Uridylylation of the Potyvirus VPg by Viral Replicase NIb Correlates with the Nucleotide Binding Capacity of VPg

Abstract

Poty- and picornaviruses share similar genome organizations and polyprotein processing strategies. By analogy to picornaviruses it has been proposed that the genome-linked protein VPg may serve as a primer for genome replication of potyviruses. The multifunctional VPg of potato virus A (PVA; genus Potyvirus) was found to be uridylylated by NIb, the RNA polymerase of PVA. The nucleotidylation activity of NIb is more efficient in the presence of Mn2+ than Mg2+ and does not require an RNA template. Our results suggest that the nucleotidylation reaction exhibits weak preference for UTP over the other NTPs. An NTP-binding experiment with oxidized [α-32P]UTP revealed that PVA VPg contains an NTP-binding site. Deletion of a 7-amino acid-long putative NTP-binding site from VPg reduced nucleotide-binding capacity and debilitated uridylylation reaction. These results provide evidence that VPg may play a similar role in RNA synthesis of potyviruses as it does in the case of picornaviruses. Poty- and picornaviruses share similar genome organizations and polyprotein processing strategies. By analogy to picornaviruses it has been proposed that the genome-linked protein VPg may serve as a primer for genome replication of potyviruses. The multifunctional VPg of potato virus A (PVA; genus Potyvirus) was found to be uridylylated by NIb, the RNA polymerase of PVA. The nucleotidylation activity of NIb is more efficient in the presence of Mn2+ than Mg2+ and does not require an RNA template. Our results suggest that the nucleotidylation reaction exhibits weak preference for UTP over the other NTPs. An NTP-binding experiment with oxidized [α-32P]UTP revealed that PVA VPg contains an NTP-binding site. Deletion of a 7-amino acid-long putative NTP-binding site from VPg reduced nucleotide-binding capacity and debilitated uridylylation reaction. These results provide evidence that VPg may play a similar role in RNA synthesis of potyviruses as it does in the case of picornaviruses. Potato virus A (PVA 1The abbreviations used are: PVA, potato virus A; PV, poliovirus; TVMV, tobacco vein mottling virus; TEV, tobacco etch virus; RdRp, RNA-dependent RNA polymerase; RT, room temperature; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; wt, wild type.1The abbreviations used are: PVA, potato virus A; PV, poliovirus; TVMV, tobacco vein mottling virus; TEV, tobacco etch virus; RdRp, RNA-dependent RNA polymerase; RT, room temperature; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; wt, wild type.; genus Potyvirus; family Potyviridae) has a single-stranded, messenger polarity RNA genome of 9,565 nucleotides. The entire PVA genome is expressed as a polyprotein, which is subsequently processed into 10 mature proteins by three different viral proteinases (1Merits A. Rajamäki M.-L. Lindholm P. Runeberg-Roos P. Kekarainen T. Puustinen P. Mäkeläinen K. Valkonen J.P.T. Saarma M. J. Gen. Virol. 2002; 83: 1211-1221Crossref PubMed Scopus (45) Google Scholar). Potyviruses resemble picornaviruses in genome organization and polyprotein processing strategy (2Sadowy E. Milner M. Hanni A.L. Adv. Virus Res. 2001; 57: 185-262Crossref PubMed Google Scholar). The genomic RNA of picornaviruses and potyviruses is covalently linked at the 5′-end to the small, virally encoded protein VPg (3Ambros V. Baltimore D. J. Biol. Chem. 1978; 253: 5263-5266Abstract Full Text PDF PubMed Google Scholar, 4Rothberg P.G. Harris T.J.R. Nomoto A. Wimmer E. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 4868-4872Crossref PubMed Scopus (160) Google Scholar, 5Oruetxebarria I. Guo D. Merits A. Mäkinen K. Saarma M. Valkonen J.P.T. Virus Res. 2001; 73: 103-112Crossref PubMed Scopus (45) Google Scholar). The N-terminal VPg domain is slowly released from NIa by the proteolytic activity of the C-terminal NIa-Pro domain in the course of potyvirus infection. It is essential for replication that proteolytic processing at the suboptimal NIa internal cleavage site occurs (6Carrington J.C. Hadelman R. Dolja V.V. Restrepo-Hartwig M.A. J. Virol. 1993; 67: 6995-7000Crossref PubMed Google Scholar). Potyviral VPg is a multifunctional protein (as reviewed in Ref. 2Sadowy E. Milner M. Hanni A.L. Adv. Virus Res. 2001; 57: 185-262Crossref PubMed Google Scholar), which is also exposed at one end of the virion (7Puustinen P. Rajamäki M.-L. Ivanov K. Valkonen J.P.T. Mäkinen K. J. Virol. 2002; 76: 12703-12711Crossref PubMed Scopus (55) Google Scholar). The size of potyviral VPg is substantially larger than that of poliovirus (PV) VPg, and they do not share any obvious sequence homology. PV VPg represents the most advanced model for understanding the role of VPg as a primer for the viral replicase. PV VPg is a small peptide containing only 22 amino acids. It is genome-linked via a bond between the hydroxyl group of a tyrosine residue and the α-phosphate group of uridylic acid. In vitro uridylylation of PV VPg catalyzed by the viral polymerase resulted in a VPg-linked poly(U) (8Paul V.A. Boom J. Filippov D. Wimmer E. Nature. 1998; 393: 280-284Crossref PubMed Scopus (297) Google Scholar). Replication of picornaviruses requires cis-acting RNA elements, termed cre (9McKnight K.L. Lemon S.M. J. Virol. 1996; 70: 1941-1952Crossref PubMed Google Scholar). In PV this small RNA hairpin structure is within the coding region of protein 2C. Its presence in the in vitro reaction mixture stimulated uridylylation as compared with a reaction with poly(A) as a template (10Paul V.A. Rieder E. Kim D.W. Boom J.H. Wimmer E. J. Virol. 2000; 74: 10359-10370Crossref PubMed Scopus (236) Google Scholar). The PV cre-dependent VPg uridylylation is required only for the synthesis of new positive strands, whereas the minus strand synthesis is VPg-dependent but cre-independent (11Murray K.E. Barton D.J. J. Virol. 2003; 77: 4739-4750Crossref PubMed Scopus (108) Google Scholar). VPg of tobacco vein mottling virus (TVMV; genus Potyvirus) is linked to the viral RNA via Tyr-60 and TVMV carrying the mutation Tyr-60 to Ala is not able to replicate in protoplasts (12Murphy J.F. Klein P.G. Hunt A.G. Shaw J.G. Virology. 1996; 220: 535-538Crossref PubMed Scopus (83) Google Scholar, 13Murphy J.F. Rychlik W. Rhoads R.A. Hunt A.G. Shaw J.G. J. Virol. 1991; 65: 511-513Crossref PubMed Google Scholar). Similarly, substitution of the corresponding Tyr with Ala in another potyvirus, tobacco etch virus (TEV), makes it amplification-defective (14Schaad M.C. Haldeman-Cahill R. Cronin S. Carrington J.C. J. Virol. 1996; 70: 7039-7048Crossref PubMed Google Scholar). Nuclear inclusion protein NIb is the viral RNA-dependent RNA polymerase (RdRp) responsible for genome replication of potyviruses (15Hong Y. Hunt A. Virology. 1996; 226: 146-151Crossref PubMed Scopus (75) Google Scholar). It contains an amino acid triplet Gly-Asp-Asp, which is universally conserved among RdRps (16Kamer G. Argos P. Nucleic Acids Res. 1984; 12: 7269-7282Crossref PubMed Scopus (603) Google Scholar), and when NIb of TEV was mutated at this motif, the virus was unable to replicate (17Li X.H. Carrington J.C. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 457-461Crossref PubMed Scopus (55) Google Scholar). The NIb of TEV interacts specifically with the protease part of NIa (18Li X.H. Valdez P. Olvera R.E. Carrington J.C. J. Virol. 1997; 71: 1598-1607Crossref PubMed Google Scholar), whereas the NIb of TVMV can interact with the NIa-VPg, as shown in vitro (19Fellers J. Wan J. Hong Y. Collins G.B. Hunt A.G. J. Gen. Virol. 1998; 79: 2043-2049Crossref PubMed Scopus (60) Google Scholar) and also in vivo with the yeast two-hybrid system (20Hong Y. Levay K. Murphy J.F. Klein P.G. Shaw J.G. Hunt A.G. Virology. 1995; 214: 159-166Crossref PubMed Scopus (77) Google Scholar). Taken together, these findings have supported the view that VPg may serve as a primer for genome replication of potyviruses. However, no direct evidence to support this hypothesis is available. Here we show that the recombinant PVA VPg can be uridylylated by purified recombinant PVA NIb in an in vitro reaction and that VPg is an NTP-binding protein. Deletion of a 7-amino acid-long putative NTP-binding site from VPg led to a reduced nucleotide-binding capacity and debilitated uridylylation reaction. These results increase our understanding of the mechanisms involved in potyvirus RNA synthesis. Recombinant Protein Expression—(His)6/pQE (Qiagen) constructs for PVA VPg and NIb expressions were made as described previously (21Merits A. Guo D. Saarma M. J. Gen. Virol. 1998; 79: 3123-3127Crossref PubMed Scopus (61) Google Scholar). Both proteins were expressed in Escherichia coli strain M15[REP4] cells. VPg was purified by using Ni2+-nitrilotriacetic acid-agarose under denaturing conditions according to standard protocol. Purified VPg proteins were refolded by a rapid dialysis procedure shown previously to recover the protein-protein binding properties of PVA proteins (22Merits A. Guo D. Järvekülg L. Saarma M. Virology. 1999; 263: 15-22Crossref PubMed Scopus (77) Google Scholar). PVA NIb was purified under native conditions according to standard protocol with the following modifications. Bacterial cell pellets were resuspended in binding buffer A (50 mm NaH2PO4, pH 8.0, 1 m NaCl, 20 mm imidazole, 10% glycerol, 0,1% Tween, and 1 μm phenylmethylsulfonyl fluoride) and then in a cell at were at for 20 at The were to a Ni2+-nitrilotriacetic acid-agarose proteins were and by in with buffer mm in buffer and buffer mm in buffer and to In was as the of from [α-32P]UTP into the purified PVA VPg in an in vitro reaction. conditions were as described previously (8Paul V.A. Boom J. Filippov D. Wimmer E. Nature. 1998; 393: 280-284Crossref PubMed Scopus (297) Google Scholar). The were at room 22 for in a of containing of [α-32P]UTP of PVA VPg, a of NIb 10 mm pH mm of and of when were with buffer they were purified for The were by in proteins were and by with a and In VPg was in vitro uridylylated as described in the presence of one of the following of the were and The reaction was 20 containing of [α-32P]UTP 10 mm pH mm of of NIb, and of poly(A) template in of the of at RT, the were and proteins were by were to with a and the into the VPg the nucleotidylation reaction was of proteins were uridylylated with [α-32P]UTP in the presence and of a poly(A) template in standard were with in and with acid in VPg were the to the protein from the The was with in and proteins were with acid into a The were by and into a buffer containing m m CHAPS, were then to pH and to with using the following for at and then at 20 1 at 1 at and 1 at by a from to for 1 and at V. the the was to the in buffer (50 mm pH m were to the pH in were and VPg was VPg was by and the were with and The containing the protein of were and with in mm acid The protein was the with 1 of in mm in 10% The were released from the by of then and with and in of pH The peptide were 20 The was at for 22 in pH The was to in the by in at were by of the in of the VPg was purified to the of [α-32P]UTP according to the that 10 of were with 0,1% acid. VPg proteins were and with Protein was in of m The were by as described in Ref. P. Rajamäki M.-L. Ivanov K. Valkonen J.P.T. Mäkinen K. J. Virol. 2002; 76: 12703-12711Crossref PubMed Scopus (55) Google acid were with and amino were from and were with a using containing of VPg protein at a of in were between the to with 10 and a at were for and the protein were with were as described and of at different of in the uridylylation reaction buffer were to the reaction The uridylylated were to and by with and by NTP-binding was oxidized with mm in the in the presence of mm at The was reduced with UTP was to PVA VPg by the used previously P. J. Biol. Chem. Full Text PDF PubMed Google Scholar, S. E. 1995; PubMed Scopus Google Scholar). mixture of oxidized 10 mm pH mm mm and of PVA VPg in a of were at for The reaction were with any reaction were and The of PVA VPg by PVA VPg and NIb were expressed as N-terminal in E. PVA VPg was purified by in denaturing conditions and was then refolded by have shown previously that PVA VPg by using this part in protein-protein (22Merits A. Guo D. Järvekülg L. Saarma M. Virology. 1999; 263: 15-22Crossref PubMed Scopus (77) Google Scholar) and can be by (7Puustinen P. Rajamäki M.-L. Ivanov K. Valkonen J.P.T. Mäkinen K. J. Virol. 2002; 76: 12703-12711Crossref PubMed Scopus (55) Google Scholar, Puustinen P. Merits A. Saarma M. Mäkinen K. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). of NIb was in native this was required for the polymerase to be in the uridylylation reaction. the proteins were purified with Ni2+-nitrilotriacetic acid they were by at the of for VPg and for NIb was in the not The purified PVA VPg was for uridylylation in an in vitro reaction containing the purified PVA NIb and [α-32P]UTP in the presence of mm The reaction were by and by with with PVA VPg was found to be uridylylated by NIb protein was in the of VPg that the to recombinant of NIb from the reaction mixture also uridylylation that the the not to UTP to VPg and that the VPg not any activity of of NIb in reaction led to uridylylation of the uridylylation reaction for an at not The NIb PVA VPg a for is that and RNA require a for Mn2+ was from the reaction mixture in a with in a no uridylylation was the presence of a was for NIb uridylylation which was required for an Mg2+ and Mn2+ were these to as of the RNA and the nucleotidylation reaction. The results that NIb has a preference for Mn2+ over Mg2+ The capacity of Mg2+ to support nucleotidylation reaction was 10 than that of Mn2+ in an were not Mn2+ can support the nucleotidylation reaction at from to mm not for a for the was for the polymerase of the and also for PV J. L. M. Nucleic Acids Res. PubMed Scopus Google Scholar, S. D. S. R. J. Virol. 2003; 77: PubMed Scopus Google Scholar). these for a binding site the presence of the of nucleotidylation A of in the presence of a of was of the nucleotidylation reaction that Mg2+ and Mn2+ do not for the site. A of the in in the and of an RNA presence of an RNA template is an for PV VPg in to virus (8Paul V.A. Boom J. Filippov D. Wimmer E. Nature. 1998; 393: 280-284Crossref PubMed Scopus (297) Google Scholar, A. M. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Our that under the conditions the PVA system not require a template. of the poly(A) template not the uridylylation reaction but the synthesis of a that not into the and The of this when more NIb (RdRp) was to the reaction mixture we that the of this resulted from a reaction catalyzed by PVA The synthesis was of the presence of VPg that this reaction was not by no VPg was by at the of the in this It is that the is a poly(U) from reaction catalyzed by An for the of the is a activity of The of PV has an activity in to TVMV NIb (15Hong Y. Hunt A. Virology. 1996; 226: 146-151Crossref PubMed Scopus (75) Google Scholar, Y. J. Virol. PubMed Google Scholar). the PV RNA the more than the poly(A) (10Paul V.A. Rieder E. Kim D.W. Boom J.H. Wimmer E. J. Virol. 2000; 74: 10359-10370Crossref PubMed Scopus (236) Google Scholar), the PVA not any for the VPg uridylylation reaction not the the presence of poly(A) PVA RNA a small in the of the uridylylated VPg which is most to a of NIb for the uridylylation reaction in the presence of a reaction and more for the of the VPg VPg, carrying more than one covalently linked has been found in Baltimore D. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). to is most of the reaction. the of uridylic acid were linked to PVA VPg the uridylylation in the presence and in the of the RNA was to VPg no VPg was in the in the presence of the poly(A) template the of [α-32P]UTP with VPg from the reaction with and RNA were found in the and as that VPg were However, of VPg protein a and of this to uridylylated VPg proteins revealed similar not within the purified VPg protein VPg uridylylated with NIb was with and to a VPg was in the and released were shown in were it is that uridylylated VPg were it is also that different amino in different were uridylylated a amino acid contains the uridylic but to cleavage it is found in part of the amino acid a to the of uridylic by NIb, we a of the VPg to carrying covalently linked by However, the of the uridylylated VPg in in vitro was and the of not only of the virus VPg was uridylylated in an in vitro reaction similar to A. M. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus (45) Google Scholar). The of the amino acid residue involved in the PVA was The uridylylated PVA VPg was A amino acid revealed that the represents the whereas the one to the with the that a tyrosine residue was uridylylated in the in vitro reaction. Tyr-60 a to viral RNA in TVMV VPg, as shown in of in vivo virus J.F. Rychlik W. Rhoads R.A. Hunt A.G. Shaw J.G. J. Virol. 1991; 65: 511-513Crossref PubMed Google Scholar). we the corresponding with in PVA mutation not have any obvious the uridylylation that another tyrosine residue was uridylylated in the in vitro reaction not of the the nucleotidylation the of of the and was in The nucleotidylation were in the presence of of [α-32P]UTP and of for [α-32P]UTP was in in the presence and of a poly(A) template. the the of the reaction for UTP In μm the other were able to with These results suggest an for UTP at the site within VPg A within PVA PVA VPg sequence motif, which contains a putative nucleotide-binding was by using the that VPg is an NTP-binding we a experiment with oxidized [α-32P]UTP in the presence of a and Mg2+ Mn2+ in similar mm that were used in the nucleotidylation The results that wild VPg oxidized UTP in the presence of Mn2+ was over Mg2+ as in the uridylylation reaction. It has been proposed that under the reaction conditions we the is between the oxidized and a residue E. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). The nucleotide-binding sequence of PVA VPg contains three Deletion of this 7-amino acid-long region in VPg binding it by as compared with the wild of the oxidized to a protein may via exposed binding was S. E. 1995; PubMed Scopus Google Scholar). the of the putative NTP-binding site the uridylylation the VPg was in the in vitro uridylylation reaction. The of uridylylation of VPg was only of that of wild VPg that the VPg region containing the amino has role in the uridylylation reaction. The peptide one tyrosine residue in a sequence that the uridylylated tyrosine in PV However, a mutation to not have any PVA VPg uridylylation not The for uridylylation be the of an NTP-binding domain a in the structure of PVA The structure of PVA VPg is not it is not to the of VPg to the structure of the that the mutation of the VPg we compared the structure of the with that of the wild PVA VPg by using in the the were in is at the However, we that the structure of PVA VPg is in the VPg The of the PVA VPg in multifunctional protein of PV, domain the VPg, has been shown to W. A. D. K. Wimmer E. J. Virol. 1998; PubMed Google Scholar). PVA VPg I. Guo D. Merits A. Mäkinen K. Saarma M. Valkonen J.P.T. Virus Res. 2001; 73: 103-112Crossref PubMed Scopus (45) Google Scholar). A experiment was to the PVA VPg is the nucleotidylation of were to the reaction In a similar of was in the not with and in the that the were to in denaturing of the reaction in and the revealed that most of the uridylylated VPg was in a The of was and were not of with NIb in a that it catalyzed the nucleotidylation reaction. of the uridylic acid into VPg the of PVA encoded is the responsible for the genomic RNA of RNA replication of as synthesis of a RNA using the viral RNA genome as template and synthesis of the viral RNA using the RNA as template. In the group of picornaviruses of RNA synthesis is a The revealed that this is also the case with the multifunctional VPg of PVA was found to be uridylylated by NIb, the viral It was that the mutation to within the recombinant PVA VPg not have any the uridylylation evidence that this residue VPg to the genomic RNA in TVMV and TEV (12Murphy J.F. Klein P.G. Hunt A.G. Shaw J.G. Virology. 1996; 220: 535-538Crossref PubMed Scopus (83) Google Scholar, 13Murphy J.F. Rychlik W. Rhoads R.A. Hunt A.G. Shaw J.G. J. Virol. 1991; 65: 511-513Crossref PubMed Google Scholar, M.C. Haldeman-Cahill R. Cronin S. Carrington J.C. J. Virol. 1996; 70: 7039-7048Crossref PubMed Google Scholar). The of this site in the genomic RNA of PVA to VPg is not and it is not that another tyrosine in PVA VPg may the support to this is that the acid to of VPg in the of PVA no T. Valkonen J.P.T. Res. 2002; 12: PubMed Scopus Google Scholar). However, other more the synthesis may require a different from that of the synthesis the in vitro system may not in vivo potyviral proteins essential for virus at a cell T. Valkonen J.P.T. Res. 2002; 12: PubMed Scopus Google Scholar), and it that the potyviral replication is via a of protein-protein The protein the to the Virus Res. 1995; PubMed Scopus Google Scholar, M.A. Carrington J.C. J. Virol. PubMed Google Scholar). the with the replication the for the putative in vivo uridylylation reaction. These provide more and reaction conditions than in an poly(A) the PVA RNA was required as to the of the in In the case of PV, the residue of the cre RNA sequence is shown to be the template for VPg uridylylation and to the reaction E. Kim D.W. Boom J.H. Wimmer E. J. Virol. 2000; 74: PubMed Scopus Google Scholar). within PV RNA the uridylylation to the viral It is that a in potyvirus but this not The of the viral genome may by other than via an RNA from the uridylylated was It may require other and viral not in our in vitro reaction is not to of It was that RNA polymerase the from to the of 2002; PubMed Scopus Google Scholar). The a is required in PVA NIb to the of to be RdRps require for in as of the group of the The structure of the revealed an site from the Nature. 2001; PubMed Scopus Google Scholar). Our results that PVA NIb requires Mn2+ for in vitro VPg nucleotidylation but was by Mg2+ of and it has been that virus RdRps have a for Mn2+ than Mg2+ S. D. S. R. J. Virol. 2003; 77: PubMed Scopus Google Scholar). The of NIb may also Mg2+ for RNA synthesis in Mg2+ be required in the activity from a to the RNA synthesis as proposed S. D. S. R. J. Virol. 2003; 77: PubMed Scopus Google Scholar). The of Mn2+ as the of the polymerase stimulated the but it was not of the replication of the genome J. L. M. Nucleic Acids Res. PubMed Scopus Google Scholar). The revealed that UTP is over the other three in the used in our uridylylation The preference for UTP not a template However, the for of the was when the of any of the three was over a that the of UTP to the hydroxyl group within VPg is to than with the other nucleotides. In we can only the between the and the of tyrosine is NIb the and by with VPg the to the of the tyrosine for the binding of the is to VPg, and the is required for the NIb over the VPg to be an NTP-binding protein in the of the direct role of VPg in The NTP-binding capacity of PVA VPg was in an experiment with a specifically a bond between the and an Mn2+ stimulated the NTP-binding activity of VPg, and this may that the is for and of the to Deletion of and in the VPg the NTP-binding the region was amino it was not to the role of this region for the structure of of the VPg, that no in the VPg structure to the be in the to this NTP-binding site. the VPg only of the VPg in vitro uridylylation the mutation to not this region have other in in Our NTP-binding with the region that is to be the of TEV A of the region within TEV VPg revealed that in of NIa debilitated also to genome (14Schaad M.C. Haldeman-Cahill R. Cronin S. Carrington J.C. J. Virol. 1996; 70: 7039-7048Crossref PubMed Google Scholar). TEV containing a mutation of the corresponding to PVA VPg and resulted in a in of the virus in tobacco The that the RNA activity of VPg was by the site The in vitro used in this revealed that the region in PVA VPg is essential for VPg also found a between PV VPg and the NTP-binding region in PVA VPg the PV VPg in vivo and in vitro uridylylation is also to the mutation to to in a virus J. J. J. Boom J.H. Wimmer E. J. Virol. 2003; 77: PubMed Scopus Google Scholar). In an binding was by the for the amino and in PV Taken together, we that the NTP-binding site of VPg has an essential in potyvirus infection. also that the of share similar in the of for with Ivanov for in protein and with the in the and for and nucleotides. also and Valkonen for of the