Poliovirus 2C Protein Forms Homo-oligomeric Structures Required for ATPase Activity
2009; Elsevier BV; Volume: 284; Issue: 33 Linguagem: Inglês
10.1074/jbc.m109.031807
ISSN1083-351X
AutoresPeter L. Adams, Eaazhisai Kandiah, Grégory Effantin, Alasdair C. Steven, Ellie Ehrenfeld,
Tópico(s)Viral gastroenteritis research and epidemiology
ResumoThe poliovirus protein 2C plays an essential role in viral RNA replication, although its precise biochemical activities or structural requirements have not been elucidated. The protein has several distinctive properties, including ATPase activity and membrane and RNA binding, that are conserved among orthologs of many positive-strand RNA viruses. Sequence alignments have placed these proteins in the SF3 helicase family, a subset of the AAA+ ATPase superfamily. A feature common to AAA+ proteins is the formation of oligomeric rings that are essential for their catalytic functions. Here we show that a recombinant protein, MBP-2C, in which maltose-binding protein was fused to 2C, formed soluble oligomers and that ATPase activity was restricted to oligomer-containing fractions from gel-filtration chromatography. The active fraction was visualized by negative-staining electron microscopy as ring-like particles composed of 5–8 protomers. This conclusion was confirmed by mass measurements obtained by scanning transmission electron microscopy. Mutation of amino acid residues in the 2C nucleotide-binding domain demonstrated that loss of the ability to bind or hydrolyze ATP did not affect oligomerization. Co-expression of active MBP-2C and inactive mutant proteins generated mixed oligomers that exhibited little ATPase activity, suggesting that incorporation of inactive subunits eliminates the function of the entire particle. Finally, deletion of the N-terminal 38 amino acids blocked oligomerization of the fusion protein and eliminated ATPase activity, despite retention of an unaltered nucleotide-binding domain. The poliovirus protein 2C plays an essential role in viral RNA replication, although its precise biochemical activities or structural requirements have not been elucidated. The protein has several distinctive properties, including ATPase activity and membrane and RNA binding, that are conserved among orthologs of many positive-strand RNA viruses. Sequence alignments have placed these proteins in the SF3 helicase family, a subset of the AAA+ ATPase superfamily. A feature common to AAA+ proteins is the formation of oligomeric rings that are essential for their catalytic functions. Here we show that a recombinant protein, MBP-2C, in which maltose-binding protein was fused to 2C, formed soluble oligomers and that ATPase activity was restricted to oligomer-containing fractions from gel-filtration chromatography. The active fraction was visualized by negative-staining electron microscopy as ring-like particles composed of 5–8 protomers. This conclusion was confirmed by mass measurements obtained by scanning transmission electron microscopy. Mutation of amino acid residues in the 2C nucleotide-binding domain demonstrated that loss of the ability to bind or hydrolyze ATP did not affect oligomerization. Co-expression of active MBP-2C and inactive mutant proteins generated mixed oligomers that exhibited little ATPase activity, suggesting that incorporation of inactive subunits eliminates the function of the entire particle. Finally, deletion of the N-terminal 38 amino acids blocked oligomerization of the fusion protein and eliminated ATPase activity, despite retention of an unaltered nucleotide-binding domain. Poliovirus is the prototype member of the Picornaviridae family. The 7.5-kb positive sense RNA genome encodes both capsid and noncapsid proteins that are necessary for virus replication. Translation of the viral genome into a single polyprotein yields both functionally distinct precursors and final products that are required for productive viral replication via an orchestrated series of co- and post-translational cleavage events catalyzed by viral proteinases. Replication of the viral RNA occurs in the cytoplasm, localized on the surfaces of newly formed membranous structures that develop after infection. Viral and host proteins involved in viral RNA replication form a poorly characterized, nuclease-resistant replication complex associated with the remodeled membrane structures. Numerous studies have demonstrated that viral protein 2C and its precursor 2BC play key roles in viral RNA replication, yet their actual biochemical functions in this complex reaction remain undefined (1Pincus S.E. Wimmer E. J. Virol. 1986; 60: 793-796Crossref PubMed Google Scholar, 2Mirzayan C. Wimmer E. Virology. 1992; 189: 547-555Crossref PubMed Scopus (66) Google Scholar, 3Johnson K.L. Sarnow P. J. Virol. 1991; 65: 4341-4349Crossref PubMed Google Scholar). Protein 2C has been shown to interact with other elements of the viral replication apparatus, including 3AB (4Yin J. Liu Y. Wimmer E. Paul A.V. J. Gen Virol. 2007; 88: 2259-2267Crossref PubMed Scopus (31) Google Scholar), 3C proteinase (5Banerjee R. Dasgupta A. J. Gen Virol. 2001; 82: 2621-2627Crossref PubMed Scopus (27) Google Scholar), and the cloverleaf structure at the 5′-end of the viral genome (6Banerjee R. Weidman M.K. Echeverri A. Kundu P. Dasgupta A. J. Virol. 2004; 78: 9243-9256Crossref PubMed Scopus (31) Google Scholar). More recently it was shown that reticulon-3, a cellular protein involved in membrane trafficking and endoplasmic reticulum structure, binds polioviral as well as other picornaviral 2C proteins, and this plays an essential albeit undefined role in the process of virus replication (7Tang W.F. Yang S.Y. Wu B.W. Jheng J.R. Chen Y.L. Shih C.H. Lin K.H. Lai H.C. Tang P. Horng J.T. J. Biol. Chem. 2007; 282: 5888-5898Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar).Poliovirus 2C is tightly associated with intracellular membranes and can be cross-linked to actively replicating viral RNA isolated from infected cells (8Bienz K. Egger D. Troxler M. Pasamontes L. J. Virol. 1990; 64: 1156-1163Crossref PubMed Google Scholar). In addition, individual expression of 2C or 2BC in mammalian cells induces the formation of reorganized membrane structures from the endoplasmic reticulum, and thus these proteins have been implicated in the formation of virus-induced replication complexes (9Cho M.W. Teterina N. Egger D. Bienz K. Ehrenfeld E. Virology. 1994; 202: 129-145Crossref PubMed Scopus (258) Google Scholar, 10Aldabe R. Carrasco L. Biochem. Biophys. Res. Commun. 1995; 206: 64-76Crossref PubMed Scopus (109) Google Scholar).Poliovirus 2C is a 329-amino acid protein that is relatively highly conserved among members of the Picornaviridae family and is predicted to contain at least three domains (see Fig. 3) (11Argos P. Kamer G. Nicklin M.J. Wimmer E. Nucleic Acids Res. 1984; 12: 7251-7267Crossref PubMed Scopus (160) Google Scholar, 12Teterina N.L. Gorbalenya A.E. Egger D. Bienz K. Ehrenfeld E. J. Virol. 1997; 71: 8962-8972Crossref PubMed Google Scholar). A centrally located nucleotide-binding domain (NBD) 2The abbreviations used are: NBDnucleotide-binding domainMBPmaltose-binding proteinHRVhuman rhinovirusTEVtobacco etch virusEMelectron microscopySTEMscanning transmission electron microscopyHCVhepatitis C virusGSTglutathione S-transferase. 2The abbreviations used are: NBDnucleotide-binding domainMBPmaltose-binding proteinHRVhuman rhinovirusTEVtobacco etch virusEMelectron microscopySTEMscanning transmission electron microscopyHCVhepatitis C virusGSTglutathione S-transferase. is the most highly conserved region of the protein. Flanking the NBD, at the N terminus of poliovirus 2C, is a region (amino acids 1–54) that contains a predicted amphipathic helix (amino acids 19–36) (13Paul A.V. Molla A. Wimmer E. Virology. 1994; 199: 188-199Crossref PubMed Scopus (64) Google Scholar, 14Teterina N.L. Gorbalenya A.E. Egger D. Bienz K. Rinaudo M.S. Ehrenfeld E. Virology. 2006; 344: 453-467Crossref PubMed Scopus (38) Google Scholar) that specifies localization of 2C to the membrane (15Echeverri A.C. Dasgupta A. Virology. 1995; 208: 540-553Crossref PubMed Scopus (86) Google Scholar). The C-terminal portion of the protein contains a small Cys-rich region that binds zinc (16Pfister T. Jones K.W. Wimmer E. J. Virol. 2000; 74: 334-343Crossref PubMed Scopus (70) Google Scholar), and a region that is thought to be involved in RNA binding and also may interact with membranes (12Teterina N.L. Gorbalenya A.E. Egger D. Bienz K. Ehrenfeld E. J. Virol. 1997; 71: 8962-8972Crossref PubMed Google Scholar, 17Rodríguez P.L. Carrasco L. J. Biol. Chem. 1995; 270: 10105-10112Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). Biochemical studies of purified poliovirus 2C fusion proteins have demonstrated that the protein manifests an ATPase and a much weaker GTPase activity (18Mirzayan C. Wimmer E. Virology. 1994; 199: 176-187Crossref PubMed Scopus (87) Google Scholar, 19Rodríguez P.L. Carrasco L. J. Biol. Chem. 1993; 268: 8105-8110Abstract Full Text PDF PubMed Google Scholar). Mutations in NBD signature sequences result in impairment or abrogation of viral RNA replication when introduced into full-length or replicon viral RNAs (2Mirzayan C. Wimmer E. Virology. 1992; 189: 547-555Crossref PubMed Scopus (66) Google Scholar, 20Teterina N.L. Kean K.M. Gorbalenya A.E. Agol V.I. Girard M. J. Gen. Virol. 1992; 73: 1977-1986Crossref PubMed Scopus (69) Google Scholar). The sensitivity of poliovirus RNA replication to millimolar concentrations of guanidine-HCl has been attributed to 2C: guanidine inhibits the ATPase activity of purified 2C protein in vitro, and viral mutants that are resistant or dependent for growth in the presence of guanidine harbor alterations in the NBD (21Tershak D.R. Can. J. Microbiol. 1985; 31: 1166-1168Crossref PubMed Scopus (2) Google Scholar, 22Tolskaya E.A. Romanova L.I. Kolesnikova M.S. Gmyl A.P. Gorbalenya A.E. Agol V.I. J. Mol. Biol. 1994; 236: 1310-1323Crossref PubMed Scopus (68) Google Scholar). In other domains of the protein, mutagenesis by insertion of sequences in the region C-terminal to the NBD causes temperature-dependent packaging defects (23Li J.P. Baltimore D. J. Virol. 1990; 64: 1102-1107Crossref PubMed Google Scholar), whereas gross changes to the upstream sequence in the region encompassing the predicted amphipathic helix also affect viral RNA replication (13Paul A.V. Molla A. Wimmer E. Virology. 1994; 199: 188-199Crossref PubMed Scopus (64) Google Scholar). Taken together, these data imply that 2C is a multifunctional protein.Comparative sequence alignments of the central NBD of picornaviral 2C proteins have grouped these proteins within the SF3 helicase family (24Gorbalenya A.E. Koonin E.V. Nucleic Acids Res. 1989; 17: 8413-8440Crossref PubMed Scopus (340) Google Scholar), although there is no evidence that poliovirus 2C protein has helicase activity or that such activity is necessary for virus replication (19Rodríguez P.L. Carrasco L. J. Biol. Chem. 1993; 268: 8105-8110Abstract Full Text PDF PubMed Google Scholar, 25Pfister T. Wimmer E. J. Biol. Chem. 1999; 274: 6992-7001Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). The SF3 helicase family was originally identified in and appears to be limited to the genomes of DNA and small RNA viruses (26Gorbalenya A.E. Koonin E.V. Donchenko A.P. Blinov V.M. FEBS Lett. 1988; 235: 16-24Crossref PubMed Scopus (210) Google Scholar, 27Gorbalenya A.E. Koonin E.V. Wolf Y.I. FEBS Lett. 1990; 262: 145-148Crossref PubMed Scopus (295) Google Scholar). The NBDs of SF3 family members include the characteristic Walker A and B motifs along with a distinguishing C-motif (sensor) within a 100- to 120-amino acid region. The Walker A motif is specified by a GXXXGK(T/S) signature, which interacts with the phosphates of ATP; the Walker B signature is defined by MDD, where the Asp (or Glu) residues interact with Mg2+ or water and contribute to nucleotide hydrolysis activity. Motif C consists of an Asn residue preceded by a run of hydrophobic amino acids located C-terminal to the Walker B motif (27Gorbalenya A.E. Koonin E.V. Wolf Y.I. FEBS Lett. 1990; 262: 145-148Crossref PubMed Scopus (295) Google Scholar).Although few biochemical or structural data are available for the picornaviral 2C proteins or other SF3 ATPases from RNA viruses, some SF3 helicase members encoded by DNA viruses have been quite well characterized (28Hickman A.B. Dyda F. Curr. Opin. Struct. Biol. 2005; 15: 77-85Crossref PubMed Scopus (80) Google Scholar, 29Enemark E.J. Joshua-Tor L. Curr. Opin. Struct. Biol. 2008; 18: 243-257Crossref PubMed Scopus (161) Google Scholar). These data indicate that DNA virus SF3 family proteins belong to the AAA+ superfamily, a functionally diverse group of proteins whose biological activities include protein folding, cytoskeletal regulation, and DNA replication (30Iyer L.M. Leipe D.D. Koonin E.V. Aravind L. J. Struct. Biol. 2004; 146: 11-31Crossref PubMed Scopus (628) Google Scholar). A common feature is the formation of higher order oligomers (predominantly hexamers and heptamers) that are essential for function. Indeed, structures of SV40 and papillomavirus E1 proteins resolved by x-ray crystallography revealed hexameric rings whose formation is dependent on the presence of ATP and magnesium (31Gai D. Zhao R. Li D. Finkielstein C.V. Chen X.S. Cell. 2004; 119: 47-60Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar, 32Enemark E.J. Joshua-Tor L. Nature. 2006; 442: 270-275Crossref PubMed Scopus (413) Google Scholar). These analyses showed that elements of two adjoining subunits are used to form the nucleotide binding site in the cleft between them. The nucleotide thus facilitates the formation and stability of a functional oligomer (33Gai D. Li D. Finkielstein C.V. Ott R.D. Taneja P. Fanning E. Chen X.S. J. Biol. Chem. 2004; 279: 38952-38959Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). Structure-based sequence alignment of these DNA SF3 helicases highlighted additional conserved residues that contribute to the nucleotide binding site in an adjacent protomer (29Enemark E.J. Joshua-Tor L. Curr. Opin. Struct. Biol. 2008; 18: 243-257Crossref PubMed Scopus (161) Google Scholar).Poliovirus 2C differs significantly from SF3 DNA helicases, both in terms of overall size and properties such as membrane association. The low but significant homology among members of the SF3 family may indicate that there is commonality in biophysical properties such as oligomerization. The accumulated biochemical and genetic data for 2C oligomerization are contradictory. Yeast two-hybrid analysis of 2C proteins from polio (34Cuconati A. Xiang W. Lahser F. Pfister T. Wimmer E. J. Virol. 1998; 72: 1297-1307Crossref PubMed Google Scholar) and related coxsackievirus (35de Jong A.S. Schrama I.W. Willems P.H. Galama J.M. Melchers W.J. van Kuppeveld F.J. J. Gen Virol. 2002; 83: 783-793Crossref PubMed Scopus (46) Google Scholar) and teschovirus (36Zell R. Seitz S. Henke A. Munder T. Wutzler P. J. Gen Virol. 2005; 86: 2763-2768Crossref PubMed Scopus (12) Google Scholar) failed to demonstrate a strong propensity for oligomerization of 2C, whereas biochemical studies (34Cuconati A. Xiang W. Lahser F. Pfister T. Wimmer E. J. Virol. 1998; 72: 1297-1307Crossref PubMed Google Scholar) and a mammalian two-hybrid study (37Teterina N.L. Levenson E. Rinaudo M.S. Egger D. Bienz K. Gorbalenya A.E. Ehrenfeld E. J. Virol. 2006; 80: 5327-5337Crossref PubMed Scopus (39) Google Scholar) of poliovirus proteins suggested stable 2C-2C interactions may occur. Furthermore, genetic studies examining the mechanism of virus sensitivity or resistance to guanidine were interpreted as indicating that poliovirus 2C functioned as an oligomer (22Tolskaya E.A. Romanova L.I. Kolesnikova M.S. Gmyl A.P. Gorbalenya A.E. Agol V.I. J. Mol. Biol. 1994; 236: 1310-1323Crossref PubMed Scopus (68) Google Scholar). As part of our effort to understand the contribution(s) of 2C in the poliovirus replication cycle, we sought to determine the oligomerization status of the 2C protein and potential commonality with other related SF3 DNA helicases. Poliovirus is the prototype member of the Picornaviridae family. The 7.5-kb positive sense RNA genome encodes both capsid and noncapsid proteins that are necessary for virus replication. Translation of the viral genome into a single polyprotein yields both functionally distinct precursors and final products that are required for productive viral replication via an orchestrated series of co- and post-translational cleavage events catalyzed by viral proteinases. Replication of the viral RNA occurs in the cytoplasm, localized on the surfaces of newly formed membranous structures that develop after infection. Viral and host proteins involved in viral RNA replication form a poorly characterized, nuclease-resistant replication complex associated with the remodeled membrane structures. Numerous studies have demonstrated that viral protein 2C and its precursor 2BC play key roles in viral RNA replication, yet their actual biochemical functions in this complex reaction remain undefined (1Pincus S.E. Wimmer E. J. Virol. 1986; 60: 793-796Crossref PubMed Google Scholar, 2Mirzayan C. Wimmer E. Virology. 1992; 189: 547-555Crossref PubMed Scopus (66) Google Scholar, 3Johnson K.L. Sarnow P. J. Virol. 1991; 65: 4341-4349Crossref PubMed Google Scholar). Protein 2C has been shown to interact with other elements of the viral replication apparatus, including 3AB (4Yin J. Liu Y. Wimmer E. Paul A.V. J. Gen Virol. 2007; 88: 2259-2267Crossref PubMed Scopus (31) Google Scholar), 3C proteinase (5Banerjee R. Dasgupta A. J. Gen Virol. 2001; 82: 2621-2627Crossref PubMed Scopus (27) Google Scholar), and the cloverleaf structure at the 5′-end of the viral genome (6Banerjee R. Weidman M.K. Echeverri A. Kundu P. Dasgupta A. J. Virol. 2004; 78: 9243-9256Crossref PubMed Scopus (31) Google Scholar). More recently it was shown that reticulon-3, a cellular protein involved in membrane trafficking and endoplasmic reticulum structure, binds polioviral as well as other picornaviral 2C proteins, and this plays an essential albeit undefined role in the process of virus replication (7Tang W.F. Yang S.Y. Wu B.W. Jheng J.R. Chen Y.L. Shih C.H. Lin K.H. Lai H.C. Tang P. Horng J.T. J. Biol. Chem. 2007; 282: 5888-5898Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). Poliovirus 2C is tightly associated with intracellular membranes and can be cross-linked to actively replicating viral RNA isolated from infected cells (8Bienz K. Egger D. Troxler M. Pasamontes L. J. Virol. 1990; 64: 1156-1163Crossref PubMed Google Scholar). In addition, individual expression of 2C or 2BC in mammalian cells induces the formation of reorganized membrane structures from the endoplasmic reticulum, and thus these proteins have been implicated in the formation of virus-induced replication complexes (9Cho M.W. Teterina N. Egger D. Bienz K. Ehrenfeld E. Virology. 1994; 202: 129-145Crossref PubMed Scopus (258) Google Scholar, 10Aldabe R. Carrasco L. Biochem. Biophys. Res. Commun. 1995; 206: 64-76Crossref PubMed Scopus (109) Google Scholar). Poliovirus 2C is a 329-amino acid protein that is relatively highly conserved among members of the Picornaviridae family and is predicted to contain at least three domains (see Fig. 3) (11Argos P. Kamer G. Nicklin M.J. Wimmer E. Nucleic Acids Res. 1984; 12: 7251-7267Crossref PubMed Scopus (160) Google Scholar, 12Teterina N.L. Gorbalenya A.E. Egger D. Bienz K. Ehrenfeld E. J. Virol. 1997; 71: 8962-8972Crossref PubMed Google Scholar). A centrally located nucleotide-binding domain (NBD) 2The abbreviations used are: NBDnucleotide-binding domainMBPmaltose-binding proteinHRVhuman rhinovirusTEVtobacco etch virusEMelectron microscopySTEMscanning transmission electron microscopyHCVhepatitis C virusGSTglutathione S-transferase. 2The abbreviations used are: NBDnucleotide-binding domainMBPmaltose-binding proteinHRVhuman rhinovirusTEVtobacco etch virusEMelectron microscopySTEMscanning transmission electron microscopyHCVhepatitis C virusGSTglutathione S-transferase. is the most highly conserved region of the protein. Flanking the NBD, at the N terminus of poliovirus 2C, is a region (amino acids 1–54) that contains a predicted amphipathic helix (amino acids 19–36) (13Paul A.V. Molla A. Wimmer E. Virology. 1994; 199: 188-199Crossref PubMed Scopus (64) Google Scholar, 14Teterina N.L. Gorbalenya A.E. Egger D. Bienz K. Rinaudo M.S. Ehrenfeld E. Virology. 2006; 344: 453-467Crossref PubMed Scopus (38) Google Scholar) that specifies localization of 2C to the membrane (15Echeverri A.C. Dasgupta A. Virology. 1995; 208: 540-553Crossref PubMed Scopus (86) Google Scholar). The C-terminal portion of the protein contains a small Cys-rich region that binds zinc (16Pfister T. Jones K.W. Wimmer E. J. Virol. 2000; 74: 334-343Crossref PubMed Scopus (70) Google Scholar), and a region that is thought to be involved in RNA binding and also may interact with membranes (12Teterina N.L. Gorbalenya A.E. Egger D. Bienz K. Ehrenfeld E. J. Virol. 1997; 71: 8962-8972Crossref PubMed Google Scholar, 17Rodríguez P.L. Carrasco L. J. Biol. Chem. 1995; 270: 10105-10112Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). Biochemical studies of purified poliovirus 2C fusion proteins have demonstrated that the protein manifests an ATPase and a much weaker GTPase activity (18Mirzayan C. Wimmer E. Virology. 1994; 199: 176-187Crossref PubMed Scopus (87) Google Scholar, 19Rodríguez P.L. Carrasco L. J. Biol. Chem. 1993; 268: 8105-8110Abstract Full Text PDF PubMed Google Scholar). Mutations in NBD signature sequences result in impairment or abrogation of viral RNA replication when introduced into full-length or replicon viral RNAs (2Mirzayan C. Wimmer E. Virology. 1992; 189: 547-555Crossref PubMed Scopus (66) Google Scholar, 20Teterina N.L. Kean K.M. Gorbalenya A.E. Agol V.I. Girard M. J. Gen. Virol. 1992; 73: 1977-1986Crossref PubMed Scopus (69) Google Scholar). The sensitivity of poliovirus RNA replication to millimolar concentrations of guanidine-HCl has been attributed to 2C: guanidine inhibits the ATPase activity of purified 2C protein in vitro, and viral mutants that are resistant or dependent for growth in the presence of guanidine harbor alterations in the NBD (21Tershak D.R. Can. J. Microbiol. 1985; 31: 1166-1168Crossref PubMed Scopus (2) Google Scholar, 22Tolskaya E.A. Romanova L.I. Kolesnikova M.S. Gmyl A.P. Gorbalenya A.E. Agol V.I. J. Mol. Biol. 1994; 236: 1310-1323Crossref PubMed Scopus (68) Google Scholar). In other domains of the protein, mutagenesis by insertion of sequences in the region C-terminal to the NBD causes temperature-dependent packaging defects (23Li J.P. Baltimore D. J. Virol. 1990; 64: 1102-1107Crossref PubMed Google Scholar), whereas gross changes to the upstream sequence in the region encompassing the predicted amphipathic helix also affect viral RNA replication (13Paul A.V. Molla A. Wimmer E. Virology. 1994; 199: 188-199Crossref PubMed Scopus (64) Google Scholar). Taken together, these data imply that 2C is a multifunctional protein. nucleotide-binding domain maltose-binding protein human rhinovirus tobacco etch virus electron microscopy scanning transmission electron microscopy hepatitis C virus glutathione S-transferase. nucleotide-binding domain maltose-binding protein human rhinovirus tobacco etch virus electron microscopy scanning transmission electron microscopy hepatitis C virus glutathione S-transferase. Comparative sequence alignments of the central NBD of picornaviral 2C proteins have grouped these proteins within the SF3 helicase family (24Gorbalenya A.E. Koonin E.V. Nucleic Acids Res. 1989; 17: 8413-8440Crossref PubMed Scopus (340) Google Scholar), although there is no evidence that poliovirus 2C protein has helicase activity or that such activity is necessary for virus replication (19Rodríguez P.L. Carrasco L. J. Biol. Chem. 1993; 268: 8105-8110Abstract Full Text PDF PubMed Google Scholar, 25Pfister T. Wimmer E. J. Biol. Chem. 1999; 274: 6992-7001Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). The SF3 helicase family was originally identified in and appears to be limited to the genomes of DNA and small RNA viruses (26Gorbalenya A.E. Koonin E.V. Donchenko A.P. Blinov V.M. FEBS Lett. 1988; 235: 16-24Crossref PubMed Scopus (210) Google Scholar, 27Gorbalenya A.E. Koonin E.V. Wolf Y.I. FEBS Lett. 1990; 262: 145-148Crossref PubMed Scopus (295) Google Scholar). The NBDs of SF3 family members include the characteristic Walker A and B motifs along with a distinguishing C-motif (sensor) within a 100- to 120-amino acid region. The Walker A motif is specified by a GXXXGK(T/S) signature, which interacts with the phosphates of ATP; the Walker B signature is defined by MDD, where the Asp (or Glu) residues interact with Mg2+ or water and contribute to nucleotide hydrolysis activity. Motif C consists of an Asn residue preceded by a run of hydrophobic amino acids located C-terminal to the Walker B motif (27Gorbalenya A.E. Koonin E.V. Wolf Y.I. FEBS Lett. 1990; 262: 145-148Crossref PubMed Scopus (295) Google Scholar). Although few biochemical or structural data are available for the picornaviral 2C proteins or other SF3 ATPases from RNA viruses, some SF3 helicase members encoded by DNA viruses have been quite well characterized (28Hickman A.B. Dyda F. Curr. Opin. Struct. Biol. 2005; 15: 77-85Crossref PubMed Scopus (80) Google Scholar, 29Enemark E.J. Joshua-Tor L. Curr. Opin. Struct. Biol. 2008; 18: 243-257Crossref PubMed Scopus (161) Google Scholar). These data indicate that DNA virus SF3 family proteins belong to the AAA+ superfamily, a functionally diverse group of proteins whose biological activities include protein folding, cytoskeletal regulation, and DNA replication (30Iyer L.M. Leipe D.D. Koonin E.V. Aravind L. J. Struct. Biol. 2004; 146: 11-31Crossref PubMed Scopus (628) Google Scholar). A common feature is the formation of higher order oligomers (predominantly hexamers and heptamers) that are essential for function. Indeed, structures of SV40 and papillomavirus E1 proteins resolved by x-ray crystallography revealed hexameric rings whose formation is dependent on the presence of ATP and magnesium (31Gai D. Zhao R. Li D. Finkielstein C.V. Chen X.S. Cell. 2004; 119: 47-60Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar, 32Enemark E.J. Joshua-Tor L. Nature. 2006; 442: 270-275Crossref PubMed Scopus (413) Google Scholar). These analyses showed that elements of two adjoining subunits are used to form the nucleotide binding site in the cleft between them. The nucleotide thus facilitates the formation and stability of a functional oligomer (33Gai D. Li D. Finkielstein C.V. Ott R.D. Taneja P. Fanning E. Chen X.S. J. Biol. Chem. 2004; 279: 38952-38959Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). Structure-based sequence alignment of these DNA SF3 helicases highlighted additional conserved residues that contribute to the nucleotide binding site in an adjacent protomer (29Enemark E.J. Joshua-Tor L. Curr. Opin. Struct. Biol. 2008; 18: 243-257Crossref PubMed Scopus (161) Google Scholar). Poliovirus 2C differs significantly from SF3 DNA helicases, both in terms of overall size and properties such as membrane association. The low but significant homology among members of the SF3 family may indicate that there is commonality in biophysical properties such as oligomerization. The accumulated biochemical and genetic data for 2C oligomerization are contradictory. Yeast two-hybrid analysis of 2C proteins from polio (34Cuconati A. Xiang W. Lahser F. Pfister T. Wimmer E. J. Virol. 1998; 72: 1297-1307Crossref PubMed Google Scholar) and related coxsackievirus (35de Jong A.S. Schrama I.W. Willems P.H. Galama J.M. Melchers W.J. van Kuppeveld F.J. J. Gen Virol. 2002; 83: 783-793Crossref PubMed Scopus (46) Google Scholar) and teschovirus (36Zell R. Seitz S. Henke A. Munder T. Wutzler P. J. Gen Virol. 2005; 86: 2763-2768Crossref PubMed Scopus (12) Google Scholar) failed to demonstrate a strong propensity for oligomerization of 2C, whereas biochemical studies (34Cuconati A. Xiang W. Lahser F. Pfister T. Wimmer E. J. Virol. 1998; 72: 1297-1307Crossref PubMed Google Scholar) and a mammalian two-hybrid study (37Teterina N.L. Levenson E. Rinaudo M.S. Egger D. Bienz K. Gorbalenya A.E. Ehrenfeld E. J. Virol. 2006; 80: 5327-5337Crossref PubMed Scopus (39) Google Scholar) of poliovirus proteins suggested stable 2C-2C interactions may occur. Furthermore, genetic studies examining the mechanism of virus sensitivity or resistance to guanidine were interpreted as indicating that poliovirus 2C functioned as an oligomer (22Tolskaya E.A. Romanova L.I. Kolesnikova M.S. Gmyl A.P. Gorbalenya A.E. Agol V.I. J. Mol. Biol. 1994; 236: 1310-1323Crossref PubMed Scopus (68) Google Scholar). As part of our effort to understand the contribution(s) of 2C in the poliovirus replication cycle, we sought to determine the oligomerization status of the 2C protein and potential commonality with other related SF3 DNA helicases. We thank Dr. Martha Simon at the Brookhaven STEM facility for the provision of STEM data. Dr. Natalya Teterina provided plasmids encoding chimeric 2C proteins.
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