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• W2082834676 abstract "Reoviruses are double-stranded RNA viruses that infect the mammalian respiratory and gastrointestinal tract. Reovirus infection elicits production of type I interferons (IFNs), which trigger antiviral pathways through the induction of interferon-stimulated genes (ISGs). Although hundreds of ISGs have been identified, the functions of many of these genes are unknown. The interferon-inducible transmembrane (IFITM) proteins are one class of ISGs that restrict the cell entry of some enveloped viruses, including influenza A virus. One family member, IFITM3, localizes to late endosomes, where reoviruses undergo proteolytic disassembly; therefore, we sought to determine whether IFITM3 also restricts reovirus entry. IFITM3-expressing cell lines were less susceptible to infection by reovirus, as they exhibited significantly lower percentages of infected cells in comparison to control cells. Reovirus replication was also significantly reduced in IFITM3-expressing cells. Additionally, cells expressing an shRNA targeting IFITM3 exhibited a smaller decrease in infection after IFN treatment than the control cells, indicating that endogenous IFITM3 restricts reovirus infection. However, IFITM3 did not restrict entry of reovirus infectious subvirion particles (ISVPs), which do not require endosomal proteolysis, indicating that restriction occurs in the endocytic pathway. Proteolysis of outer capsid protein μ1 was delayed in IFITM3-expressing cells in comparison to control cells, suggesting that IFITM3 modulates the function of late endosomal compartments either by reducing the activity of endosomal proteases or delaying the proteolytic processing of virions. These data provide the first evidence that IFITM3 restricts infection by a nonenveloped virus and suggest that IFITM3 targets an increasing number of viruses through a shared requirement for endosomes during cell entry.Background: The interferon-stimulated gene (ISG) IFITM3 restricts endosomal entry of enveloped viruses.Results: IFITM3 also restricts entry of reovirus, a nonenveloped virus.Conclusion: IFITM3 alters endosomal function, either by delaying acidification or modulating proteolytic activity.Significance: IFITM3 may restrict other clinically relevant nonenveloped viruses that require endosomes for entry. Reoviruses are double-stranded RNA viruses that infect the mammalian respiratory and gastrointestinal tract. Reovirus infection elicits production of type I interferons (IFNs), which trigger antiviral pathways through the induction of interferon-stimulated genes (ISGs). Although hundreds of ISGs have been identified, the functions of many of these genes are unknown. The interferon-inducible transmembrane (IFITM) proteins are one class of ISGs that restrict the cell entry of some enveloped viruses, including influenza A virus. One family member, IFITM3, localizes to late endosomes, where reoviruses undergo proteolytic disassembly; therefore, we sought to determine whether IFITM3 also restricts reovirus entry. IFITM3-expressing cell lines were less susceptible to infection by reovirus, as they exhibited significantly lower percentages of infected cells in comparison to control cells. Reovirus replication was also significantly reduced in IFITM3-expressing cells. Additionally, cells expressing an shRNA targeting IFITM3 exhibited a smaller decrease in infection after IFN treatment than the control cells, indicating that endogenous IFITM3 restricts reovirus infection. However, IFITM3 did not restrict entry of reovirus infectious subvirion particles (ISVPs), which do not require endosomal proteolysis, indicating that restriction occurs in the endocytic pathway. Proteolysis of outer capsid protein μ1 was delayed in IFITM3-expressing cells in comparison to control cells, suggesting that IFITM3 modulates the function of late endosomal compartments either by reducing the activity of endosomal proteases or delaying the proteolytic processing of virions. These data provide the first evidence that IFITM3 restricts infection by a nonenveloped virus and suggest that IFITM3 targets an increasing number of viruses through a shared requirement for endosomes during cell entry. Background: The interferon-stimulated gene (ISG) IFITM3 restricts endosomal entry of enveloped viruses. Results: IFITM3 also restricts entry of reovirus, a nonenveloped virus. Conclusion: IFITM3 alters endosomal function, either by delaying acidification or modulating proteolytic activity. Significance: IFITM3 may restrict other clinically relevant nonenveloped viruses that require endosomes for entry. The innate immune system must recognize signatures of pathogen infection (such as the presence of double-stranded RNA (dsRNA) for RNA viruses) and then initiate mechanisms that hinder the ability of the pathogen to replicate (1Mogensen T.H. Pathogen recognition and inflammatory signaling in innate immune defenses.Clin. Micro. Rev. 2009; 22: 240-273Crossref PubMed Scopus (1925) Google Scholar). Innate detection mechanisms must, practically, be nonspecific. However, after initial detection, the restriction mechanisms, exemplified by the type I interferon (IFN) system, need not be general. Initiating a wide variety of anti-pathogen responses may give an organism the greatest chance to contain an infection whether or not each individual mechanism is effective against the specific pathogen. Type I IFNs (IFN-α/β) are major antiviral cytokines and can be induced by several different pattern recognition receptors, including Toll-like receptors and cytoplasmic RIG-I-like helicases (2Jensen S. Thomsen A.R. Sensing of RNA viruses. A review of innate immune receptors involved in recognizing RNA virus invasion.J. Virol. 2012; 86: 2900-2910Crossref PubMed Scopus (403) Google Scholar). After induction, type I IFNs signal through the type I IFN receptor (IFNAR) to induce a number of ISGs. 2The abbreviations used are: ISG, interferon-stimulated gene; IFITM, interferon-inducible transmembrane protein; shIFITM3, short hairpin RNA targeting IFITM3; T1L, reovirus type 1 Lang; T3D, reovirus type 3 Dearing; rsT3D, T3D reovirus strain; ISVP, infectious subvirion particle; EGFP, enhanced GFP; RILP, Rab-interacting lysosomal protein; m.o.i., multiplicity(s) of infection; pfu, plaque forming units; qPCR, quantitative reverse transcriptase PCR; A546, Alexa Fluor 546; Bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol. Hundreds of ISGs have been identified, of which only a handful have a characterized function (3Borden E.C. Williams B.R. Interferon-stimulated genes and their protein products. what and how?.J. Interferon Cytokine Res. 2011; 31: 1-4Crossref PubMed Scopus (42) Google Scholar). Some induce broad antiviral responses, such as inhibition of cellular protein synthesis or degradation of RNA (4Farrell P.J. Balkow K. Hunt T. Jackson R.J. Trachsel H. Phosphorylation of initiation factor eIF-2 and the control of reticulocyte protein synthesis.Cell. 1977; 11: 187-200Abstract Full Text PDF PubMed Scopus (446) Google Scholar, 5Ratner L. Wiegand R.C. Farrell P.J. Sen G.C. Cabrer B. Lengyel P. Interferon, double-stranded RNA, and RNA degradation. Fractionation of the endonuclease INT system into two macromolecular components. Role of a small molecule in nuclease activation.Biochem. Biophys. Res. Commun. 1978; 81: 947-954Crossref PubMed Scopus (66) Google Scholar). However, other ISGs, such as TRIM79α, target specific viruses (6Taylor R.T. Lubick K.J. Robertson S.J. Broughton J.P. Bloom M.E. Bresnahan W.A. Best S.M. TRIM79α, an interferon-stimulated gene product, restricts tick-borne encephalitis virus replication by degrading the viral RNA polymerase.Cell Host Microbe. 2011; 10: 185-196Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Understanding how individual ISGs restrict virus infection is of broad interest for designing antiviral therapeutics that can be targeted to specific or more general classes of pathogens. One family of ISGs that is known to restrict specific classes of virus is the interferon-inducible transmembrane proteins (IFITM), which are conserved across numerous vertebrate species. In humans, the IFITM1, -2, -3, and -5 genes are located on chromosome 11 and have previously been shown to function in cell signaling, adhesion, and bone mineralization (7Evans S.S. Collea R.P. Leasure J.A. Lee D.B. IFN-α induces homotypic adhesion and Leu-13 expression in human B lymphoid cells.J. Immunol. 1993; 150: 736-747PubMed Google Scholar8Imai T. Yoshie O. C33 antigen and M38 antigen recognized by monoclonal antibodies inhibitory to syncytium formation by human T cell leukemia virus type 1 are both members of the transmembrane 4 superfamily and associate with each other and with CD4 or CD8 in T cells.J. Immunol. 1993; 151: 6470-6481PubMed Google Scholar, 9Lange U.C. Saitou M. Western P.S. Barton S.C. Surani M.A. The fragilis interferon-inducible gene family of transmembrane proteins is associated with germ cell specification in mice.BMC Dev. Biol. 2003; 3: 1Crossref PubMed Scopus (116) Google Scholar10Smith R.A. Young J. Weis J.J. Weis J.H. Expression of the mouse fragilis gene products in immune cells and association with receptor signaling complexes.Genes Immun. 2006; 7: 113-121Crossref PubMed Scopus (43) Google Scholar). These proteins contain two intramembrane domains linked by a highly conserved intracellular loop, features shared among the more than 200 CD225 protein family members. Family members IFITM1, -2, and -3 restrict the cell entry of influenza A, flaviviruses, dengue virus, West Nile virus, severe acute respiratory syndrome coronavirus, and filoviruses (11Brass A.L. Huang I.-C. Benita Y. John S.P. Krishnan M.N. Feeley E.M. Ryan B.J. Weyer J.L. van der Weyden L. Fikrig E. Adams D.J. Xavier R.J. Farzan M. Elledge S.J. The IFITM proteins mediate cellular resistance to influenza A H1N1 virus, West Nile virus, and dengue virus.Cell. 2009; 139: 1243-1254Abstract Full Text Full Text PDF PubMed Scopus (956) Google Scholar, 12Huang I.-C. Bailey C.C. Weyer J.L. Radoshitzky S.R. Becker M.M. Chiang J.J. Brass A.L. Ahmed A. A Chi X. Dong L. Longobardi L.E. Boltz D. Kuhn J.H. Elledge S.J. Bavari S. Denison M.R. Choe H. Farzan M. Distinct patterns of IFITM-mediated restriction of filoviruses, SARS coronavirus, and influenza A virus.PLoS Pathog. 2011; 7: e1001258Crossref PubMed Scopus (435) Google Scholar13Jiang D. Weidner J.M. Qing M. Pan X.-B. Guo H. Xu C. Zhang X. Birk A. Chang J. Shi P.-Y. Block T.M. Guo J.-T. Identification of five interferon-induced cellular proteins that inhibit west nile virus and dengue virus infections.J. Virol. 2010; 84: 8332-8341Crossref PubMed Scopus (268) Google Scholar). These viruses are enveloped and enter cells via membrane fusion in endosomal compartments; indeed, the IFITM proteins restrict viruses independent of receptor usage but are dependent upon processes that occur in late endosomes (12Huang I.-C. Bailey C.C. Weyer J.L. Radoshitzky S.R. Becker M.M. Chiang J.J. Brass A.L. Ahmed A. A Chi X. Dong L. Longobardi L.E. Boltz D. Kuhn J.H. Elledge S.J. Bavari S. Denison M.R. Choe H. Farzan M. Distinct patterns of IFITM-mediated restriction of filoviruses, SARS coronavirus, and influenza A virus.PLoS Pathog. 2011; 7: e1001258Crossref PubMed Scopus (435) Google Scholar, 14Feeley E.M. Sims J.S. John S.P. Chin C.R. Pertel T. Chen L.-M. Gaiha G.D. Ryan B.J. Donis R.O. Elledge S.J. Brass A.L. IFITM3 inhibits influenza A virus infection by preventing cytosolic entry.PLoS Pathog. 2011; 7: e1002337Crossref PubMed Scopus (284) Google Scholar). IFITM3 is the most potent IFITM family member in restricting influenza A replication in cell culture (11Brass A.L. Huang I.-C. Benita Y. John S.P. Krishnan M.N. Feeley E.M. Ryan B.J. Weyer J.L. van der Weyden L. Fikrig E. Adams D.J. Xavier R.J. Farzan M. Elledge S.J. The IFITM proteins mediate cellular resistance to influenza A H1N1 virus, West Nile virus, and dengue virus.Cell. 2009; 139: 1243-1254Abstract Full Text Full Text PDF PubMed Scopus (956) Google Scholar), and recently it has been shown to reduce the pathogenesis of influenza in mice (15Bailey C.C. Huang I.-C. Kam C. Farzan M. Ifitm3 limits the severity of acute influenza in mice.PLoS Pathogens. 2012; 8: e1002909Crossref PubMed Scopus (176) Google Scholar, 16Everitt A.R. Clare S. Pertel T. John S.P. Wash R.S. Smith S.E. Chin C.R. Feeley E.M. Sims J.S. Adams D.J. Wise H.M. Kane L. Goulding D. Digard P. Anttila V. Baillie J.K. Walsh T.S. Hume D.A. Palotie A. Xue Y. Colonna V. Tyler-Smith C. Dunning J. Gordon S.B. GenISIS Investigators, MOSAIC Investigators Smyth R.L. Openshaw P.J. Dougan G. Brass A.L. Kellam P. IFITM3 restricts the morbidity and mortality associated with influenza.Nature. 2012; 484: 519-523Crossref PubMed Scopus (553) Google Scholar) and in infected humans (16Everitt A.R. Clare S. Pertel T. John S.P. Wash R.S. Smith S.E. Chin C.R. Feeley E.M. Sims J.S. Adams D.J. Wise H.M. Kane L. Goulding D. Digard P. Anttila V. Baillie J.K. Walsh T.S. Hume D.A. Palotie A. Xue Y. Colonna V. Tyler-Smith C. Dunning J. Gordon S.B. GenISIS Investigators, MOSAIC Investigators Smyth R.L. Openshaw P.J. Dougan G. Brass A.L. Kellam P. IFITM3 restricts the morbidity and mortality associated with influenza.Nature. 2012; 484: 519-523Crossref PubMed Scopus (553) Google Scholar). Our recent data suggest a model where IFITM3 prevents influenza A virus fusion, thereby trapping virions within the endocytic pathway ultimately leading to their destruction in lysosomes and autolysosomes (14Feeley E.M. Sims J.S. John S.P. Chin C.R. Pertel T. Chen L.-M. Gaiha G.D. Ryan B.J. Donis R.O. Elledge S.J. Brass A.L. IFITM3 inhibits influenza A virus infection by preventing cytosolic entry.PLoS Pathog. 2011; 7: e1002337Crossref PubMed Scopus (284) Google Scholar). Whether IFITM3 restricts non-enveloped viruses that also require endosomal access for cell entry is not known. Mammalian orthoreoviruses (reoviruses) are non-enveloped viruses containing a segmented genome composed of dsRNA (17.Schiff, L. A., Nibert, M. L., Tyler, K. L., in Field's Virology (Knipe, D. M., Howley, P. M., eds.) 5th Ed., pp. 1853–1915, Lippincott Williams and Wilkins ET, Philadelphia,Google Scholar). Reoviruses utilize a multistep entry process, first binding to cell surface carbohydrates before engagement with the proteinaceous receptor junction adhesion molecule-A (JAM-A) (18Barton E.S. Forrest J.C. Connolly J.L. Chappell J.D. Liu Y. Schnell F.J. Nusrat A. Parkos C.A. Dermody T.S. Junction adhesion molecule is a receptor for reovirus.Cell. 2001; 104: 441-451Abstract Full Text Full Text PDF PubMed Scopus (516) Google Scholar). Subsequent interactions with β1-integrins target virions to late endosomal compartments (19Maginnis M.S. Forrest J.C. Kopecky-Bromberg S.A. Dickeson S.K. Santoro S.A. Zutter M.M. Nemerow G.R. Bergelson J.M. Dermody T.S. β1 integrin mediates internalization of mammalian reovirus.J. Virol. 2006; 80: 2760-2770Crossref PubMed Scopus (127) Google Scholar), where the actions of acid-dependent cathepsin proteases are required for uncoating (20Ebert D.H. Deussing J. Peters C. Dermody T.S. Cathepsin L and cathepsin B mediate reovirus disassembly in murine fibroblast cells.J. Biol. Chem. 2002; 277: 24609-24617Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar). These proteases cleave outer capsid protein σ3, revealing the membrane penetration protein μ1 (21Sturzenbecker L.J. Nibert M. Furlong D. Fields B.N. Intracellular digestion of reovirus particles requires a low pH and is an essential step in the viral infectious cycle.J. Virol. 1987; 61: 2351-2361Crossref PubMed Google Scholar22Chandran K. Farsetta D.L. Nibert M.L. Strategy for nonenveloped virus entry. A hydrophobic conformer of the reovirus membrane penetration protein m1 mediates membrane disruption.J. Virol. 2002; 76: 9920-9933Crossref PubMed Scopus (141) Google Scholar, 23Chandran K. Parker J.S. Ehrlich M. Kirchhausen T. Nibert M.L. The delta region of outer-capsid protein m1 undergoes conformational change and release from reovirus particles during cell entry.J. Virol. 2003; 77: 13361-13375Crossref PubMed Scopus (78) Google Scholar, 24Nibert M.L. Odegard A.L. Agosto M.A. Chandran K. Schiff L.A. Putative autocleavage of reovirus m1 protein in concert with outer-capsid disassembly and activation for membrane permeabilization.J. Mol. Biol. 2005; 345: 461-474Crossref PubMed Scopus (73) Google Scholar25Odegard A.L. Chandran K. Zhang X. Parker J.S. Baker T.S. Nibert M.L. Putative autocleavage of outer capsid protein m1, allowing release of myristoylated peptide “μ1N during particle uncoating, is critical for cell entry by reovirus.J. Virol. 2004; 78: 8732-8745Crossref PubMed Scopus (97) Google Scholar). Cleavages of μ1 into the particle-associated fragments δ and φ and the dissociated fragment μ1N allow for conformational changes and subsequent membrane association of the viral capsid that mediates penetration of the viral core into the cytoplasm (23Chandran K. Parker J.S. Ehrlich M. Kirchhausen T. Nibert M.L. The delta region of outer-capsid protein m1 undergoes conformational change and release from reovirus particles during cell entry.J. Virol. 2003; 77: 13361-13375Crossref PubMed Scopus (78) Google Scholar, 26Nibert M.L. Fields B.N. A carboxyl-terminal fragment of protein m1/m1C is present in infectious subvirion particles of mammalian reoviruses and is proposed to have a role in penetration.J. Virol. 1992; 66: 6408-6418Crossref PubMed Google Scholar). Although reovirus particles traverse a number of endocytic compartments, correct targeting to late endosomes containing Rab7 and Rab9 is crucial for establishing a productive infection (27Mainou B.A. Dermody T.S. Transport to late endosomes is required for efficient reovirus infection.J. Virol. 2012; 86: 8346-8358Crossref PubMed Scopus (81) Google Scholar). Because IFITM3 also localizes to late endosomes, we sought to test whether IFITM3 restricts reovirus infection. In this study we found that IFITM3 expression significantly restricts infection and replication by reovirus. IFITM3-mediated restriction occurs at the level of endosomal penetration, as neither receptor binding nor RNA synthesis was affected. Targeting of reovirus particles to Rab7-containing late endosomes occurred with similar kinetics in the presence or absence of IFITM3. However, the kinetics of acidification and subsequent proteolysis of the reovirus capsid were delayed in IFITM3-expressing cells. Thus, IFITM3 likely alters the dynamics of endosomal uncoating, either leading to inefficient membrane penetration or lysosomal degradation of viral particles. This represents the first evidence that IFITM3 can restrict the infection of non-enveloped viruses that utilize endosome-dependent cell entry mechanisms and provides further mechanistic evidence for how IFITM3 restricts an increasing number of viruses by targeting a shared requirement for endosomes for cell entry. HeLa, U2OS, and A549 vector control and IFITM3- and shIFITM3-expressing cells were characterized previously (11Brass A.L. Huang I.-C. Benita Y. John S.P. Krishnan M.N. Feeley E.M. Ryan B.J. Weyer J.L. van der Weyden L. Fikrig E. Adams D.J. Xavier R.J. Farzan M. Elledge S.J. The IFITM proteins mediate cellular resistance to influenza A H1N1 virus, West Nile virus, and dengue virus.Cell. 2009; 139: 1243-1254Abstract Full Text Full Text PDF PubMed Scopus (956) Google Scholar, 14Feeley E.M. Sims J.S. John S.P. Chin C.R. Pertel T. Chen L.-M. Gaiha G.D. Ryan B.J. Donis R.O. Elledge S.J. Brass A.L. IFITM3 inhibits influenza A virus infection by preventing cytosolic entry.PLoS Pathog. 2011; 7: e1002337Crossref PubMed Scopus (284) Google Scholar) and were maintained in Dulbecco's modified Eagle's medium supplemented to contain 10% fetal bovine serum, 2 mm l-glutamine, 100 units/ml penicillin,100 μg/ml streptomycin, and 250 ng/ml amphotericin B (Sigma). L929 cells were maintained in Joklik's minimum essential medium supplemented to contain 5% fetal bovine serum, 2 mm l-glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin, and 25 ng/ml amphotericin B. Reovirus strains type 1 Lang (T1L) and type 3 Dearing (T3D) are laboratory stock. Reovirus strain rsT3D-σ1T249I was recovered by plasmid rescue (28Kobayashi T. Ooms L.S. Ikizler M. Chappell J.D. Dermody T.S. An improved reverse genetics system for mammalian orthoreoviruses.Virology. 2010; 398: 194-200Crossref PubMed Scopus (114) Google Scholar). rsT3D-σ1T249I is isogenic to T3D with the exception of a single amino acid substitution in the σ1 protein rendering it insensitive to protease cleavage and, therefore, capable of generating infectious subvirion particles (ISVPs) with infectivity equivalent to that of T3D virions (29Kobayashi T. Antar A.A. Boehme K.W. Danthi P. Eby E.A. Guglielmi K.M. Holm G.H. Johnson E.M. Maginnis M.S. Naik S. Skelton W.B. Wetzel J.D. Wilson G.J. Chappell J.D. Dermody T.S. A plasmid-based reverse genetics system for animal double-stranded RNA viruses.Cell Host Microbe. 2007; 1: 147-157Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar). Purified reovirus virions were generated using second or third passage L-cell lysates of twice-plaque-purified reovirus as described (30Furlong D.B. Nibert M.L. Fields B.N. Sigma 1 protein of mammalian reoviruses extends from the surfaces of viral particles.J. Virol. 1988; 62: 246-256Crossref PubMed Google Scholar). Viral particles were Freon-extracted from infected cell lysates, layered onto 1.2–1.4-g/cm3 CsCl gradients, and centrifuged at 62,000 × g for 18 h. Bands corresponding to virions (1.36 g/cm3) were collected and dialyzed in virion storage buffer (150 mm NaCl, 15 mm MgCl2, 10 mm Tris-HCl, pH 7.4). Concentrations of reovirus virions in purified preparations were determined from an equivalence of 1 absorbance unit at 260 nm equals 2.1 × 1012 virions (31Smith R.E. Zweerink H.J. Joklik W.K. Polypeptide components of virions, top component and cores of reovirus type 3.Virology. 1969; 39: 791-810Crossref PubMed Scopus (439) Google Scholar). Viral titer was determined by a plaque assay using murine L929 cells, and all indications of m.o.i. are based on L929 cell titer (32Virgin 4th, H.W. Bassel-Duby R. Fields B.N. Tyler K.L. Antibody protects against lethal infection with the neurally spreading reovirus type 3 (Dearing).J. Virol. 1988; 62: 4594-4604Crossref PubMed Google Scholar). ISVPs were generated as described (33Connolly J.L. Dermody T.S. Virion disassembly is required for apoptosis induced by reovirus.J. Virol. 2002; 76: 1632-1641Crossref PubMed Scopus (83) Google Scholar) and confirmed by SDS-PAGE and Coomassie Brilliant Blue staining. Guinea pig anti-reovirus σNS and rabbit anti-T3D antisera were generously provided by Dr. Terence S. Dermody (Vanderbilt University Medical Center). Plasmids encoding Rab7-EGFP and Rab-interacting lysosomal protein (RILP)-EGFP (27Mainou B.A. Dermody T.S. Transport to late endosomes is required for efficient reovirus infection.J. Virol. 2012; 86: 8346-8358Crossref PubMed Scopus (81) Google Scholar) and guinea pig anti-reovirus σNS antiserum were obtained from Dr. Terence S. Dermody (Vanderbilt University Medical Center). Reovirus virions were labeled with succinimidyl ester Alexa Fluor 546 (A546) or pHrodo S.E. (pHrodo; Invitrogen) as described previously (27Mainou B.A. Dermody T.S. Transport to late endosomes is required for efficient reovirus infection.J. Virol. 2012; 86: 8346-8358Crossref PubMed Scopus (81) Google Scholar, 34Fecek R.J. Busch R. Lin H. Pal K. Cunningham C.A. Cuff C.F. Production of Alexa Fluor 488-labeled reovirus and characterization of target cell binding, competence, and immunogenicity of labeled virions.J. Immunol. Methods. 2006; 314: 30-37Crossref PubMed Scopus (13) Google Scholar). Succinimidyl esters preferentially label reovirus proteins λ2, μ1, σ2, and σ3 (34Fecek R.J. Busch R. Lin H. Pal K. Cunningham C.A. Cuff C.F. Production of Alexa Fluor 488-labeled reovirus and characterization of target cell binding, competence, and immunogenicity of labeled virions.J. Immunol. Methods. 2006; 314: 30-37Crossref PubMed Scopus (13) Google Scholar). Reovirus particles (3 × 1012) were diluted into fresh 0.05 m sodium bicarbonate, pH 8.5, and incubated with 10 μm succinimidyl ester A546 or pHrodo at room temperature for 90 min in the dark. Virus particles were dialyzed against phosphate-buffered saline (PBS) at 4 °C overnight and stored at 4 °C. Cells (4 × 104) were grown in 24-well tissue culture plates and adsorbed with reovirus strains at various m.o.i. for 1 h at 4 °C. After adsorption, 0.5 ml of fresh medium was added, and the cells were incubated at 37 °C for 18 h. Cells were fixed with 2% paraformaldehyde for 30 min, washed twice with PBS, and permeabilized and blocked in PBS + 2% bovine serum albumin + 0.1% Triton-X100 (PBS-T) for at least 1 h at 4 °C. Cells were then incubated with guinea pig anti-reovirus σNS antiserum (1:1000) in PBS-T for at least 1 h at 4 °C, washed 3× with PBS-T, and incubated with an anti-guinea pig Alexa 568-conjugated antibody (1:2000) in PBS-T for at least 1 h at 4 °C. Cells were washed 3× with PBS and visualized using fluorescence microscopy. Reovirus antigen-positive cells were quantified by counting fluorescent cells in at least two random fields of view in triplicate wells at a magnification of 113×. Total cell number was quantified using background fluorescence. Cells (4 × 104) grown in 24-well tissue culture plates were adsorbed with reovirus strain T3D at an m.o.i. of 1 or 100 pfu/cell for 1 h at 4 °C. After adsorption, cells were washed 2× with PBS and incubated in 0.5 ml of fresh medium at 37 °C. After different time intervals, cells were freeze-thawed twice, and viral titer was determined by plaque assay. The viral yields were calculated by dividing viral titers at the indicated times by the viral titer at 0 h. Cells (4 × 104) grown in 24-well tissue culture plates were either mock-treated or treated with IFN-α (100 IU/ml) for 6 h before inoculation with either T1L or T3D at an m.o.i. of 10 pfu/cell for 1 h at 4 °C. After inoculation, 0.5 ml of fresh medium was added, and the cells were incubated at 37 °C for 18 h before performing a fluorescent focus assay. Alternatively, viral titers at various times were determined by plaque assay. Cells (4 × 104) grown in 24-well tissue culture plates were adsorbed with either rsT3D-σ1T249I virions or ISVPs at the indicated m.o.i. for 45 min at 4 °C. After adsorption, cells were either mock-treated or treated with 10 mm ammonium chloride and incubated at 37 °C for 18 h and analyzed by fluorescent focus assay. Cells (4 × 104) grown in 24-well tissue culture plates were inoculated with rsT3D at an m.o.i. of 25 pfu/cell for 1 h at 4 °C. The inoculum was removed, and the cells were washed 2× with PBS and incubated in fresh prewarmed media at 37 °C. At various times after adsorption, ammonium chloride was added to the medium for a final concentration of 25 mm. Cells were then incubated for 18 h post-adsorption, fixed with 2% paraformaldehyde, and stained for a fluorescent focus assay. Cells (5 × 105) grown in 60-mm dishes were inoculated with T3D at an m.o.i. of 100 pfu/cell for 1 h at 4 °C. The inoculum was removed, and the cells were washed 2× with PBS and incubated in fresh prewarmed media at 37 °C. At various times after adsorption, cells were scraped into 1 ml of ice-cold PBS and pelleted at 3000 × g for 5 min at 4 °C. The supernatant was aspirated, and the pellet was resuspended in 100 μl of ice-cold modified radioimmunoprecipitation assay buffer (50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 1 mm EDTA, 1% sodium deoxycholate, 1% IGEPAL CA-630, 1 mm PMSF) supplemented with protease inhibitor mixture (Roche Applied Science). The lysate was clarified by centrifugation at 13,000 × g for 10 min at 4 °C, and the supernatant was removed to a fresh tube and frozen at −20 °C. Extracts (10 μg of total protein) were resolved by electrophoresis in 4–12% Bis-Tris gels and transferred to PVDF membranes. The membranes were blocked overnight at room temperature in PBS + 1% Tween 20 (PBS-T) containing 5% milk and incubated with a rabbit anti-T3D antiserum (1:500) in PBS-T plus milk at room temperature for 3 h. The membranes were washed 3× for 10 min with PBS-T and incubated with an alkaline phosphatase-conjugated goat anti-rabbit antibody (Bio-Rad) diluted 1:2000 for 3 h. After 3 washes with PBS-T, the membranes were incubated for 5 min with chemiluminescent alkaline phosphate substrate (Bio-Rad) and visualized using a ChemiDock XRS+ molecular imager (Bio-Rad). Band densities were analyzed using Image J software. Cells (5 × 105) grown in 60-mm dishes were adsorbed with T3D in PBS at an m.o.i. of 100 pfu/cell at 4 °C for 1 h. Cells were incubated in medium at 37 °C for various intervals, removed from plates with a scraper, washed once with PBS, and centrifuged at 500 × g for 5 min. The supernatant was removed, and the cell pellet was frozen at −20 °C. RNA was extracted by using an RNeasy Plus RNA extraction minikit (Qiagen) according to the manufacturer's instructions. RNA was converted to cDNA by using an Omniscript RT cDNA synthesis kit (Qiagen) with an oligo(dT) primer according to the manufacturer's instructions. qPCR was performed using the Express SYBR Green ER system (Invitrogen). Primers specific for human GAPDH (forward primer 5′-GATCATCAGCAATGCCTCCT-3′ and reverse primer 5′-TGTGGTCATGAGTCCTTCCA-3′) or human IFITM3 (forward primer 5′-ATGTCGTCTGGTCCCTGTTC-3′ and reverse primer 5′-GTCATGAGGATGCCCAGAAT-3′) were used at a final concentration of 0.2 μm. Quantification and melt curve analyses were performed according to the manufacturer's protocol. For each sample, the CT (threshold cycle) for the RNA of interest was normalized to that for GAPDH. -Fold induction was calculated by comparing normalized CT values (ΔΔCT) of duplicate cDN" @default.
• W2082834676 created "2016-06-24" @default.
• W2082834676 creator A5031301336 @default.
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• W2082834676 date "2013-06-01" @default.
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• W2082834676 title "Interferon-inducible Transmembrane Protein 3 (IFITM3) Restricts Reovirus Cell Entry" @default.
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• W2082834676 doi "https://doi.org/10.1074/jbc.m112.438515" @default.
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