Coronavirus Replication Complex Formation Utilizes Components of Cellular Autophagy

Abstract

The coronavirus mouse hepatitis virus (MHV) performs RNA replication on double membrane vesicles (DMVs) in the cytoplasm of the host cell. However, the mechanism by which these DMVs form has not been determined. Using genetic, biochemical, and cell imaging approaches, the role of autophagy in DMV formation and MHV replication was investigated. The results demonstrated that replication complexes co-localize with the autophagy proteins, microtubule-associated protein light-chain 3 and Apg12. MHV infection induces autophagy by a mechanism that is resistant to 3-methyladenine inhibition. MHV replication is impaired in autophagy knockout, APG5–/–, embryonic stem cell lines, but wild-type levels of MHV replication are restored by expression of Apg5 in the APG5–/–cells. In MHV-infected APG5–/–cells, DMVs were not detected; rather, the rough endoplasmic reticulum was dramatically swollen. The results of this study suggest that autophagy is required for formation of double membrane-bound MHV replication complexes and that DMV formation significantly enhances the efficiency of replication. Furthermore, the rough endoplasmic reticulum is implicated as the possible source of membranes for replication complexes. The coronavirus mouse hepatitis virus (MHV) performs RNA replication on double membrane vesicles (DMVs) in the cytoplasm of the host cell. However, the mechanism by which these DMVs form has not been determined. Using genetic, biochemical, and cell imaging approaches, the role of autophagy in DMV formation and MHV replication was investigated. The results demonstrated that replication complexes co-localize with the autophagy proteins, microtubule-associated protein light-chain 3 and Apg12. MHV infection induces autophagy by a mechanism that is resistant to 3-methyladenine inhibition. MHV replication is impaired in autophagy knockout, APG5–/–, embryonic stem cell lines, but wild-type levels of MHV replication are restored by expression of Apg5 in the APG5–/–cells. In MHV-infected APG5–/–cells, DMVs were not detected; rather, the rough endoplasmic reticulum was dramatically swollen. The results of this study suggest that autophagy is required for formation of double membrane-bound MHV replication complexes and that DMV formation significantly enhances the efficiency of replication. Furthermore, the rough endoplasmic reticulum is implicated as the possible source of membranes for replication complexes. Autophagy is a cellular stress response that functions to recycle proteins and organelles (1Klionsky D.J. Emr S.D. Science. 2000; 290: 1717-1721Crossref PubMed Scopus (2988) Google Scholar, 2Reggiori F. Klionsky D.J. Eukaryot. Cell. 2002; 1: 11-21Crossref PubMed Scopus (474) Google Scholar). The mechanism of autophagy has been extensively studied in yeast, with more than 15 genes identified that are required for functional autophagy (1Klionsky D.J. Emr S.D. Science. 2000; 290: 1717-1721Crossref PubMed Scopus (2988) Google Scholar, 2Reggiori F. Klionsky D.J. Eukaryot. Cell. 2002; 1: 11-21Crossref PubMed Scopus (474) Google Scholar). Autophagy has been most widely studied as a response to amino acid starvation; however, the role that autophagy may play in development, disease pathogenesis, and microbial infections is only beginning to be examined. Studies with Sindbis virus and herpes simplex virus-1 have demonstrated that autophagy may be an important defense mechanism against infection with those viruses (3Talloczy Z. Jiang W. Virgin IV, H.W. Leib D.A. Scheuner D. Kaufman R.J. Eskelinen E.L. Levine B. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 190-195Crossref PubMed Scopus (647) Google Scholar, 4Liang X.H. Kleeman L.K. Jiang H.H. Gordon G. Goldman J.E. Berry G. Herman B. Levine B. J. Virol. 1998; 72: 8586-8596Crossref PubMed Google Scholar). Cellular autophagy has been proposed to be a mechanism of replication complex formation for the positive sense RNA viruses, poliovirus, equine arteritis virus, and mouse hepatitis virus (MHV) 1The abbreviations used are: MHV, mouse hepatitis virus; DMV, double membrane vesicles; 3-MA, 3-methyladenine; LC3, light chain 3 (microtubule-associated protein); ES, embryonic stem; SARS, severe acute respiratory syndrome; CoV, coronavirus; p.i., postinfection; DBT, delayed brain tumor; N, nucleocapsid; M, membrane; hel, helicase; pfu, plaque-forming unit; DMEM, Dulbecco’s modified Eagle’s medium; FCS, fetal calf serum; PBS, phosphate-buffered saline; BSA, bovine serum albumin; ER, endoplasmic reticulum; RER, rough endoplasmic reticulum. (5Pederson K. van der Meer Y. Roos N. Snijer E.J. J. Virol. 1999; 73: 2016-2026Crossref PubMed Google Scholar, 6Suhy D.A. Giddings Jr., T.H. Kirkegaard K. J. Virol. 2000; 74: 8953-8965Crossref PubMed Scopus (425) Google Scholar, 7Schlegel A. Giddings T.J. Ladinsky M.S. Kirkegaard K. J. Virol. 1996; 70: 6576-6588Crossref PubMed Google Scholar, 8Gosert R. Kanjanahaluethai A. Egger D. Bienz K. Baker S.C. J. Virol. 2002; 76: 3697-3708Crossref PubMed Scopus (339) Google Scholar). For all of these viruses, replication complexes have been shown to form as double membrane vesicles (DMVs) in the cytoplasm, which is suggestive of an autophagic origin. For poliovirus and MHV, multiple organelle markers have been reported to co-localize or co-fractionate with replication complexes, also consistent with an autophagy-like process (6Suhy D.A. Giddings Jr., T.H. Kirkegaard K. J. Virol. 2000; 74: 8953-8965Crossref PubMed Scopus (425) Google Scholar, 7Schlegel A. Giddings T.J. Ladinsky M.S. Kirkegaard K. J. Virol. 1996; 70: 6576-6588Crossref PubMed Google Scholar, 9Shi S.T. Schiller J.J. Kanjanahaluethai A. Baker S.C. Oh J.W. Lai M.M. J. Virol. 1999; 73: 5957-5969Crossref PubMed Google Scholar, 10Sims A.C. Ostermann J. Denison M.R. J. Virol. 2000; 74: 5647-5654Crossref PubMed Scopus (64) Google Scholar). In addition, poliovirus and MHV replication complexes have been reported to acquire lysosomal markers over the course of infection similar to the maturation of autophagosomes. These studies have demonstrated that replication complexes formed by these viruses share features of autophagosomes; however, it is not known whether cellular autophagy is required for the formation of replication complexes. Recently, markers for autophagic vacuoles in mammalian cells have been described: microtubule-associated protein light chain 3 (LC3) and Apg12 (14Drosten C. Gunther S. Preiser W. Van Der Werf S. Brodt H.R. Becker S. Rabenau H. Panning M. Kolesnikova L. Fouchier R.A. Berger A. Burguiere A.M. Cinatl J. Eickmann M. Escriou N. Grywna K. Kramme S. Manuguerra J.C. Muller S. Rickerts V. Sturmer M. Vieth S. Klenk H.D. Osterhaus A.D. Schmitz H. Doerr H.W. N. Engl. J. Med. 2003; 348: 1967-1976Crossref PubMed Scopus (3455) Google Scholar, 15Poutanen S.M. Low D.E. Henry B. Finkelstein S. Rose D. Green K. Tellier R. Draker R. Adachi D. Ayers M. Chan A.K. Skowronski D.M. Salit I. Simor A.E. Slutsky A.S. Doyle P.W. Krajden M. Petric M. Brunham R.C. McGeer A.J. N. Engl. J. Med. 2003; 348: 1995-2005Crossref PubMed Scopus (1019) Google Scholar). In addition, murine embryonic stem (ES) cells lacking a critical gene product in the pathway of cellular autophagy (Apg5) have been established (12Mizushima N. Yamamoto A. Hatano M. Kobayashi Y. Kabeya Y. Suzuki K. Tokuhisa T. Ohsumi Y. Yoshimori T. J. Cell Biol. 2001; 152: 657-668Crossref PubMed Scopus (1161) Google Scholar). Together, these advances provide new approaches to investigating the role of cellular autophagy in viral infections. Coronaviruses are enveloped positive sense RNA viruses that replicate entirely in the cytoplasm of cells. Coronaviruses are important causes of disease in many domesticated animals and are responsible for up to 30% of human colds. In addition, a newly recognized human coronavirus has recently been identified as the causative agent of severe acute respiratory syndrome (SARS) (13Ksiazek T.G. Erdman D. Goldsmith C. Zaki S.R. Peret T. Emery S. Tong S. Urbani C. Comer J.A. Lim W. Rollin P.E. Nghiem K.H. Dowell S. Ling A.E. Humphrey C. Shieh W.J. Guarner J. Paddock C.D. Rota P. Fields B. DeRisi J. Yang J.Y. Cox N. Hughes J. LeDuc J.W. Bellini W.J. Anderson L.J. N. Engl. J. Med. 2003; 348: 1953-1966Crossref PubMed Scopus (3375) Google Scholar, 14Drosten C. Gunther S. Preiser W. Van Der Werf S. Brodt H.R. Becker S. Rabenau H. Panning M. Kolesnikova L. Fouchier R.A. Berger A. Burguiere A.M. Cinatl J. Eickmann M. Escriou N. Grywna K. Kramme S. Manuguerra J.C. Muller S. Rickerts V. Sturmer M. Vieth S. Klenk H.D. Osterhaus A.D. Schmitz H. Doerr H.W. N. Engl. J. Med. 2003; 348: 1967-1976Crossref PubMed Scopus (3455) Google Scholar, 15Poutanen S.M. Low D.E. Henry B. Finkelstein S. Rose D. Green K. Tellier R. Draker R. Adachi D. Ayers M. Chan A.K. Skowronski D.M. Salit I. Simor A.E. Slutsky A.S. Doyle P.W. Krajden M. Petric M. Brunham R.C. McGeer A.J. N. Engl. J. Med. 2003; 348: 1995-2005Crossref PubMed Scopus (1019) Google Scholar). Coronaviruses and arteriviruses are the two families within the order Nidovirales. Viruses in this order have similar genome organization and express the proteins required for RNA replication as polyproteins (5Pederson K. van der Meer Y. Roos N. Snijer E.J. J. Virol. 1999; 73: 2016-2026Crossref PubMed Google Scholar, 9Shi S.T. Schiller J.J. Kanjanahaluethai A. Baker S.C. Oh J.W. Lai M.M. J. Virol. 1999; 73: 5957-5969Crossref PubMed Google Scholar, 16Hegyi A. Ziebuhr J. J. Gen. Virol. 2002; 83: 595-599Crossref PubMed Scopus (208) Google Scholar, 17Hegyi A. Friebe A. Gorbalenya A.E. Ziebuhr J. J. Gen. 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MHV replication complexes form in the cytoplasm of infected cells and are first detectable at 4–5 by in MHV-infected delayed brain cells S.T. Schiller J.J. Kanjanahaluethai A. Baker S.C. Oh J.W. Lai M.M. J. Virol. 1999; 73: 5957-5969Crossref PubMed Google Scholar, 22Denison M.R. Spaan W.J. van der Meer Y. Gibson C.A. Sims A.C. Prentice E. Lu X. J. Virol. 1999; 73: 6862-6871Crossref PubMed Google Scholar, Lu X. Denison M.R. J. Virol. 2000; 74: PubMed Scopus Google Scholar, Prentice E. Denison M.R. Virology. 2001; PubMed Scopus Google Scholar). complexes as with the and of replication complexes over the course of infection S.T. Schiller J.J. Kanjanahaluethai A. Baker S.C. Oh J.W. Lai M.M. J. Virol. 1999; 73: 5957-5969Crossref PubMed Google Scholar, 22Denison M.R. Spaan W.J. van der Meer Y. Gibson C.A. Sims A.C. Prentice E. Lu X. J. Virol. 1999; 73: 6862-6871Crossref PubMed Google Scholar, Lu X. Denison M.R. J. Virol. 2000; 74: PubMed Scopus Google Scholar, Prentice E. Denison M.R. Virology. 2001; PubMed Scopus Google Scholar, W. Hughes S. S.R. J. 1998; PubMed Scopus Google Scholar). These complexes are in viral RNA and all proteins to as as the protein S.T. Schiller J.J. Kanjanahaluethai A. Baker S.C. Oh J.W. Lai M.M. J. Virol. 1999; 73: 5957-5969Crossref PubMed Google Scholar, 22Denison M.R. Spaan W.J. van der Meer Y. Gibson C.A. Sims A.C. Prentice E. Lu X. J. Virol. 1999; 73: 6862-6871Crossref PubMed Google Scholar, Lu X. Denison M.R. J. Virol. 2000; 74: PubMed Scopus Google Scholar, Prentice E. Denison M.R. Virology. 2001; PubMed Scopus Google Scholar). have shown that of the replication the and proteins, and of RNA replication to of viral in the Prentice E. Denison M.R. Virology. 2001; PubMed Scopus Google Scholar). In this to whether cellular autophagy was and required MHV replication complex formation and viral replication. Cell imaging and and approaches were used to that MHV replication complexes were with the autophagic markers and Apg12 and that MHV proteins known to replication complexes also with over Furthermore, MHV infection and of autophagy MHV These results that the process of autophagy an important role in the replication of and was used for infections this infections were at a of infection of plaque-forming cells were used in and protein N. K. N. PubMed Scopus Google Scholar, N. K. S. M. PubMed Scopus Google Scholar). For this to the an by to the was in and cells by of the in cells and were as (12Mizushima N. Yamamoto A. Hatano M. Kobayashi Y. Kabeya Y. Suzuki K. Tokuhisa T. Ohsumi Y. Yoshimori T. J. Cell Biol. 2001; 152: 657-668Crossref PubMed Scopus (1161) Google Scholar). The was and were by of the cells (12Mizushima N. Yamamoto A. Hatano M. Kobayashi Y. Kabeya Y. Suzuki K. Tokuhisa T. Ohsumi Y. Yoshimori T. J. Cell Biol. 2001; 152: 657-668Crossref PubMed Scopus (1161) Google Scholar). cells were on in Dulbecco’s modified Eagle’s with fetal calf serum amino and of protein was as (3Talloczy Z. Jiang W. Virgin IV, H.W. Leib D.A. Scheuner D. Kaufman R.J. Eskelinen E.L. Levine B. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 190-195Crossref PubMed Scopus (647) Google Scholar). are of in S.D. cells were for in of and The cells were and in for to for of These cells were amino acid or in and infection or were for of the cell was at the of the and the of protein at was by the at of at results are of in with in of cells were infected with MHV at a of infection of and the virus was to at for The was and the cells were with with of the for cell for all of were at and with of of at The of virus in the at was by The results viral which is as and have been for in cell cell the of in all and have been M.R. Spaan W.J. van der Meer Y. Gibson C.A. Sims A.C. Prentice E. Lu X. J. Virol. 1999; 73: 6862-6871Crossref PubMed Google Scholar, Lu X. Denison M.R. J. Virol. 2000; 74: PubMed Scopus Google Scholar). The and mouse were by of The LC3, and Apg12 were in as Y. N. T. Yamamoto A. T. T. E. Ohsumi Y. Yoshimori T. J. 2000; PubMed Scopus Google Scholar, A. N. N. Ohsumi Y. J. Biol. 2002; PubMed Scopus Google Scholar). The against was were with a in with was as (12Mizushima N. Yamamoto A. Hatano M. Kobayashi Y. Kabeya Y. Suzuki K. Tokuhisa T. Ohsumi Y. Yoshimori T. J. Cell Biol. 2001; 152: 657-668Crossref PubMed Scopus (1161) Google Scholar). cells were on were at a of infection at in The was at p.i., and the cells were with and with the the cells were with and and by the of The cells were in bovine serum For in which was mouse and the the were in a PBS, and were to for at and with PBS, and The cells were for at with a of to or The cells were with PBS, and and with to in and with For the in which were in was and to a for was as was in PBS, and and to for at The cells were with PBS, and BSA, with PBS, and with to was at and a on a of were with For protein and of were and for The of infected was were to for of In were the and been for these by of to cells and with the The was and the was to that The was to were on the and the was to for of course and cells were infected for or for and with The cells were with in at for and at Cell were with in for at and with The were with for at and in for of were a at with for whether MHV replication complexes were on the of the of replication complexes and markers for autophagic microtubule-associated protein and were by in MHV-infected cells Y. N. T. Yamamoto A. T. T. E. Ohsumi Y. Yoshimori T. J. 2000; PubMed Scopus Google Scholar). In was as the cell cytoplasm cells were infected with MHV, was in The of complexes has been shown to to of with autophagic vacuoles Y. N. T. Yamamoto A. T. T. E. Ohsumi Y. Yoshimori T. J. 2000; PubMed Scopus Google Scholar). MHV-infected cells were at for and MHV proteins known to to replication complexes and and with a of of that and were and were and were with the of that were of autophagosomes. was with a protein demonstrated to to autophagic membranes Thus, MHV replication complexes were with two protein markers for autophagic whether the with markers was to replication complexes, the of was with the MHV membrane and is known to to of virus in the and were that was not significantly with membranes or or with of virus has also been reported to be with However, not all of the in an infected cell with replication complexes. Thus, to whether replication complexes and the replication were with the of to in MHV-infected cells. was only with that the of replication complexes and were not with with shown that replication complexes with LC3, with replication complexes the course of The of LC3, hel, N, and was at and and p.i., and were and but by p.i., the two proteins were and by p.i., and were the of the and proteins were and were the infection Together, these results of that the and proteins replication complexes to of viral to newly RNA for newly Prentice E. Denison M.R. Virology. 2001; PubMed Scopus Google Scholar). was with and at and p.i., and were but by p.i., and were only and by and were only the of and were and were the course of the infection Thus, not with and to of that with replication complexes, the of was with the replication Prentice E. Denison M.R. Virology. 2001; PubMed Scopus Google Scholar). and the course of infection These demonstrated that not over but with proteins in viral replication complexes. Autophagy by MHV of proteins with that replication complexes were on autophagic possible mechanism for of proteins to autophagic vacuoles be the of cellular autophagy by The of of proteins is a shown to provide an of the of autophagy in cells (1Klionsky D.J. Emr S.D. Science. 2000; 290: 1717-1721Crossref PubMed Scopus (2988) Google Scholar, Z. Jiang W. Virgin IV, H.W. Leib D.A. Scheuner D. Kaufman R.J. Eskelinen E.L. Levine B. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 190-195Crossref PubMed Scopus (647) Google Scholar, N. Yamamoto A. Hatano M. Kobayashi Y. Kabeya Y. Suzuki K. Tokuhisa T. Ohsumi Y. Yoshimori T. J. Cell Biol. 2001; 152: 657-668Crossref PubMed Scopus (1161) Google Scholar). whether MHV infection the of protein was in cells that were amino and or with cells that were protein to with cells with In MHV-infected protein was significantly to over and cells were and protein was to with cells. cells were in the of the autophagy 3-methyladenine the in protein was In protein to in infected that protein was MHV in a not However, is a that may with viral RNA replication and results suggest that MHV autophagy a pathway or was to the in functional whether an autophagic pathway was required for the of protein of protein was in murine embryonic stem cell Apg5 or in cells the used for cells. The of has been shown to the formation of autophagic vacuoles (12Mizushima N. Yamamoto A. Hatano M. Kobayashi Y. Kabeya Y. Suzuki K. Tokuhisa T. Ohsumi Y. Yoshimori T. J. Cell Biol. 2001; 152: 657-668Crossref PubMed Scopus (1161) Google Scholar). Furthermore, these cells were shown to have of of proteins (12Mizushima N. Yamamoto A. Hatano M. Kobayashi Y. Kabeya Y. Suzuki K. Tokuhisa T. Ohsumi Y. Yoshimori T. J. Cell Biol. 2001; 152: 657-668Crossref PubMed Scopus (1161) Google Scholar, S. 1997; PubMed Scopus Google Scholar, A. K. S. Y. S. J. Virol. 1996; 70: PubMed Google Scholar). cells were or infected with MHV, protein were and In cells were or and was in levels of protein with cells was to or infected in protein was with cells Thus, an autophagic pathway to be required for protein MHV in the of demonstrated that MHV infection autophagy and that MHV replication complexes markers for autophagic whether autophagy was required for MHV replication. MHV was in cell that were and and in cells that were by Apg5 expression that Apg5 was in and but not in the or cell lines, with Apg12 and Apg5 was has been shown that all Apg5 and Apg12 is as a and that of a is an of the of Apg5 expression in a cell (12Mizushima N. Yamamoto A. Hatano M. Kobayashi Y. Kabeya Y. Suzuki K. Tokuhisa T. Ohsumi Y. Yoshimori T. J. Cell Biol. 2001; 152: 657-668Crossref PubMed Scopus (1161) Google Scholar). with to Apg12 the of the in the and cells but not in the or cells the membrane was and with against proteins were in the and but not in the or The levels of Apg5 expression in wild-type and cells were that Apg5 was in and cells and not in and these cells were used to whether in autophagy the of MHV to MHV infection of in cells was at MHV to and at in the and lines, a of of the by expression of viral to that the of MHV replication in the and cell was of the of Apg5 expression and that the be by expression of DMV results demonstrated that functional autophagy was critical for wild-type levels of MHV replication. whether formation of DMVs was to autophagy in MHV-infected were of MHV-infected cells. In a of rough endoplasmic reticulum was but membrane was In the MHV infection in the of DMVs similar to those in MHV-infected cells In the demonstrated of MHV to replicate in DMVs were not in the MHV-infected cells. the of the membranes in MHV-infected cells was with membranes the of cells These membranes with the and with an in the in multiple vesicles to be by the were with membranes that the of autophagic The in MHV-infected cells were entirely in or cells that the membrane were by MHV The in this that cellular autophagy an important role in MHV replication and that infection with MHV induces cellular this is the first to that of the autophagic pathway are required for formation of a viral replication complex and for viral The autophagy by MHV infection was not to treatment, of viral by up to not that an pathway of autophagy is by MHV infection or that viral proteins, to treatment, may or autophagic The mechanism by which MHV induces autophagy to be determined. has been shown to be for autophagic (3Talloczy Z. Jiang W. Virgin IV, H.W. Leib D.A. Scheuner D. Kaufman R.J. Eskelinen E.L. Levine B. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 190-195Crossref PubMed Scopus (647) Google Scholar). RNA is known to be a of protein it is possible that RNA viral replication may protein and is that of MHV to the cellular may that in infection was not to autophagy in that the proteins for autophagic formation are required for The of double membrane vesicles in MHV-infected cells was in light of the of MHV to replicate in these to that DMV formation may not be required for viral replication. However, viral was by in the lines, and with and cells with an that the efficiency of replication is by DMV The of infected cells was the in these cells that to multiple of these vesicles were by and the of cytoplasm and may have been of cytoplasm the of the that the of vesicles in it is possible that the may of the The is similar to the in cells with a in however, Apg5 is not known to play role in was only in MHV-infected cells and not in cells or in infected or that MHV is with or a process that The mechanism by which form replication complexes has not been determined. have shown that multiple MHV proteins that with membranes R. Kanjanahaluethai A. Egger D. Bienz K. Baker S.C. J. Virol. 2002; 76: 3697-3708Crossref PubMed Scopus (339) Google Scholar, 10Sims A.C. Ostermann J. Denison M.R. J. Virol. 2000; 74: 5647-5654Crossref PubMed Scopus (64) Google Scholar, M.R. Sims A.C. Med. Biol. 2001; PubMed Scopus Google Scholar). The that MHV infection of cells results in of the and a of DMVs that the may be the source membranes for formation of viral replication complexes. Furthermore, the is the viral proteins in the of the ER, the mechanism by which these proteins are is is to that of the autophagy pathway are required for formation of double membrane vesicles and that formation of DMVs to and MHV on the the mechanisms by which MHV with the process of autophagy to be this that MHV may have to a cellular process to replication The of the mechanisms of coronavirus cell may be of critical in the and possible of coronavirus The of a new human coronavirus as the of has dramatically the to coronavirus virus cell and mechanisms of replication (13Ksiazek T.G. Erdman D. Goldsmith C. Zaki S.R. Peret T. Emery S. Tong S. Urbani C. Comer J.A. Lim W. Rollin P.E. Nghiem K.H. Dowell S. Ling A.E. Humphrey C. Shieh W.J. Guarner J. Paddock C.D. Rota P. Fields B. DeRisi J. Yang J.Y. Cox N. Hughes J. LeDuc J.W. Bellini W.J. 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