Picornavirus Genome Replication

Abstract

All picornaviruses have a protein, VPg, covalently linked to the 5′-ends of their genomes. Uridylylated VPg (VPg-pUpU) is thought to serve as the protein primer for RNA synthesis. VPg-pUpU can be produced in vitro by the viral polymerase, 3Dpol, in a reaction in which a single adenylate residue of a stem-loop structure, termed oriI, templates processive incorporation of UMP into VPg by using a “slide-back” mechanism. This reaction is greatly stimulated by viral precursor protein 3CD or its processed derivative, 3C; both contain RNA-binding and protease activities. We show that the 3C domain encodes specificity for oriI, and the 3D domain enhances the overall affinity for oriI. Thus, 3C(D) stimulation exhibits an RNA length dependence. By using a minimal system to evaluate the mechanism of VPg uridylylation, we show that the active complex contains polymerase, oriI, and 3C(D) at stoichiometry of 1:1:2. Dimerization of 3C(D) is supported by physical and structural data. Polymerase recruitment to and retention in this complex require a protein-protein interaction between the polymerase and 3C(D). Physical and functional data for this interaction are provided for three picornaviruses. VPg association with this complex is weak, suggesting that formation of a complex containing all necessary components of the reaction is rate-limiting for the reaction. We suggest that assembly of this complex in vivo would be facilitated by use of precursor proteins instead of processed proteins. These data provide a glimpse into the organization of the ribonucleoprotein complex that catalyzes this key step in picornavirus genome replication. All picornaviruses have a protein, VPg, covalently linked to the 5′-ends of their genomes. Uridylylated VPg (VPg-pUpU) is thought to serve as the protein primer for RNA synthesis. VPg-pUpU can be produced in vitro by the viral polymerase, 3Dpol, in a reaction in which a single adenylate residue of a stem-loop structure, termed oriI, templates processive incorporation of UMP into VPg by using a “slide-back” mechanism. This reaction is greatly stimulated by viral precursor protein 3CD or its processed derivative, 3C; both contain RNA-binding and protease activities. We show that the 3C domain encodes specificity for oriI, and the 3D domain enhances the overall affinity for oriI. Thus, 3C(D) stimulation exhibits an RNA length dependence. By using a minimal system to evaluate the mechanism of VPg uridylylation, we show that the active complex contains polymerase, oriI, and 3C(D) at stoichiometry of 1:1:2. Dimerization of 3C(D) is supported by physical and structural data. Polymerase recruitment to and retention in this complex require a protein-protein interaction between the polymerase and 3C(D). Physical and functional data for this interaction are provided for three picornaviruses. VPg association with this complex is weak, suggesting that formation of a complex containing all necessary components of the reaction is rate-limiting for the reaction. We suggest that assembly of this complex in vivo would be facilitated by use of precursor proteins instead of processed proteins. These data provide a glimpse into the organization of the ribonucleoprotein complex that catalyzes this key step in picornavirus genome replication. Picornaviruses are the etiologic agent of numerous diseases of medical and veterinary importance. Poliomyelitis, the common cold, summer flu, hepatitis, and foot-and-mouth disease can all be caused by picornaviruses (1.Racaniello V.R. Knipe D.M. Howley P.M. Griffin D.E. Lamb R.A. Martin M.A. Roizman B. Straus S.E. Fields Virology. 4th Ed. Vol. 1. Lippincott-Raven Publishers, Philadelphia, PA2001: 685-722Google Scholar). These viruses have a single-stranded RNA genome of positive polarity that is on the order of 7500 nt 3The abbreviations used are: nt, nucleotide(s); PV, poliovirus; CVB3, coxsackievirus B3; HRV14, human rhinovirus type 14; HRV16, human rhinovirus type 16; SLd, stem-loop d; β-ME, β-mercaptoethanol; ATPαS, adenosine 5′-O-(1-thiotriphosphate); Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.3The abbreviations used are: nt, nucleotide(s); PV, poliovirus; CVB3, coxsackievirus B3; HRV14, human rhinovirus type 14; HRV16, human rhinovirus type 16; SLd, stem-loop d; β-ME, β-mercaptoethanol; ATPαS, adenosine 5′-O-(1-thiotriphosphate); Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine. in length (1.Racaniello V.R. Knipe D.M. Howley P.M. Griffin D.E. Lamb R.A. Martin M.A. Roizman B. Straus S.E. Fields Virology. 4th Ed. Vol. 1. Lippincott-Raven Publishers, Philadelphia, PA2001: 685-722Google Scholar). A protein, VPg (virion protein genome-linked), is covalently linked to the 5′-end of the viral genome, the so-called plus-strand, and a poly(rA) tail is present at the 3′-end (1.Racaniello V.R. Knipe D.M. Howley P.M. Griffin D.E. Lamb R.A. Martin M.A. Roizman B. Straus S.E. Fields Virology. 4th Ed. Vol. 1. Lippincott-Raven Publishers, Philadelphia, PA2001: 685-722Google Scholar). Genome replication occurs in a process that uses the plus-strand as a template for minus-strand synthesis, which, in turn, is used as a template for production of an excess of plus-strands (2.Paul A.V. Semler B.L. Wimmer E. Molecular Biology of Picornaviruses. 1st Ed. American Society for Microbiology Press, Washington, D. C.2002: 227-246Google Scholar). Initiation of both plus- and minus-strand RNA synthesis is thought to be primed by a uridylylated form of VPg, VPg-pUpU (2.Paul A.V. Semler B.L. Wimmer E. Molecular Biology of Picornaviruses. 1st Ed. American Society for Microbiology Press, Washington, D. C.2002: 227-246Google Scholar). Several years ago, Paul and Wimmer made the paradigm-shifting observation that an RNA stem-loop structure in the 2C-coding region of the poliovirus (PV) genome was capable of templating production of VPg-pUpU (3.Paul A.V. Rieder E. Kim D.W. van Boom J.H. Wimmer E. J. Virol. 2000; 74: 10359-10370Crossref PubMed E. Paul A.V. Kim D.W. van Boom J.H. Wimmer E. J. Virol. 2000; 74: PubMed the poly(rA) tail A.V. van Boom J.H. D. Wimmer E. PubMed Scholar). this that all picornaviruses to use a for production of Wimmer E. Paul A.V. J. Virol. PubMed J. Virol. PubMed Wimmer E. Paul Martin J. Virol. PubMed A.V. J. J. Rieder E. Wimmer E. J. PubMed J. PubMed J. Virol. PubMed Paul J. van Wimmer E. J. Virol. PubMed Scholar). All picornaviruses have a RNA capable of templating the production of the of this in the genome This termed (2.Paul A.V. Semler B.L. Wimmer E. Molecular Biology of Picornaviruses. 1st Ed. American Society for Microbiology Press, Washington, D. C.2002: 227-246Google Scholar). is that is for genome replication Wimmer E. Paul A.V. J. Virol. PubMed J. Virol. PubMed Wimmer E. Paul Martin J. Virol. PubMed PubMed J. PubMed J. J. Virol. 2000; 74: PubMed suggest that is for production of for plus-strand synthesis J. Virol. PubMed J. J. Virol. PubMed J. Virol. PubMed suggest that are for both plus- and minus-strand synthesis Paul J. van Wimmer E. J. Virol. PubMed Scholar). VPg reaction can be in vitro by using the viral RNA polymerase the VPg and or (3.Paul A.V. Rieder E. Kim D.W. van Boom J.H. Wimmer E. J. Virol. 2000; 74: 10359-10370Crossref PubMed E. Paul A.V. Kim D.W. van Boom J.H. Wimmer E. J. Virol. 2000; 74: PubMed Wimmer E. Paul A.V. J. Virol. PubMed J. Virol. PubMed Paul J. van Wimmer E. J. Virol. PubMed Scholar). reaction is stimulated by a viral precursor protein, 3CD both RNA-binding and protease the RNA-binding is for stimulation of the VPg reaction (3.Paul A.V. Rieder E. Kim D.W. van Boom J.H. Wimmer E. J. Virol. 2000; 74: 10359-10370Crossref PubMed E. Paul A.V. Kim D.W. van Boom J.H. Wimmer E. J. Virol. 2000; 74: PubMed Scholar). We have that the processed viral protein 3C VPg D.W. Paul A.V. J. PubMed Scholar). mechanism for 3C(D) stimulation is of a in which 3C(D) to and polymerase to D.W. Paul A.V. J. PubMed Scholar). we that this recruitment was by an interaction between the of and of 3C D.W. Paul A.V. J. PubMed Scholar). This was on the of that polymerase on primed RNA templates and VPg that be stimulated by 3C(D) D.W. Paul A.V. J. PubMed Scholar). the was made that of a interaction be as an to the Semler B.L. J. Virol. PubMed Scholar). this we the of a minimal VPg system for that we use to evaluate the and functional and structural organization of the active VPg with D.W. Paul A.V. J. PubMed polymerase is to and in the VPg complex by a physical interaction with 3C(D) to oriI. This is the for coxsackievirus and human rhinovirus type of 3C(D) to is by the 3C the 3D to affinity on in the specificity of 3CD to 3C is in the of RNA 3C(D) to at a 3C in in the and of oriI. of the 3C of 3C(D) to the in a the that would of the 3C to both and the of oriI, affinity for the single of the is that for the suggesting an step complex association of VPg with this complex is and is rate-limiting for This the to of assembly and organization of the picornavirus VPg polymerase and was was was A was was was and VPg VPg was a all and or or as of 3C region was by using all used in this or to using the J. Virol. PubMed as region was into the D.W. B. PubMed using and to the region was by using to using the J.H. B. J. Virol. PubMed as was into the D.W. Paul A.V. J. PubMed using the and to the of the 3D is by van Paul J. van Wimmer E. J. Virol. PubMed of 3C and 3D be by B. J. J. and E. in 3C a was made by using and and D.W. Paul A.V. J. PubMed as was into D.W. B. PubMed Scholar). at the was used to the of the used in this in a and of 3C of the 3C proteins using the system for D.W. B. PubMed Scholar). with the or and containing at at and at used to of with and at was at to an of 1. to and by the of to a of for at and and at on and in at a of of of using a by a at a of and to of and by in a for at at was to and with A β-ME, and containing to the was in using A containing and was by using the protein of the and the and at of 3D and was D.W. Paul A.V. J. PubMed Scholar). of 3D was by van Paul J. van Wimmer E. J. Virol. PubMed of 3D be by 3C containing an was by as a of J. and E. and of region to the in was an the E. using and was using and a reaction and RNA was at for was by for was with of for to the RNA was a to protein the RNA to the used to RNA was with and in and RNA was by at was for the three that are at the 5′-end of to be PubMed Scholar). These the of the as by the RNA on the at PubMed J. J. PubMed Scholar). region to the was using and and the as was using and RNA as was as using the that at the 5′-end and a and adenosine at the a of the These the of the as by of the is of the was D.W. Paul A.V. J. PubMed Scholar). and of the for the and the as RNA the D. J. PubMed J. J. Virol. 2000; 74: PubMed with of of and of RNA at for by of with was used to the of the RNA was used to RNA was by at that an of was to RNA of the of with and RNA as and in by RNA was to use in the VPg was by of the RNA in and at for by the of an of and at for as used for are and an containing and a to be used for by the was by a reaction of the and RNA at for was by for was with of for to the by a to the the RNA was with and with and the was in This provided the for was a was at for to the to the of the by this as by the of and was the by using using a with a was into and in an RNA was with with and in This was RNA was using and RNA was the RNA was by RNA was by at was for the three that are at the 5′-end of as D.W. Paul A.V. J. PubMed Scholar). was at for in and to for was reaction as RNA was with of and at for of in was at for the was with of reaction protein, and was by the RNA in and at for on to the reaction. to an of was to RNA was at for and on on a at of was by using a in the and by using using a system as J. J. PubMed Scholar). was with the RNA in was in the in the for at of 3C was by the in All in VPg as D.W. Paul A.V. J. PubMed with reaction for VPg system 3Dpol, or as template as and VPg in reaction or β-ME, and All to a of of the components are in All components to to at for the and with an of in containing by using and Tricine, and the at a for by using a in the and by using by and as 3D and 3C a or as in reaction β-ME, for at was to a of and the at for All to a of All components to to was provided in this was and at in single use used to of the which is to by the of an of and containing by by using or to was using the for at in in was used to the and at for produced in proteins and 3C by used to for the capable of at of protein at in for the 3D was used at a in 3D and 3D used at in 3C was used at a of in or at a in was used as the and used to the was used in the for the and was used for the 3CD for with that 3C for 3CD in VPg that a template D.W. Paul A.V. J. PubMed Scholar). These data produced reaction is for at in in containing 3CD or 3C is in a was 3CD or 3C was present at the of 3C this as D.W. Paul A.V. J. PubMed Scholar). for 3C to for 3CD in all is that templates the of 3C to for in the of the genome in we the of RNA by the length of the RNA would the of 3CD or the 2C-coding in the genome the was the RNA D. J. PubMed J. J. Virol. 2000; 74: PubMed Scholar). templates supported VPg uridylylation, VPg-pUpU as the to by using the the of VPg to VPg as the RNA length was This a in the of 3C stimulation by using the These are with 3C containing the for stimulation of the VPg the 3D of 3CD the affinity specificity of 3C to in the of A and 3C to and VPg in the of RNA to with the assembly of the VPg complex in of the and mechanism of the VPg reaction would be facilitated by using a minimal order to the minimal template for VPg uridylylation, the of the was at the to that or nt in length be that stem-loop the as the RNA by for the formation of a the of of RNA to nt caused an in caused a in RNA was have a on the of the to the of the be by using as the in the reaction J. PubMed Scholar). use of a in the of to with for RNA in that be to a in the of the polymerase D.W. PubMed Scholar). in was in the of in to nt, suggesting that the in the of was to the of the the of a in which the of the be the for the and 3C as as the of on the RNA to that on the RNA in the of 3C to 3CD the use of of both proteins and a of 3CD to 3C by using of and 3C of 3C in the of the RNA to the made for the the in with the in data show that a and 3C are for in with J. Paul A.V. Wimmer E. Rieder E. J. Virol. PubMed Scholar). and 3C(D) of the VPg reaction by using the in the of of the stoichiometry of the components in the reaction. is to that of polymerase, 3C(D) and would be for assembly of an active this was in which 3Dpol, present at a of VPg was present at a of with a RNA in the of a of D.W. Paul A.V. J. PubMed Scholar). of and the are in the was suggesting that the for components in the active complex is formation with the 3CD was for 3D or suggesting that 3CD was in the active complex 3D or of for for and for by the These data are with a stoichiometry of of of VPg this complex was weak, the was of 3C a of 3C to is at the of the 3C reaction was to that for 3CD in This that the 3D domain of 3CD the 3D domain of 3CD the of the reaction containing 3C that of a reaction containing the of 3C is to that of is that in the active of 3C are 3C or 3C the to is data and in the that 3C D. Wimmer E. J. Virol. PubMed Scholar). structural data for 3C a that the formation of in J. PubMed Scholar). order to evaluate this we with 3C protein in a and of 3C order to that the was to the 3C protein was and the for this protein to was and with the 3C in 3C and as 3C and of 3C on in the the single-stranded region of in both and RNA this to be in structure by 3C data have 3C(D) to J. Paul A.V. Wimmer E. Rieder E. J. Virol. PubMed Scholar). a we in order to 3C to single-stranded We the for 3C to of length and data for in the of or are in 3C to all single-stranded the minimal was in the 3C to was the by in to and was to a RNA this as 3C to RNA this 3C to RNA was on the order of to single-stranded RNA of length 3C to RNA was RNA by using in a for 3C to to both and RNA to or stem-loop both of which contain and is an stem-loop structure in the and as a positive D. J. PubMed D. PubMed Semler B.L. J. Virol. PubMed J. Virol. PubMed E. Semler B.L. E. Paul Wimmer E. J. Virol. PubMed J. E. PubMed J. PubMed D. PubMed Scholar). to was the of the was a to that the 3C of the VPg reaction this was for a of RNA We the of the into the single-stranded components as and in and of 3C to of single-stranded was to the RNA and suggesting that 3C the in 3C to was in the of with the of to this interaction by and D. J. PubMed D. PubMed Scholar). between 3C and 3D that an interaction between 3C and is for VPg D.W. Paul A.V. J. PubMed Scholar). This was on a in which and of to or VPg by was stimulated by 3CD to for for the as in the of and for D.W. Paul A.V. J. PubMed Scholar). of supported replication in D.W. Paul A.V. J. PubMed Scholar). order to for the of a interaction and to the of this interaction we the interaction of all of the picornaviruses to viruses that with at or and an interaction between 3C and 3C proteins viruses with a would be to VPg as as in and and show to in the region of the We in vitro for and for VPg and and the proteins are in all VPg of the polymerase was the 3C was of the of the VPg and 3D was stimulated by 3C to a by of the 3C proteins the and VPg or made for the 3D and 3C and and the 3D and 3C and We that and 3C proteins would for 3C the and to 3Dpol, three This was supported by the data These data are all with that an functional between and a physical components in this VPg of the are of is the for the reaction. structural are the three stem-loop the of and have to be (3.Paul A.V. Rieder E. Kim D.W. van Boom J.H. Wimmer E. J. Virol. 2000; 74: 10359-10370Crossref PubMed Wimmer E. Paul Martin J. Virol. PubMed J. PubMed J. Paul A.V. Wimmer E. Rieder E. J. Virol. PubMed suggesting that structure is the for by the templating in the is in an 3C and proteins. of of 3C and of 3D is and for a are at VPg of the of the VPg and oriI. VPg or of 3C and is as is a of the of UMP incorporation are in S.E. of the in β-ME, and at for using 3D for and CVB3, for 3C VPg and Physical between 3C and 3D order to a physical interaction between 3C and we by and by and for of in the of 3C produced that by the and at a with the of and By the using a 3C we that of to 3C proteins with the of the 3C proteins to VPg with interaction for was for and the of for proteins with and with and provide for a physical interaction between a and a 3C that is for VPg Picornaviruses are RNA viruses with a single-stranded This of viruses contains numerous human and veterinary and contain a protein linked to the is thought to by using uridylylated VPg (VPg-pUpU) as primer for the RNA polymerase an that is at of RNA synthesis J. PubMed Scholar). VPg-pUpU can be produced by the polymerase by using a RNA stem-loop structure (3.Paul A.V. Rieder E. Kim D.W. van Boom J.H. Wimmer E. J. Virol. 2000; 74: 10359-10370Crossref PubMed E. Paul A.V. Kim D.W. van Boom J.H. Wimmer E. J. Virol. 2000; 74: PubMed or a poly(rA) tail A.V. van Boom J.H. D. Wimmer E. PubMed Scholar). of VPg is for (3.Paul A.V. Rieder E. Kim D.W. van Boom J.H. Wimmer E. J. Virol. 2000; 74: 10359-10370Crossref PubMed E. Paul A.V. Kim D.W. van Boom J.H. Wimmer E. J. Virol. 2000; 74: PubMed Scholar). and the use of for VPg picornavirus genome replication (3.Paul A.V. Rieder E. Kim D.W. van Boom J.H. Wimmer E. J. Virol. 2000; 74: 10359-10370Crossref PubMed E. Paul A.V. Kim D.W. van Boom J.H. Wimmer E. J. Virol. 2000; 74: PubMed Wimmer E. Paul A.V. J. Virol. PubMed Wimmer E. Paul Martin J. Virol. PubMed J. Virol. PubMed Paul J. van Wimmer E. J. Virol. PubMed Scholar). is the use of VPg-pUpU produced by using for both plus- and minus-strand synthesis Paul J. van Wimmer E. J. Virol. PubMed J. Virol. PubMed J. J. Virol. PubMed J. Virol. PubMed Scholar). for and of the VPg VPg reaction in vitro components (3.Paul A.V. Rieder E. Kim D.W. van Boom J.H. Wimmer E. J. Virol. 2000; 74: 10359-10370Crossref PubMed E. Paul A.V. Kim D.W. van Boom J.H. Wimmer E. J. Virol. 2000; 74: PubMed Scholar). the structural assembly and functional for are (2.Paul A.V. Semler B.L. Wimmer E. Molecular Biology of Picornaviruses. 1st Ed. American Society for Microbiology Press, Washington, D. C.2002: 227-246Google Scholar). We provided a glimpse into the structural organization years by an interaction between protein 3C(D) and that was for VPg uridylylation, to the by D.W. Paul A.V. J. PubMed Scholar). are with the for VPg in A of 3C(D) to the of in the of the by 3C to the This complex to the of the the to of is to and on the by a protein-protein interaction with or both of the 3C of of VPg with this complex is and be to the with 3Dpol, or of three a and in and a in contains adenylate for templating and processive by using a mechanism Wimmer E. Paul A.V. J. Virol. PubMed A.V. J. J. Rieder E. Wimmer E. J. PubMed Scholar). contains for uridylylation, in this region Wimmer E. Paul Martin J. Virol. PubMed J. Paul A.V. Wimmer E. Rieder E. J. Virol. PubMed Scholar). and for to this region J. PubMed Scholar). that contains the and the in the of the genome J. PubMed Scholar). replication J. PubMed Scholar). These data are with the the specificity for to proteins for and the a structural the of to would be by the the of the in the of the genome be 3C(D) to to a minimal system for that was by a and 3C that 3C with the This interaction be by These data are with data that of the with 3CD J. Paul A.V. Wimmer E. Rieder E. J. Virol. PubMed Scholar). of 3C to the have a on the of the minimal 3C and 3CD on 3CD is This the for 3CD to to 3C We that 3C specificity and 3D enhances with this observation is the that in 3C can specificity J. PubMed Scholar). that of 3CD of and for of 3C be in 3CD was in this is that the 3D domain in of PubMed Scholar). of 3C is the observation that 3C as a J. PubMed Scholar). of the 3C to be by using the system or in the is D. Wimmer E. J. Virol. PubMed Scholar). We that a of 3C(D) to the of oriI. of the of the picornavirus are and have single-stranded and as as (2.Paul A.V. Semler B.L. Wimmer E. Molecular Biology of Picornaviruses. 1st Ed. American Society for Microbiology Press, Washington, D. C.2002: 227-246Google Scholar). in 3C to both and for to single-stranded RNA is nt RNA affinity is and with the of the VPg reaction of 3C to the was to the single that form the with and in We the 3C of the the and the This of the is a mechanism for the into the in an structure for a minimal mechanism for interaction of the with PubMed Scholar). This of the be in the of to the and have for an interaction between and 3C that is for D.W. Paul A.V. J. PubMed Scholar). By at and on the of the of to or VPg was to 3Dpol, be stimulated by 3C or 3CD at all D.W. Paul A.V. J. PubMed Scholar). that the all VPg by using of 3C and of the was the of VPg or of the was a of the for a 3C to with that the of the with 3C D.W. Paul A.V. J. PubMed Scholar). the of VPg-pUpU to by the 3D and 3C between and genome replication is is that a interaction was supported by A and in to a a was and a 3C in the form of the complex that of with the of a 3C to VPg is that the structure for 3CD D.W. J. Virol. PubMed Scholar). between 3C and 3D are that are for VPg We that both recruitment of to the VPg complex and retention of are by a physical interaction between 3C and that of a interaction have as on the active Semler B.L. J. Virol. PubMed Scholar). These data this in this the that are of VPg with the with the complex in order for VPg to VPg to the complex VPg is A structure for a picornavirus VPg to the RNA-binding of in an with numerous is that is a complex that is to oriI. We have that of a protein to the by to 3C and this protein can be uridylylated by is that VPg to in the by a by that be in the on Genome in the in vitro VPg reaction E. Paul A.V. Kim D.W. van Boom J.H. Wimmer E. J. Virol. 2000; 74: PubMed Wimmer E. Paul Martin J. Virol. PubMed D.W. Paul A.V. J. PubMed the reaction are to genome replication in Initiation J. 2000; PubMed a at the of components that to and in the formation of a complex containing all of the components is and the use of of VPg would VPg association VPg as and the precursor protein in is that or with by with or uridylylation, the overall of the process in a for Initiation of the of Genome that the VPg as both the VPg and recruitment for foot-and-mouth disease J. J. Virol. PubMed Scholar). of and is and of as a for Initiation of the of Genome Scholar). B. and E. We J. van and J. for with We Paul and for on the