, room temperature; HRP, horseradish peroxidase; OPD, Ophenylenediamine; NP40, Nonidet P-40; NETN, Tris-EDTA-buffered saline with NP40; FITC, fluorescein isothiocyanate. ⁎ Corresponding author at: Department of Marine Sciences, Ningbo University, Fenghua Road No. 818, 315211 Ningbo, PR China. E-mail address: [email protected] (D. Li). 1 J.F. and D.L. contributed equally to this work.
http://dx.doi.org/10.1016/j.aquaculture.2015.06.001 0044-8486/© 2015 Elsevier B.V. All rights reserved.
Intact SCRV virions were purified as described previously with some modifications (Li, 2013; Xie et al., 2005) from the tissues of artificially infected P. trituberculatus. Briefly, tissues were homogenized in precooled TNE2 buffer [50 mM Tris–HCl; 400 mM NaCl; 5 mM ethylenediaminetetraacetic acid (EDTA), pH 8.5; and 1 mM phenylmethylsulfonyl fluoride (PMSF), which was freshly added] at 4 °C and then centrifuged at 8000 × g for 10 minutes (min) at 4 °C. The collected supernatant fluid was centrifuged again as described above. The supernatant was then collected and centrifuged at 38,000 × g for 1.5 hours (h) at 4 °C. The sediment was immersed in TNE2 buffer containing 1 mM PMSF at
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2.4. Mass spectrometry (MS) analysis of the SCRV-binding protein To investigate the elements comprising the identified SCRV-binding protein, a protein band that corresponded to the viral binding band on the VOPBA was excised from the 8% SDS-PAGE gel and sent to Guangzhou Fitgene Biotechnology Company Limited (PR China) for Micromass Maldi Time-of-Flight Mass Spectrometry (MALDI-TOF-MS) analysis. To confirm their validity, assays including gel excision and MALDI-TOF-MS were repeated twice. 2.5. Preparation of polyclonal antibodies against the SCRV-binding protein
Fig. 1. Electron micrograph of purified SCRV virions negatively stained with 2% uranyl acetate.
4 °C overnight. The sediment was gently resuspended and then centrifuged at 3500 ×g for 5 min at 4 °C. The supernatant was centrifuged at 38,000 × g for 1.5 h at 4 °C. Following this, the upper loose pellet was rinsed out carefully in TM2 buffer (50 mM Tris–HCl; 10 mM MgCl2; 1.5% NaCl; and 1 mM PMSF, pH 7.5–7.6) at 4 °C, and the lower white pellet was immersed again in TNE2 buffer at 4 °C overnight as described above. After centrifugation at 5000 × g for 5 min, the virus particles were sedimented by centrifugation at 38,000 × g for 1.5 h at 4 °C and then resuspended in phosphate-buffered saline (PBS, 8 g of NaCl, 0.2 g of KCl, 1.44 g of NaHPO4 and 0.24 g of KH2PO4, dissolved in 1 l distilled water at pH 7.4) and stored at −80 °C until use. The purified virus sample was detected by negative staining under a transmission electron microscope (TEM).
The SCRV-binding protein identified from swimming crab gill was excised from the 8% SDS-PAGE gel. After homogenization and suspension in 0.9% NaCl, the mixture was emulsified in an equal volume of complete Freund's adjuvant (Sigma) and was used to subcutaneously immunize ICR mice. After 2 weeks, the first booster immunization was administered to each mouse, which included the protein suspended in incomplete Freund's adjuvant (Sigma). The other two booster injections were given at 1-week intervals. Mouse sera were obtained 7 days after the last injection and stored at − 80 °C for later use. Non-immunized mouse serum was also prepared as a control. The specificity and presence of murine antibodies against the SCRV-binding protein were identified by western blot. The titers of the mouse sera were determined by ELISA according to a previous method (Yang et al., 2013). 2.6. Identification of the SCRV-binding protein by western blot analysis Mouse membrane proteins were separated on 8% SDS-PAGE gels and electrophoretically transferred to PVDF membranes (Millipore). Mouse anti-SCRV-binding protein polyclonal sera and a commercial mouse anti-β-tubulin antibody (purchased based on the MS results) were used as primary antibodies in western blot analysis. 2.7. Coimmunoprecipitation assays
2.2. Membrane protein preparation Cell membrane preparations were acquired as previously described with minor modifications (Jindadamrongwech et al., 2004). Briefly, healthy swimming crab was dissected, and gill tissue was homogenized in 10 volumes of ice-cold buffer M (100 mM Tris–HCl, pH 8.0; 2 mM MgCl2; 1 mM EDTA; 0.2% Triton X-100; and 1 mM PMSF). Nuclei and cell debris were removed by centrifugation at 600 × g for 3 min at 4 °C. The supernatant was then centrifuged at 6000 × g for 5 min at 4 °C to remove membranous organelles and then again at 35,000 × g for 2 h to pellet membrane proteins. The pellet was resuspended in buffer M and stored at −80 °C for later use. 2.3. Virus overlay protein binding assay (VOPBA) Membrane proteins were separated on an 8% gel using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a 0.45 μm polyvinylidene fluoride (PVDF) membrane (Millipore). The membrane was blocked overnight at 4 °C in 5% lowfat milk in PBST (PBS containing 0.5% Tween-20) and washed three times with PBST. The membrane was then incubated with 12.9 μg purified SCRV in PBS for 1 h at room temperature (RT). Following this, the membrane was washed three times with PBST and incubated with a specific chicken polyclonal anti-SCRV antibody at room temperature for 2 h. After washing three times with PBST, the membrane was incubated with horseradish peroxidase (HRP)-conjugated sheep antichicken IgG (ComWin Biotech, CWBIO) (diluted 1:5000 in 0.5% skim milk in PBST) for 1 h at RT. After three washes with PBST, the viral binding bands on the membrane were developed with O-phenylenediamine (OPD, Invitrogen) and then photographed.
The binding activity of tubulin to SCRV was detected by coimmunoprecipitation analysis. Coimmunoprecipitation assays were carried out according to a previously described method (Li et al., 2007) with some modifications. A 500 μl aliquot of membrane protein from swimming crab gill was incubated with 30 μl purified SCRV on a rocker for 8 h at 4 °C. A 10 μl aliquot of either mouse anti-tubulin polyclonal antibody or IgG against an irrelevant protein (as a negative control) was added to each tube and incubated overnight at 4 °C. Subsequently, 60 μl of agarose beads conjugated with protein A-G (Santa Cruz) was added to the mixture and incubated for 3 h at 4 °C with gentle rocking. Following this, the agarose beads were collected by centrifugation and washed four times with high-salt NETN buffer (20 mM Tris–HCl, 0.5% NP-40, 500 mM NaCl, 1 mM EDTA, 25 μg/ml PMSF, pH 7.6) and once with NETN buffer (20 mM Tris–HCl, 0.5% NP-40, 100 mM NaCl, 1 mM EDTA, 25 μg/ml PMSF, pH 7.6). The bound proteins were dissociated from the antibodies by boiling in loading buffer for 4 min and were then separated on 12% SDS-PAGE gels. SCRV was then detected by standard western blotting techniques with an anti-SCRV polyclonal antibody. Similarly, an immunoprecipitation of tubulin with the anti-SCRV polyclonal antibody was performed. Either 500 μl membrane protein from swimming crab gill or 500 μl PBS (as a negative control) was incubated with 30 μl purified SCRV on a rocker for 8 h at 4 °C. A 10 μl aliquot of mouse anti-SCRV polyclonal antibody was added to each tube and incubated overnight at 4 °C. Subsequently, a process was performed following the steps of the immunoprecipitation assay on SCRV, except that bound proteins were separated on 8% SDS-PAGE gels, and tubulin was detected by standard western blotting techniques with an antitubulin polyclonal antibody.
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2.8. SCRV infection inhibition assay in vitro A virus infection inhibition assay was carried out in vitro with hemocytes from swimming crab. Hemocyte suspensions were placed into coated 96-well cell culture (BIOFIL, Canada) plates with either antitubulin polyclonal antibodies (1/10, 1/20 and 1/40 dilutions) or PBS (virus control) for 1 h at RT with gentle shaking. Simultaneously, SCRV was labeled with fluorescein isothiocyanate (FITC) as previously described (Li et al., 2007). Briefly, purified virions were incubated in 1 mg/ml FITC at room temperature for 1 h, followed by centrifugation at 10,000 × g for 10 min. Pellets of the labeled virions were resuspended in TM2 buffer (50 mM Tris–HCl, 10 mM MgCl2, 1.5% NaCl, pH 7.5) and then sedimented again by centrifugation at 10,000 × g for 10 min to remove free FITC. After washing three times, labeled virions were suspended in PBS. FITC-labeled SCRV (FITC-SCR) was added to each well and incubated for 30 min at RT with gentle shaking. Subsequently, the mixtures were removed, and unbound SCRV was washed off with PBS. The treated cells were observed under a microscope.
3. Results 3.1. Virus purification Fig. 2. VOPBA analysis of SCRV binding to P. Trituberculatus cell membrane proteins. (A) SDS-PAGE profile of P. Trituberculatus cell membrane proteins. (B) Detection of the positions of the SCRV-binding proteins by VOPBA. The prominent binding bands with molecular weights of approximately 550 kDa are indicated with black arrows on the right.
Purified SCRV was negatively stained with 2% uranyl acetate and observed under a transmission electron microscope (TEM). The results indicated that the virions were pure and intact (Fig. 1).
Fig. 3. Mass spectrographic analysis of the SCRV-binding protein for the first time. (A) Fingerprint of the protein; matched peaks are marked by asterisks. (B) Mass spectrographic analysis of the fingerprint of the protein using Mascot; protein scores greater than 41 are significant (p b 0.05). Accession information is as follows. NCBI accession no: gi|338224308, score: 48, description: beta-I tubulin (Scylla paramamosain).
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Fig. 4. Mass spectrographic analysis of the SCRV-binding protein for the second time. (A) Fingerprint of the protein; matched peaks are marked by asterisks. (B) Mass spectrographic analysis of the fingerprint of the protein using Mascot; protein scores greater than 47 are significant (p b 0.05). Accession information is as follows. NCBI accession no: gi|338224308, score: 48, description: beta-I tubulin (Scylla paramamosain).
3.2. Screening of the virus binding protein in swimming crab gill cells by VOPBA
3.5. Co-immunoprecipitation assay confirms that swimming crab tubulin interacts with SCRV proteins
Swimming crab gill cell-membrane proteins were prepared and separated by SDS-PAGE (Fig. 2A, Lane 1) and then transferred onto PVDF membranes, followed by incubation with SCRV. The membranes were subsequently incubated with polyclonal antibodies against SCRV, and HRP-conjugated antibody was added to visualize the reactive bands. The VOPBA results showed that SCRV could specifically bind to a swimming crab cell membrane protein with a molecular mass of approximately 550 kDa (Fig. 2B, Lane 1).
To demonstrate the interaction between tubulin and SCRV protein, a coimmunoprecipitation of SCRV and swimming crab tubulin was performed. The results showed that SCRV was precipitated with an Ab against tubulin as a membrane protein of swimming crab, while SCRV could not be precipitated with an Ab against an irrelevant protein (Fig. 6). In turn, tubulin and SCRV were both immunoprecipitated with an anti-SCRV Ab.
3.6. Neutralization of cell surface tubulin blocked SCRV infection in vitro 3.3. MALDI-TOF MS–MS analysis Based on searching NCBI BLAST with the peptide mass fingerprints, MALDI-TOF analysis of the 550 kDa protein twice exhibited homology to β-tubulin (Figs. 3, 4).
To test if tubulin plays a role in mediating the infection of SCRV, infection-blocking assays were carried out on hemocytes of P. Trituberculatus in vitro. Hemocytes pretreated with different dilutions of anti-tubulin polyclonal antibodies were infected with SCRV (Fig. 7). Hemocytes pretreated with PBS were employed as a control. The results
3.4. Western blot analysis To verify that the 550 kDa SCRV-binding protein was tubulin, mouse membrane proteins were respectively separated on 8% SDSPAGE gels and electrophoretically transferred to 0.45 μm PVDF membranes. Mouse anti-SCRV-binding-protein polyclonal sera and a commercial mouse anti-β-tubulin antibody were used as primary antibodies respectively in western blot analysis. The results showed that the immunized sera against the 550 kDa SCRV-binding protein, coincident with the commercial mouse anti-β-tubulin antibody (Fig. 5A, lane 1), could specifically recognize mouse tubulin (Fig. 5B, lane 1).
Fig. 5. Identification of SCRV-binding protein by western blot analysis. (A) A commercial mouse anti-β-tubulin antibody was used as a primary antibody in western blot analysis. Lane 1: mouse membrane proteins + mouse-anti-β-tubulin antibody. (B) Mouse antiSCRV-binding-protein polyclonal sera were used as primary antibodies in western blot analysis. Lane 1: mouse membrane proteins + immunized sera.
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Fig. 6. Co-immunoprecipitation of SCRV and tubulin. Membrane proteins from swimming crabs were mixed with SCRV and immunoprecipitated with anti-tubulin antibodies (lane 1) or an antibody against an irrelevant protein (negative control, lane 3), in addition to protein A-G agarose. The immunoprecipitates were subjected to western blot analysis using antibodies against SCRV as outlined in Materials and methods section. Lane 1: swimming crab membrane proteins + SCRV + anti-tubulin Ab. Lane 2: SCRV. Lane 3: swimming crab membrane proteins + SCRV + Ab against irrelevant protein.
demonstrated that the neutralization of cell surface tubulin with antitubulin antibodies (1/10 dilution) significantly inhibited the infection of SCRV in vitro. 4. Discussion The swimming crab P. Trituberculatus (Miers, 1876) (Crustacea: Decapoda: Brachyura), also known as gazami crab, horse crab or Japanese blue crab, is the most widely fished species of crab in the world, producing a yield of over 300,000 tons annually. In recent years, P. trituberculatus crab farms in China suffered from an outbreak epidemic that led to mass mortality, which heavily impacted the industry. SCRV is a primary lethal pathogen, which spreads widely and causes losses of millions of dollars in the crab cultivation industry annually. The similarity of nucleic acid sequences between SCRV and mud crab S. Serrata reovirus was 99%, although SCRV cannot infect mud crab (unpublished). No effective strategy for preventing or controlling SCRV is available. Studies on the interactions between SCRV and swimming crab cells, especially identification of the virus receptor, are critical for understanding its infectious mechanism, which has considerable implications for developing strategies to prevent and control infection. The purpose of this study was to screen for cell receptors of SCRV. Viral overlay protein binding assay (VOPBA) has been used
to identify the receptor proteins of various viruses, such as dengue virus (Jindadamrongwech and Smith, 2004), pancreatic necrosis virus (Orpetveit et al., 2008), fowl adenovirus (Taharaguchi et al., 2007), bovine adenovirus (Li et al., 2009), and hepatitis E virus (Zhang et al., 2011). In a VOPBA, membrane proteins are separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto polyvinylidene fluoride (PVDF) membranes; the membranes containing transferred proteins are incubated with viral particles and tested with virus-specific antibodies (Yang et al., 2013). This assay is sensitive enough to detect 5 mg of purified glycophorin or to detect the binding of less than 100 mg of protein from a crude membrane preparation (Gershoni et al., 1986). In this study, by applying VOPBA, an approximately 550 kDa protein in the membrane fraction of swimming crab gill cells was found to interact with SCRV. Twice, the results of MALDI-TOF analysis of the 550 kDa protein exhibited homology to β-tubulin. To determine whether the 550 kDa protein was tubulin, the SCRV-binding protein was identified using western blot analysis (Fig. 5). The results clearly indicated that antibodies against the 550 kDa swimming crab protein could specifically recognize the mouse protein coincident with the ability of a commercial mouse anti-β-tubulin antibody. These results indicated that the SCRV-binding protein is tubulin and suggested that tubulin may exist either as a multimeric form or bound to GTP and nucleotides in
Fig. 7. Infection inhibition assay using anti-tubulin polyclonal antibody in vitro. Hemocytes were incubated with different dilutions of anti-tubulin polyclonal antibodies for 1 h at RT and then FITC-SCRV was added to each well and incubated for 30 min at RT with gentle shaking. Cells were viewed under a microscope. Scale bar = 500 μm.
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swimming crab. Tubulin heterodimers have long been known to be involved in the assembly and transport of various viruses (Hong and Ng, 1987; Ng and Hong, 1989; Ogino et al., 2003; Ruthel et al., 2005; Smith and Enquist, 2002). It has been reported that DENV2 through DENV2E binds directly to tubulin or to a tubulin-like protein in C6/36 mosquito cells (Chee and Sazaly, 2004). Reoviruses have been reported to interact with microtubules (Babiss et al., 1979; Heinemann et al., 2011; Kim et al., 2004; Kobayashi et al., 2009; Parker et al., 2002). Reovirus cell entry requires functional microtubules, which facilitate the targeting of reovirus to acidified endosomes for viral disassembly. Microtubule inhibitors can block various stages of the reovirus life cycle (Huang et al., 2011; Mainou et al., 2013). Generally, tubulin is considered to be an intracellular protein and is a cytoskeletal element that is essential for intracellular transport and cell division in all eukaryotes (Nogales et al., 1998). Earlier, we were confused in the face of our results. Therefore, we consulted a number of relevant studies and eventually obtained references (Por et al., 1991; Quillen et al., 1985; Rubin et al., 1982; Stephens, 1981) which reported that tubulin can also act as a cell surface protein. Stephens (1981) identified that tubulin was a membrane protein. Rubin et al. (1982) reported that tubulin was a major cell surface protein in human lymphoid cells of leukemic origin. Quillen et al. (1985) found that tubulin can exist either in or on the outer surfaces of living cells. Por et al. (1991) provided evidence for the presence of tubulin on the surface of a human monocyte-like cell line, U937. Coimmunoprecipitation of tubulin and SCRV identified the interaction between them. To further confirm the role of tubulin in mediating the infection of SCRV, infection-blocking assays were carried out on hemocytes of P. Trituberculatus in vitro. Antibodies against tubulin were able to inhibit virus entry into cells. All of the above findings suggest that tubulin mediates the infection of SCRV in swimming crab. 5. Conclusion This is the first study to reveal that tubulin serves as a receptor for SCRV in swimming crab using VOPBA, mass spectrometry, western blot, coimmunoprecipitation and infectivity reduction via anti-tubulin antibodies. It proposes a new drug target for preventing or controlling SCRV. This is also the first study to find that tubulin serves as a receptor for viruses and to identify a reovirus receptor in aquatic organisms. Acknowledgments This work was financially supported by the Spark Program of China (no. 2012GA701006), the Zhejiang Provincial Natural Science Foundation (no. LY12C19004), the Science Foundation of the Department of Education of Zhejiang Province (Y201327815), the Agricultural Program of Ningbo, Zhejiang, China (no. 2011C10004), and the Open Foundation of Key Disciplines of Zhejiang Province (E02009124200) and was sponsored by the K.C. Wong Magna Fund of Ningbo University. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. References Attoui, H., Belhouchet, M., Mohd, J.F., Biagini, P., Cantaloube, J.F., de, M.P., de, L.X., 2005. Expansion of family Reoviridae to include nine-segmented dsRNA viruses: isolation and characterization of a new virus designated Aedes pseudoscutellaris reovirus assigned to a proposed genus (Dinovernavirus). Virology 343, 212–223. Babiss, L.E., Luftig, R.B., Weatherbee, J.A., Weihing, R.R., Ray, U.R., Fields, B.N., 1979. Reovirus serotypes 1 and 3 differ in their in vitro association with microtubules. J. Virol. 30, 863–874. Barton, E.S., Forrest, C., Connolly, J.L., 2001. Junction adhesion molecule is a receptor for reovirus. Cell 104, 441–451. Bernardo, A.M., Terence, S.D., 2011. Src kinase mediates productive endocytic sorting of reovirus during cell entry. J. Virol. 85, 3203–3205.
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