Reticulon 3 Binds the 2C Protein of Enterovirus 71 and Is Required for Viral Replication

Enterovirus 71 is an enterovirus of the family Picornaviridae. The 2C protein of poliovirus, a relative of enterovirus 71, is essential for viral replication. The poliovirus 2C protein is associated with host membrane vesicles, which form viral replication complexes where viral RNA synthesis takes place. We have now identified a host-encoded 2C binding protein called reticulon 3, which we found to be associated with the replication complex through direct interaction with the enterovirus 71-encoded 2C protein. We observed that the N terminus of the 2C protein, which has both RNA- and membrane-binding activity, interacted with reticulon 3. This region of interaction was mapped to its reticulon homology domain, whereas that of 2C was encoded by the 25th amino acid, isoleucine. Reticulon 3 could also interact with the 2C proteins encoded by other enteroviruses, such as poliovirus and coxsackievirus A16, implying that it is a common factor for such viral replication. Reduced production of reticulon 3 by RNA interference markedly reduced the synthesis of enterovirus 71-encoded viral proteins and replicative double-stranded RNA, reducing plaque formation and apoptosis. Furthermore, reintroduction of nondegradable reticulon 3 into these knockdown cells rescued enterovirus 71 infectivity, and viral protein and double-stranded RNA synthesis. Thus, reticulon 3 is an important component of enterovirus 71 replication, through its potential role in modulation of the sequential interactions between enterovirus 71 viral RNA and the replication complex. Enterovirus 71 is an enterovirus of the family Picornaviridae. The 2C protein of poliovirus, a relative of enterovirus 71, is essential for viral replication. The poliovirus 2C protein is associated with host membrane vesicles, which form viral replication complexes where viral RNA synthesis takes place. We have now identified a host-encoded 2C binding protein called reticulon 3, which we found to be associated with the replication complex through direct interaction with the enterovirus 71-encoded 2C protein. We observed that the N terminus of the 2C protein, which has both RNA- and membrane-binding activity, interacted with reticulon 3. This region of interaction was mapped to its reticulon homology domain, whereas that of 2C was encoded by the 25th amino acid, isoleucine. Reticulon 3 could also interact with the 2C proteins encoded by other enteroviruses, such as poliovirus and coxsackievirus A16, implying that it is a common factor for such viral replication. Reduced production of reticulon 3 by RNA interference markedly reduced the synthesis of enterovirus 71-encoded viral proteins and replicative double-stranded RNA, reducing plaque formation and apoptosis. Furthermore, reintroduction of nondegradable reticulon 3 into these knockdown cells rescued enterovirus 71 infectivity, and viral protein and double-stranded RNA synthesis. Thus, reticulon 3 is an important component of enterovirus 71 replication, through its potential role in modulation of the sequential interactions between enterovirus 71 viral RNA and the replication complex. Enterovirus 71 (EV71) 2The abbreviations used are: EV71, enterovirus 71; CA16, coxsackievirus A16; dsRNA, double-stranded RNA; MOI, multiplicity of infection; p.i., postinfection; PV, poliovirus; ER, endoplasmic reticulum; RTN, reticulon; RHD, reticulon homology domain; siRNA, small interfering RNA; GST, glutathione S-transferase; PBS, phosphate-buffered saline; RT, reverse transcription; aa, amino acids; GFP, green fluorescent protein; IFN, interferon; SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein receptor; vRNA, viral RNA; CPE, cytopathic effect. is a member of the Picornaviridae family and contains a single-stranded RNA genome of positive polarity. The genome of human enteroviruses is about 7.5 kilobases. It encodes a single polyprotein that is proteolytically cleaved to give rise to various structural and nonstructural viral proteins. The human enterovirus family is classified into five species: the poliovirus (PV), coxsackievirus A, coxsackievirus B, echovirus, and other enteroviruses (1Racaniello V. Knipe D.M. Howley P.M. Fields Virology. 2001: 685-722Google Scholar). Their genomes are highly homologous and thus share very similar biochemical properties. One of their universal features is that infection with positive-stranded RNA viruses results in a range of membrane morphologies, many of which involve complex membrane rearrangements. Vesicular structures generated in picornavirus-infected cells appear to serve as compartments where synthesis of viral RNA takes place. These structures are derived from membranes of different cellular organelles involved in secretory pathways, primarily endoplasmic reticulum (ER) but also the Golgi complex and possibly others (2Bienz K. Egger D. Pasamontes L. Virology. 1987; 160: 220-226Crossref PubMed Scopus (213) Google Scholar, 3Rust R.C. Landmann L. Gosert R. Tang B.L. Hong W. Hauri H.P. Egger D. Bienz K. J. Virol. 2001; 75: 9808-9818Crossref PubMed Scopus (189) Google Scholar, 4Bolten R. Egger D. Gosert R. Schaub G. Landmann L. Bienz K. J. Virol. 1998; 72: 8578-8585Crossref PubMed Google Scholar, 5Schlegel A. Giddings Jr., T.H. Ladinsky M.S. Kirkegaard K. J. Virol. 1996; 70: 6576-6588Crossref PubMed Google Scholar). These membrane rearrangements are clearly triggered by the viral preprotein 2BC and its mature polypeptide 2C (6Bienz K. Egger D. Rasser Y. Bossart W. Virology. 1983; 131: 39-48Crossref PubMed Scopus (138) Google Scholar, 7Cho M.W. Teterina N. Egger D. Bienz K. Ehrenfeld E. Virology. 1994; 202: 129-145Crossref PubMed Scopus (260) Google Scholar, 8Suhy D.A. Giddings Jr., T.H. Kirkegaard K. J. Virol. 2000; 74: 8953-8965Crossref PubMed Scopus (424) Google Scholar). The vesicles are associated with the above viral proteins (2Bienz K. Egger D. Pasamontes L. Virology. 1987; 160: 220-226Crossref PubMed Scopus (213) Google Scholar, 9Bienz K. Egger D. Troxler M. Pasamontes L. J. Virol. 1990; 64: 1156-1163Crossref PubMed Google Scholar) and with other viral nonstructural polypeptides (42Egger D. Pasamontes L. Bolten R. Boyko V. Bienz K. J. Virol. 1996; 70: 8675-8683Crossref PubMed Google Scholar) and exhibit a rosette-like morphology with elongated replication complexes surrounded by round vesicles (9Bienz K. Egger D. Troxler M. Pasamontes L. J. Virol. 1990; 64: 1156-1163Crossref PubMed Google Scholar). They are equipped with all the necessary components of the RNA- and translation-synthesizing machinery. It is not clear how the viral RNA is located to these complexes, and the mechanism by which these vesicles are generated is unknown. Furthermore, it has not been determined whether host-encoded proteins play any role in the formation of these replication complex-associated vesicles. The 2C protein is of the highly of the viral proteins all The 2C protein is and is of amino a of binding proteins PubMed Scopus Google Scholar, E. J. PubMed Scopus Google Scholar). both the and the of the protein RNA binding in L. J. PubMed Scopus Google Scholar, R. A. J. Virol. 2001; PubMed Scopus Google Scholar). 2C and its 2BC appear to be associated with viral RNA in cells (9Bienz K. Egger D. Troxler M. Pasamontes L. J. Virol. 1990; 64: 1156-1163Crossref PubMed Google where the 2C protein are associated with replication complex-associated vesicles (2Bienz K. Egger D. Pasamontes L. Virology. 1987; 160: 220-226Crossref PubMed Scopus (213) Google Scholar, 9Bienz K. Egger D. Troxler M. Pasamontes L. J. Virol. 1990; 64: 1156-1163Crossref PubMed Google Scholar, R. B.L. E. A. PubMed Scopus Google Scholar, J. Virol. PubMed Google Scholar). They be involved in the formation of these complexes (9Bienz K. Egger D. Troxler M. Pasamontes L. J. Virol. 1990; 64: 1156-1163Crossref PubMed Google Scholar). the 2C protein, in in to membranes A. Virology. PubMed Scopus Google Scholar). these in binding the region amino was for membrane This region is associated with a between and A. E. Virology. 1994; PubMed Scopus Google Scholar). Thus, the 2C the to RNA and cellular of region results in viral RNA synthesis. the role of 2C in RNA replication is not We a in to for cellular proteins that interact with the region of the 2C protein of EV71, which We the of reticulon 3 a member of the reticulon family of as a binding of the viral 2C protein of are of the reticulon all of which a highly reticulon homology of about amino with by a PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). the family is involved in membrane and J. Y. K. Y. N. K. PubMed Scopus Google Scholar, K. L. R. J. PubMed Scopus Google Scholar, Y. K. K. M. K. Hauri H.P. M. PubMed Scopus Google Scholar, Y. PubMed Scopus Google Scholar). of these interactions and their have been to structural of the protein and of of the to other compartments PubMed Scopus Google Scholar). their in to viral replication, interaction with the encoded viral proteins of enterovirus 71, unknown. that in in cells viral double-stranded RNA synthesis and protein the 2C proteins of and coxsackievirus to interact with that the host-encoded protein as a cellular of enterovirus infection and replication. and and cells in and and in a was used to and RNA in The cells in and The and from the of and was from the of in determined from of different cells an plaque cells in cells and with for the was with and The was and the cells with and with The of the was as PubMed Scopus Google Scholar). and as for in which the enterovirus 71 2C protein amino and to the binding of in the from in from by The into and into and of to the and for The of human with amino L. M. N. J. PubMed Scopus Google Scholar) and its from a human by and into The 2C proteins and of enterovirus 71 into an of to the and 2C was from in and into 2C and 2C from RNA by reverse and into to and that the in the and not the the was with to an human to the The positive and of the to and and proteins of human and proteins of human enterovirus 71 and 2C proteins to the and and from and and green fluorescent protein was from The was to was used to the replication complex. was used as a of dsRNA, and the was as Virology. PubMed Scopus Google Scholar, J. J. E. J. J. N. PubMed Scopus Google Scholar). was with an of in and and the was with of The was into The in the The and of the to and viral by an and not of cells with into phosphate-buffered and by was by in and for to and of The for to membrane and The membrane was with in for and for The in and cells by and with of various and for cells with and into with for and a the was for to the of with of with as an was used as a with protein for 3 with with the was and for by and by The cells with various cells in for and with in and with the of dsRNA, the cells in binding was by the cells with a and in for was by the cells with in for by with with for was a Reticulon 3 and of in and the into The knockdown is with its The was and the was the reintroduction of nondegradable into and in the region of that was to the siRNA, The as The was and the reverse was The was and the reverse was are the and the of and for by The into and the was into the to a of and was into the human was into the protein proteins and to the proteins with in and for 3 with and proteins with glutathione and by of the of 2C was to A. E. Virology. 1994; PubMed Scopus Google Scholar). the in the used to The amino have been these and into and cells with an of and RNA was from cells the The of was the reverse for was in a and reverse green and various of by green was by the the in the was used to relative the as in for 2001; The is as the which is determined to be The of to was to the p.i., to and cells an of with and by was used for The 2C with and the 2C is of the highly proteins all G. E. PubMed Scopus Google Scholar, J. R. G. D. J. PubMed Google Scholar). It with that of the PV, which to and membranes to form the replication complex. whether with membranes is also with the 2C protein, we that between and membrane proteins. cells with and by an protein was found in the in the was with various of 2C was protein 2C was soluble in and the of Y. J. PubMed Scopus Google we that the 2C protein is as has been observed for the 2C protein of universal of positive-stranded RNA viruses is the of host membranes in RNA replication complex formation and Thus, it be to whether associated with 2C is the viral RNA replication complex. have that the replication complex contains replicative viral and of the we used to the replication complex the ER, we a a fluorescent protein to its cells with by infection with and for in the 2C protein of with the for endoplasmic we whether the amino of which has a potential membrane binding similar to that of with the we of and into The of was similar to the 2C protein in that it and and with the for we of cells with and 2C the of fluorescent for and the 2C protein of membrane and the of the 2C protein to be similar to its in that it is associated with structures and be involved in the formation of the replication complex (9Bienz K. Egger D. Troxler M. Pasamontes L. J. Virol. 1990; 64: 1156-1163Crossref PubMed Google Scholar). of Reticulon 3 as a for the 2C by the viral replication of EV71, we for potential with 2C the a we used a the of 2C to a human We positive their to all of the in One was identified as reticulon 3, and was identified as reticulon we used as the of identified as This that be interaction between 2C and of the Furthermore, both and share the protein in their the binding of 2C is also involved in of the identified by the RHD, is to be involved in the interaction between 2C and the proteins. in was in such as and but was in such as and not is in with PubMed Scopus Google Scholar). all of these positive for not of the of we to for and in of the 2C with the interaction between and we an in binding proteins. and into a and a and to and protein was and with The proteins and a and was by a these to but not to that the interaction between and was direct and The interaction between 2C and was also by cells with and in with The cells in and used to the and the from the protein to and and was with with single to the proteins in the and These not was used and a was used the of the The 2C was to interact with with similar of with different human enterovirus 2C proteins. cells with and the 2C proteins of enterovirus 71 2C and 2C from RNA by reverse as cells and the to an and as of the used in the The the whether with 2C protein in we cells with by infection with The for with and the by The but not the clearly a to the of 2C a in the not The 2C with the of member of the reticulon family a and has PubMed Scopus Google Scholar). 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PubMed Scopus Google Scholar, J. 1994; PubMed Google Scholar). not with 2C but also with in The of 2C with dsRNA, which is and Thus, it that is involved in replication complex formation and that it is associated with the replication complex through direct and interaction with the 2C protein. of 2C with in results that the 2C protein of with the host-encoded protein. we that the 2C protein of also interact with all reticulon proteins their We thus in whether could interact with the 2C proteins of other the nonstructural protein 2C is highly different enteroviruses, we whether could interact with the 2C proteins of and The region of and with and PV, 2C proteins from EV71, CA16, to and with into used in The interaction between all 2C proteins and was observed we used 3, and but not in the with 3, 3, and Thus, be involved in the viral replication of of as for the amino involved in the interaction of with we in the protein 2C is the polypeptide with to amino the region is and an A. E. Virology. 1994; PubMed Scopus Google Scholar) have in the protein 2C We the in the in the we have amino for that are located the of the and These amino by The an in the and in the to in the These into the and with into cells for was with with single to the protein in the and a was not the was with the was used 3, and Thus, is for the interaction of 2C with the of The in the 2C protein viral protein and thus replication A. E. Virology. 1994; PubMed Scopus Google Scholar). The into the also in cytopathic for the not This is similar to the by and that play a role in the viral replication of and possibly other and for and results that be involved in viral replication. the role of in we used RNA interference to knockdown in knockdown called was This not the of cells not but protein reduced The in with the The Y. PubMed Scopus Google Scholar, Jr., PubMed Scopus Google Scholar). 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We have also the viral RNA synthesis by and the viral RNA synthesis was reduced in cells We the of these This is by the of viral cells with by These results by the of cells cells The of cells infection of about but of knockdown cells the infection to the of This that is necessary and for viral infection and that for RNA interference was the reintroduction of the for rescued the of viral these are with the that as a of viral replication in these host The and cells also to infection with to the of viral The that the in in knockdown cells with the cells from about to by markedly reduced viral and of and a of viral proteins enterovirus was by enterovirus 71 was the and cells a the of cells apoptosis. The enterovirus in the Thus, knockdown of enterovirus in The cells also a reduced cytopathic not and cells morphology with the cells by with to the proteins and not We the of from the through the Golgi by a K. J. PubMed Scopus Google Scholar) the cells been to a we observed that was to the the Golgi in both and cells not These that the of viral replication is not to a in the and but is a of reduced interactions between and the 2C protein. whether the RNA of RNA replication in cells could be by of the we of in and cells The was similar between these but cells about of the others not We also whether other and found that from cells replication by not We that the of the could not the in replication by protein and synthesis in knockdown and rescued A, reduced viral protein and synthesis in knockdown cells and of the and and with an of and the cells for as are in and The results are of B, viral is by These different as above and to with the cells with of as The was as cells as a of cells are from with of viral RNA in knockdown cells by and cells with an of and RNA was from cells the the in the was used to relative the The was The of to was to the p.i., to of cells A, of in The and cells a of and 3, p.i., viruses from the cells by of and determined by plaque and the of viral was to the p.i., to are from B, of by of with with a of p.i., and cells and with and was by was by the cells with The the of cells in the of the we that could cellular protein for viral interacted with protein and infection in cells with an knockdown of the to viral RNA and protein and The reduced viral and from synthesis of viral protein and RNA in as by plaque and we that the of a nondegradable form of in knockdown cells could the by we that a role in replication. has been used as a for the of positive-stranded RNA replication complex with results in a range of membrane morphologies, many of which involve complex membrane rearrangements to form replication complex-associated vesicles. These vesicles are derived from organelles involved in the secretory from but also possibly from the Golgi complex. that protein 2C to membranes and thus formation through its region in the of other viral proteins (6Bienz K. Egger D. Rasser Y. Bossart W. Virology. 1983; 131: 39-48Crossref PubMed Scopus (138) Google Scholar, 7Cho M.W. Teterina N. Egger D. Bienz K. Ehrenfeld E. Virology. 1994; 202: 129-145Crossref PubMed Scopus (260) Google Scholar, 8Suhy D.A. Giddings Jr., T.H. Kirkegaard K. J. Virol. 2000; 74: 8953-8965Crossref PubMed Scopus (424) Google Scholar, R. L. PubMed Scopus Google Scholar). We amino in region the N terminus of 2C and their the interaction with was to the of amino the that the in the of the and its to This in 2C a in RNA synthesis A. E. Virology. 1994; PubMed Scopus Google Scholar). the was for interaction between 2C and was We also observed that has of the not Thus, we that the reduced viral RNA synthesis in observed by A. E. Virology. 1994; PubMed Scopus Google Scholar) was by the of interaction between 2C and thus RNA synthesis. five of all of which a but with have of to human and have and amino L. M. N. J. PubMed Scopus Google Scholar). is about the of these The was to the and to all of the of the These cells found to be thus that is not an essential for the reintroduction of a small of amino as by L. M. N. J. PubMed Scopus Google rescued viral and Thus, it is the the that is for viral replication. the play a role in the interaction with 2C protein. We have that all of the reticulon family of with the protein the RHD, to 2C 2C and The of the interaction of with 2C is but the of the interaction of the protein with 2C is that the the binding and the of and possibly other Vesicular structures generated in the cells appear to play a role as compartments where synthesis of viral RNA takes place. These structures are derived from membranes of different cellular vesicles are in the R.C. Landmann L. Gosert R. Tang B.L. Hong W. Hauri H.P. Egger D. Bienz K. J. Virol. 2001; 75: 9808-9818Crossref PubMed Scopus (189) Google Scholar, 5Schlegel A. Giddings Jr., T.H. Ladinsky M.S. Kirkegaard K. J. Virol. 1996; 70: 6576-6588Crossref PubMed Google Scholar, Virology. PubMed Scopus Google whereas membranes from other cellular and the Golgi also to formation R. Egger D. Gosert R. Schaub G. Landmann L. Bienz K. J. Virol. 1998; 72: 8578-8585Crossref PubMed Google Scholar, 5Schlegel A. Giddings Jr., T.H. Ladinsky M.S. Kirkegaard K. J. Virol. 1996; 70: 6576-6588Crossref PubMed Google Scholar). is an protein, and its reduced in knockdown cells the of replication complex vesicles from the membrane family protein with soluble N-ethylmaleimide-sensitive factor attachment protein proteins K. L. R. J. PubMed Scopus Google Scholar) and is also involved in membrane between the endoplasmic reticulum and Golgi Y. K. K. M. K. Hauri H.P. M. PubMed Scopus Google Scholar). of a protein of and its the and membrane proteins. proteins are important for and of the vesicles to membranes in R. PubMed Scopus Google Scholar). of membrane protein in cells N. V. 1998; PubMed Scopus Google Scholar). The protein also in formation R. 1998; PubMed Scopus Google Scholar). replication complex-associated vesicles are in the by the cellular mechanism and are similar to the vesicles of the membrane R.C. Landmann L. Gosert R. Tang B.L. Hong W. Hauri H.P. Egger D. Bienz K. J. Virol. 2001; 75: 9808-9818Crossref PubMed Scopus (189) Google Scholar). proteins are for direct of and proteins into vesicles from the interaction of proteins with the proteins is thus in with the that replication complex-associated vesicles are associated with from which the vesicles are from the membrane R.C. Landmann L. Gosert R. Tang B.L. Hong W. Hauri H.P. Egger D. Bienz K. J. Virol. 2001; 75: 9808-9818Crossref PubMed Scopus (189) Google Scholar). The protein 2C has involved in RNA in the region located between amino and that 2C not any domain, and it interact with host proteins M.W. Teterina N. Egger D. Bienz K. Ehrenfeld E. Virology. 1994; 202: 129-145Crossref PubMed Scopus (260) Google Scholar, A. Virology. PubMed Scopus Google Scholar). We that is the potential host protein that with 2C for the of and thus be involved in the of 2C with viral RNA to form the replication complex a We that 2C protein, which in viral RNA for replication. synthesis of 2C and reduced to but in cells with an of and not This that the protein be involved in the of infection and replication. membranes are used to form vesicles (2Bienz K. Egger D. Pasamontes L. Virology. 1987; 160: 220-226Crossref PubMed Scopus (213) Google whereas for the Golgi with vesicles R. Egger D. Gosert R. Schaub G. Landmann L. Bienz K. J. Virol. 1998; 72: 8578-8585Crossref PubMed Google Scholar). This that the a and for structures in but membranes of the Golgi the it not that a between the and Golgi One is that the RNA and with the of its to the and as a of E. PubMed Scopus Google Scholar) for replication the of the It that the of of replication complex-associated vesicles the of to but not in knockdown cells that complex the We found viral protein synthesis in infection in knockdown One is that the viral replication a it the replication complexes from the to is that 2C protein also with other of the family 2C and we the that the other proteins for but with a of the cellular of viral replication in cells is a of cellular have been identified to interact with the viral nonstructural protein and the region and have been to replication in Virology. 1998; PubMed Scopus Google cellular protein with other nonstructural proteins has been is thus the cellular protein to be identified that with a viral nonstructural protein involved in the formation of the viral replication complex. of family of proteins how the replication complex be from its We have a role for the family of proteins in the of infection with and have that has an essential and direct role in These of how viruses the formation of the replication complex and into the host-encoded cellular We and for the and RNA, We the from the of with