The genome-linked protein of picornaviruses. VII. Genetic mapping of poliovirus VPg by protein and RNA sequence studies

The poliovirus genome-linked protein (VPg) has been subjected to radiochemical microsequence analysis. Sequence studies of virion RNA by a modification of Sanger's dideoxy method have revealed a base sequence corresponding to the amino acid analysis. This result proves that VPg is virus-encoded. The RNA sequence has allowed us to predict the total amino acid sequence of VPg and part of its precursor. VPg is, at most, 27 amino acids long. It maps within the 3' terminal segment of the viral genome that encodes the precursor polypeptide NCVP1b for the virus-specific RNA polymerase NCVP4.     

Mutational analysis of the genome-linked protein VPg of poliovirus.

Using a mutagenesis cartridge (R. J. Kuhn, H. Tada, M. F. Ypma-Wong, J. J. Dunn, B. L. Semler, and E. Wimmer, Proc. Natl. Acad. Sci. USA 85:519-523, 1988), we have generated single and multiple amino acid replacement mutants, as well as a single amino acid insertion mutant in the genome-linked protein VPg of poliovirus. Moreover, we constructed three different 5-amino-acid insertion mutants that map close to the C terminus of 3A, a viral polypeptide whose coding sequence is adjacent to VPg. Transfection of HeLa cells with RNA synthesized in vitro was used to test the effect of the mutation on viral proliferation. Mutations were either lethal or nonlethal. A temperature-sensitive phenotype was not observed. The arginine at position 17 of VPg could not be exchanged with any other amino acid without loss of viability, whereas the lysine at position 20, an amino acid conserved among all known polioviruses, coxsackieviruses, and echoviruses, was replaceable with several neutral amino acids and even with glutamic acid. Replacement of poliovirus VPg with echovirus 9 VPg yielded viable virus with impaired growth properties. Our results suggest considerable flexibility in the amino acid sequence of a functional VPg. All insertions in polypeptide 3A proved to be lethal. In vitro translation of mutated viral RNAs gave patterns of proteolytic processing that in some cases was aberrant, even though the mutation was nonlethal.     

Antibody to a synthetic nonapeptide corresponding to the NH2 terminus of poliovirus genome-linked protein VPg reacts with native VPg and inhibits in vitro replication of poliovirus RNA.

A synthetic nonapeptide corresponding to the N-terminal sequence of poliovirus genome-linked protein (VPg) was linked to bovine serum albumin and used to raise antibodies in rabbits. The antipeptide antibodies specifically precipitated the nonapeptide, native VPg, and VPg-linked poliovirion RNA. The antipeptide antibodies inhibited host factor-stimulated, poliovirus replicase-catalyzed in vitro synthesis of full-length (35S) RNA in response to virion RNA. Oligouridylic acid-stimulated RNA synthesis was not affected by the antipeptide antibodies. Preincubation of the antibodies with excess nonapeptide reversed the antipeptide antibody-mediated inhibition of host factor-stimulated RNA synthesis by the poliovirus replicase. A role for VPg in the in vitro replication of poliovirus RNA genome is discussed.     

Detection of a genome-linked protein (VPg) of hepatitis A virus and its comparison with other picornaviral VPgs.

The nucleotide sequence corresponding to the P3 region of the hepatitis A virus (HAV) polyprotein genome was determined from cloned cDNA and translated into an amino acid sequence. Comparison of the amino acid sequences of the genome-linked proteins (VPgs) of other picornaviruses with the predicted amino acid sequence of HAV was used to locate the primary structure of a putative VPg within the genome of HAV. The sequence of HAV VPg, like those of other picornaviral VPg molecules, contains a tyrosine residue as a potential binding site for HAV RNA in position 3 from its N terminus. The potential cleavage sites to generate VPg from a putative HAV polyprotein are between glutamic acid and glycine at the N terminus and glutamic acid and serine or glutamine and serine at the C terminus. A synthetic peptide corresponding to 10 amino acids of the predicted C terminus of HAV VPg induced anti-peptide antibodies in rabbits when it was conjugated to thyroglobulin as a carrier. These antibodies were specific for the peptide and precipitated VPg, linked to HAV RNA, from purified HAV and from lysates of HAV-infected cells. The precipitation reaction was blocked by the synthetic peptide (free in solution or coupled to carrier proteins) and prevented by pretreatment of VPg RNA with protease. Thus, our predicted amino acid sequence is colinear with the nucleotide sequence of the VPg gene in the HAV genome. From our results we concluded that HAV has the typical organization of picornavirus genes in this part of its genome. Similarity among hydrophobicity patterns of amino acid sequences of different picornaviral VPgs was revealed in hydropathy plots. Thus, the VPg of HAV appears to be closely related to VPg1 and VPg2 of foot-and-mouth disease virus. In contrast, HAV VPg has a unique isoelectric point (pI = 7.15) among the picornavirus VPgs.     

Allosteric effects of ligands and mutations on poliovirus RNA-dependent RNA polymerase

Protein priming of viral RNA synthesis plays an essential role in the replication of picornavirus RNA. Both poliovirus and coxsackievirus encode a small polypeptide, VPg, which serves as a primer for addition of the first nucleotide during synthesis of both positive and negative strands. This study examined the effects on the VPg uridylylation reaction of the RNA template sequence, the origin of VPg (coxsackievirus or poliovirus), the origin of 3D polymerase (coxsackievirus or poliovirus), the presence and origin of interacting protein 3CD, and the introduction of mutations at specific regions in the poliovirus 3D polymerase. Substantial effects associated with VPg origin were traced to differences in VPg-polymerase interactions. The effects of 3CD proteins and mutations at polymerase-polymerase intermolecular Interface I were most consistent with allosteric effects on the catalytic 3D polymerase molecule. In conclusion, the efficiency and specificity of VPg uridylylation by picornavirus polymerases is greatly influenced by allosteric effects of ligand binding that are likely to be relevant during the viral replicative cycle.     

A poliovirus type 1 neutralization epitope is located within amino acid residues 93 to 104 of viral capsid polypeptide VP1.

Using nuclease Bal31, deletions were generated within the poliovirus type 1 cDNA sequences, coding for capsid polypeptide VP1, within plasmid pCW119. The fusion proteins expressed in Escherichia coli by the deleted plasmids reacted with rabbit immune sera directed against poliovirus capsid polypeptide VP1 (alpha VP1 antibodies). They also reacted with a poliovirus type 1 neutralizing monoclonal antibody C3, but reactivity was lost when the deletion extended up to VP1 amino acids 90-104. Computer analysis of the protein revealed a high local density of hydrophilic amino acid residues in the region of VP1 amino acids 93-103. A peptide representing the sequence of this region was chemically synthesized. Once coupled to keyhole limpet hemocyanin, this peptide was specifically immunoprecipitated by C3 antibodies. The peptide also inhibited the neutralization of poliovirus type 1 by C3 antibodies. We thus conclude that the neutralization epitope recognized by C3 is located within the region of amino acids 93-104 of capsid polypeptide VP1.     

Two forms of VPg on poliovirus RNAs

The protein (VPg) linked to the 5' termini of poliovirus RNAs resolved into two species when subjected to non-equilibrium electrofocusing. The differently charged forms of VPg were not due to protein phosphorylation nor to variability of the number of phosphate residues associated with the nucleotide moiety remaining after RNase digestion of the nucleoprotein. Single-stranded viral RNA isolated from mature virions contained predominantly the more basic form of VPg, whereas unpackaged single-stranded RNa remaining in cells at the end of the virus replication cycle contained predominantly the more acidic form of VPg. Replicative-form (RF) molecules also contained both species of VPg, with the more acidic form representing the major species. Both plus and minus RNA strands in RF had similar VPg compositions; however, there appeared to be a strongly selective loss of VPg from only the minus strand in RF, particularly at late times postinfection.     

Characterization of poliovirus clones containing lethal and nonlethal mutations in the genome-linked protein VPg.

Viral RNA synthesis was assayed in HeLa cells transfected with nonviable poliovirus RNA mutated in the genome-linked protein VPg-coding region. The transfecting RNA was transcribed in vitro from full-length poliovirus type 1 (Mahoney) cDNA containing a VPg mutagenesis cartridge. Hybridization experiments using ribonucleotide probes specific for the 3' end of positive- and negative-sense poliovirus RNA indicated that all mutant RNAs encoding a linking tyrosine in position 3 or 4 of VPg were replicated even though no virus was produced. VPg, but no VPg precursor, was found to be linked to the 5' end of the newly synthesized RNA. Encapsidated mutant RNAs were not found in transfected-cell lysates. After extended maintenance of transfected HeLa cells, a viable revertant of one of the nonviable RNAs was recovered; the revertant lost the lethal lesion in VPg by restoring the wild-type amino acid, but it retained all other nucleotide changes introduced during construction of the mutagenesis cartridge. Mutant RNA encoding phenylalanine or serine rather than tyrosine, the linking amino acid in VPg, was not replicated in transfected cells. A chimeric mutant containing the VPg-coding region of coxsackievirus within the poliovirus genome was viable but displayed impaired multiplication. A poliovirus-coxsackievirus chimera lacking a linking tyrosine in VPg was nonviable and replication-negative. The results indicate that a linkage-competent VPg is necessary for poliovirus RNA synthesis to occur but that a step in poliovirus replication other than initiation of RNA synthesis can be interrupted by lethal mutations in VPg.     

Purification and properties of a HeLa cell enzyme able to remove the 5'-terminal protein from poliovirus RNA

Using a rapid phenol extraction assay, an enzyme was purified from uninfected HeLa cells that can cleave the 5'-terminal protein (VPg) from poliovirus RNA. Both cytoplasmic and nuclear extracts had enzymes with similar behavior. A polypeptide of molecular weight 27,000 was the major one present in the purified preparation. Assuming that this protein is the enzyme, a very low turnover number was calculated for it. The purified enzyme would cleave the tyrosine-phosphate bond linking VPg to poliovirus RNA with minimal degradation of the RNA or of VPg. If the RNA was first treated with proteinase K to degrade VPg, leaving a small peptide on the RNA, this peptide could also be removed by the enzyme. If the RNA was degraded with T1 RNase, leaving VPg attached to a nonanucleotide, the enzyme still would cleave off VPg, although incompletely. If the RNA was degraded completely, leaving either pUp or pU attached to VPg, the enzyme would not remove the nucleotides from the protein. Thus, for the enzyme to be active requires some length of polynucleotide attached to the protein but only a short peptide need be present for the enzyme to act.     

Homologous sequences in non-structural proteins from cowpea mosaic virus and picornaviruses

Computer analyses have revealed sequence homology between two non-structural proteins encoded by cowpea mosaic virus (CPMV), and corresponding proteins encoded by two picornaviruses, poliovirus and foot-and-mouth disease virus. A region of 535 amino acids in the 87-K polypeptide from CPMV was found to be homologous to the RNA-dependent RNA polymerases from both picornaviruses, the best matches being found where the picornaviral proteins most resemble each other. Additionally, the 58-K polypeptide from CPMV and polypeptide P2-X from poliovirus contain a conserved region of 143 amino acids. Based on the homology observed, a genetic map of the CPMV genome has been constructed in which the 87-K polypeptide represents the core polymerase domain of the CPMV replicase. These results have implications for the evolution of RNA viruses, and mechanisms are discussed which may explain the existence of homology between picornaviruses (animal viruses with single genomic RNAs) and comoviruses (plant viruses with two genomic RNAs).     

Cowpea mosaic virus VPg: sequencing of radiochemically modified protein allows mapping of the gene on B RNA

A partial amino acid sequence of cowpea mosaic virus (CPMV) VPg radiochemically modified by chloramine-T and Bolton-Hunter reagent has been determined. VPg covalently bound to viral RNA chains (VPg-RNA) was iodinated with chloramine-T and Bolton-Hunter reagent to label tyrosine and lysine residues, respectively. [¹²⁵I]VPg-RNA was digested with nuclease P1 and the resulting [¹²⁵I]VPg-pU was purified by SDS-polyacrylamide gel electrophoresis and subjected to automated Edman degradation. Control experiments with chemically synthesized poliovirus VPg showed the feasibility of radiochemical microsequence analysis of protein that had been radiochemically modified by chloramine-T and Bolton-Hunter reagent. Analysis of CPMV [¹²⁵I]VPg-pU revealed the presence of tyrosine residues at position 12 and 14, and of lysine residues at position 3 and 20, respectively. In combination with Edman degradation of unlabeled CPMV VPg, which showed serine and arginine residues to be present at position 1 and 2, respectively, the data obtained allow the precise positioning of VPg within the 200 000 dalton (200 K) polyprotein encoded by CPMV B RNA and the prediction of its entire amino acid sequence. VPg is located at the COOH terminus of its 60 K, membrane-bound,precursor and proximal to the amino terminus of the protease-polymerase domain of the polyprotein. A processing scheme for the 200 K polyprotein is discussed in which Gln-Ser amino acid pairs act as the major signal for proteolytic cleavage.     

Molecular cloning of a protein associated with soybean seed oil bodies that is similar to thiol proteases of the papain family

A 34,000-Da protein (P34) is one of the four major soybean oil body proteins observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of isolated organic solvent-extracted oil bodies from mature seeds. P34 is processed during seedling growth to a 32,000-Da polypeptide (P32) by the removal of an amino-terminal decapeptide (Herman, E.M., Melroy, D.L., and Buckhout, T.J. (1990) Plant Physiol, in press). A soybean lambda ZAP II cDNA library constructed from RNA isolated from midmaturation seeds was screened with monoclonal antibodies directed against two different epitopes of P34. The isolated cDNA clone encoding P34 contains 1,350 base pairs terminating in a poly(A)+ tail and an open reading frame 1,137 base pairs in length. The open reading frame includes a deduced amino acid sequence which matches 23 of 25 amino-terminal amino acids determined by automated Edman degradation of P34 and P32. The cDNA predicts a mature protein of 257 amino acids and of 28,641 Da. The open reading frame extends 5' from the known amino terminus of P34 encoding a possible precursor and signal sequence segments with a combined additional 122 amino acids. Prepro-P34 is deduced to be a polypeptide of 42,714 Da, indicating that the cDNA clone apparently encodes a polypeptide of 379 amino acids. A comparison of the nucleotide and deduced amino acid sequences in the GenBank Data Bank with the sequence of P34 has shown considerable sequence similarity to the thiol proteases of the papain family. Southern blot analysis of genomic DNA indicated that the P34 gene has a low copy number.     

Intramolecular and intermolecular uridylylation by poliovirus RNA-dependent RNA polymerase

The 22-amino-acid protein VPg can be uridylylated in solution by purified poliovirus 3D polymerase in a template-dependent reaction thought to mimic primer formation during RNA amplification in infected cells. In the cell, the template used for the reaction is a hairpin RNA termed 2C-cre and, possibly, the poly(A) at the 3' end of the viral genome. Here, we identify several additional substrates for uridylylation by poliovirus 3D polymerase. In the presence of a 15-nucleotide (nt) RNA template, the poliovirus polymerase uridylylates other polymerase molecules in an intermolecular reaction that occurs in a single step, as judged by the chirality of the resulting phosphodiester linkage. Phosphate chirality experiments also showed that VPg uridylylation can occur by a single step; therefore, there is no obligatory uridylylated intermediate in the formation of uridylylated VPg. Other poliovirus proteins that could be uridylylated by 3D polymerase in solution were viral 3CD and 3AB proteins. Strong effects of both RNA and protein ligands on the efficiency and the specificity of the uridylylation reaction were observed: uridylylation of 3D polymerase and 3CD protein was stimulated by the addition of viral protein 3AB, and, when the template was poly(A) instead of the 15-nt RNA, the uridylylation of 3D polymerase itself became intramolecular instead of intermolecular. Finally, an antiuridine antibody identified uridylylated viral 3D polymerase and 3CD protein, as well as a 65- to 70-kDa host protein, in lysates of virus-infected human cells.     

Characterization of protein-protein interactions critical for poliovirus replication: analysis of 3AB and VPg binding to the RNA-dependent RNA polymerase

Two critical interactions within the poliovirus RNA replication complex are those of the RNA-dependent RNA polymerase 3D with the viral proteins 3AB and VPg. 3AB is a membrane-binding protein responsible for the localization of the polymerase to the membranous vesicles at which replication occurs. VPg (a peptide comprising the 3B region of 3AB) is the 22-residue soluble product of 3AB cleavage and serves as the protein primer for RNA replication. The detailed interactions of these proteins with the RNA-dependent RNA polymerase 3D were analyzed to elucidate the precise roles of 3AB and VPg in the viral RNA replication complex. Using a membrane-based pull-down assay, we have identified a binding "hot-spot" spanning residues 100 to 104 in the 3B (VPg) region of 3AB which plays a critical role in mediating the interaction of 3AB with the polymerase. Isothermal titration calorimetry shows that the interaction of VPg with 3D is enthalpically driven, with a dissociation constant of 11 microM. Mutational analyses of VPg indicate that a subset of the residues important for 3AB-3D binding are also important for VPg-3D binding. Two residues in particular, P14 and R17, were shown to be absolutely critical for the binding interaction. This work provides the direct characterization of two binding interactions critical for the replication of this important class of viruses and identifies a conserved polymerase binding sequence responsible for targeting the polymerase.     

Poliovirus CRE-dependent VPg uridylylation is required for positive-strand RNA synthesis but not for negative-strand RNA synthesis

The cis-acting replication element (CRE) is a 61-nucleotide stem-loop RNA structure found within the coding sequence of poliovirus protein 2C. Although the CRE is required for viral RNA replication, its precise role(s) in negative- and positive-strand RNA synthesis has not been defined. Adenosine in the loop of the CRE RNA structure functions as the template for the uridylylation of the viral protein VPg. VPgpUpU(OH), the predominant product of CRE-dependent VPg uridylylation, is a putative primer for the poliovirus RNA-dependent RNA polymerase. By examining the sequential synthesis of negative- and positive-strand RNAs within preinitiation RNA replication complexes, we found that mutations that disrupt the structure of the CRE prevent VPg uridylylation and positive-strand RNA synthesis. The CRE mutations that inhibited the synthesis of VPgpUpU(OH), however, did not inhibit negative-strand RNA synthesis. A Y3F mutation in VPg inhibited both VPgpUpU(OH) synthesis and negative-strand RNA synthesis, confirming the critical role of the tyrosine hydroxyl of VPg in VPg uridylylation and negative-strand RNA synthesis. trans-replication experiments demonstrated that the CRE and VPgpUpU(OH) were not required in cis or in trans for poliovirus negative-strand RNA synthesis. Because these results are inconsistent with existing models of poliovirus RNA replication, we propose a new four-step model that explains the roles of VPg, the CRE, and VPgpUpU(OH) in the asymmetric replication of poliovirus RNA.     

Carboxy-terminal analysis of poliovirus proteins: termination of poliovirus RNA translation and location of unique poliovirus polyprotein cleavage sites.

The carboxy-terminal amino acids of a number of poliovirus proteins were determined by carboxypeptidase A analysis. The nonstructural proteins P3-2, P3-4b and their precursor. P3-1b, were found to be coterminal with a sequence of -Ser-Phe-COOH. As these proteins are coded for at the extreme 3' end of the viral RNA, it is possible to establish the termination site of translation at nucleotide 7,361, 73 nucleotides before the start of the polyadenylic acid tract of the RNA. Two additional nonstructural proteins, P2-X and its precursor, P2-3b, were also found to be coterminal with a sequence of -Phe-Gln-COOH. This result confirms the existence of at least one Gln-Gly proteolytic cleavage site. These Gln-Gly cleavage sites are predicted from the nucleotide sequence to be ubiquitous throughout the poliovirus genome. The only exceptions are the cleavage sites at the carboxy termini of the structural protein VP4 and VP1. Carboxypeptidase A analysis of VP1 establishes a terminal sequence of -Thr-Tyr-COOH, and similar analysis of VP4 shows Asn to be the terminal amino acid residue, observations that prove the existence of the exceptional C-terminal amino acids. In none of the analyzed cases has C-terminal trimming after cleavage been observed.     

Anti-VPg antibody inhibition of the poliovirus replicase reaction and production of covalent complexes of VPg-related proteins and RNA

Anti-VPg antibodies inhibited host-factor-dependent RNA synthesis by the poliovirus replicase but not oligo(U)-primed synthesis, implicating VPg in the de novo initiation of replicase products. Complexes of VPg-related polypeptide and newly made RNA could be immunoprecipitated by anti-VPg antibody from the host-factor-stimulated products of the replicase reaction. The complexes appeared to be covalently linked and involved 50 to 150 nucleotide chains of RNA that were RNAase-T1-resistant and could be largely poly(U).     

The C-terminal hydrophobic domain of hepatitis C virus RNA polymerase NS5B can be replaced with a heterologous domain of poliovirus protein 3A

Replication of the plus-stranded RNA genome of hepatitis C virus (HCV) occurs in a membrane-bound replication complex consisting of viral and cellular proteins and viral RNA. NS5B, the RNA polymerase of HCV, is anchored to the membranes via a C-terminal 20-amino-acid-long hydrophobic domain, which is flanked on each side by a highly conserved positively charged arginine. Using a genotype 1b subgenomic replicon (V. Lohmann, F. Korner, J. O. Koch, U. Herian, L. Theilmann, and R. Bartensclager, Science 285:110-113, 1999), we determined the effect of mutations of some highly conserved residues in this domain. The replacement of arginine 570 with alanine completely abolished the colony-forming ability by the replicon, while a R591A change was found to be highly detrimental to replication, viability, and membrane binding by the mutant NS5B protein. Mutations of two other highly conserved amino acids (L588A and P589A) reduced but did not eliminate colony formation. It was of interest, if specific amino acid residues play a role in membrane anchoring of NS5B and replication, to determine whether a complete exchange of the NS5B hydrophobic domain with a domain totally unrelated to NS5B would ablate replication. We selected the 22-amino-acid-long hydrophobic domain of poliovirus polypeptide 3A that is known to adopt a transmembrane configuration, thereby anchoring 3A to membranes. Surprisingly, either partial or full replacement of the NS5B hydrophobic domain with the anchor sequences of poliovirus polypeptide 3A resulted in the replication of replicons whose colony-forming abilities were reduced compared to that of the wild-type replicon. Upon continued passage of the replicon in Huh-7 cells in the presence of neomycin, the replication efficiency of the replicon increased. However, the sequence of the poliovirus polypeptide 3A hydrophobic domain, in the context of the subgenomic HCV replicon, was stably maintained throughout 40 passages. Our results suggest that anchoring NS5B to membranes is necessary but that the amino acid sequence of the anchor per se does not require HCV origin. This suggests that specific interactions between the NS5B hydrophobic domain and other membrane-bound factors may not play a decisive role in HCV replication.     

Removal of the genome-linked protein of foot-and-mouth disease virus by rabbit reticulocyte lysate.

Rabbit reticulocyte lysate cleaves the genome-linked protein VPg from foot-and-mouth disease virus (FMDV) RNA. This activity could be reliably monitored since removal of the protein resulted in a change in migration in polyacrylamide gels of the small specific 5' and fragment of the RNA (S fragment). The unlinking activity cleaved the bond between the tyrosine residue of VPg and the RNA to leave a 5' phosphate on the RNA. The 5' sequence of the RNA from which VPg had been removed by rabbit reticulocyte lysate was the same as that of FMDV mRNA isolated from infected cells. VPg released from the RNA was rapidly degraded by the rabbit reticulocyte lysate to material which eluted with the inclusion volume of a Sepharose 6B column and partitioned to the aqueous phase during phenol extraction. The unlinking activity was inhibited by heating the lysate to 56 degrees C, by sodium dodecyl sulfate (SDS), EDTA, and Zn2+ ions but was unaffected by reducing agents, a translation inhibitor, and a number of protease and RNase inhibitors.     

Genetic variation of the poliovirus genome with two VPg coding units.

Amongst the picornaviruses, poliovirus encodes a single copy of the genome-linked protein, VPg wheras foot-and-mouth disease virus uniquely encodes three copies of VPg. We have previously shown that a genetically engineered poliovirus genome containing two tandemly arranged VPgs is quasi-infectious (qi) that, upon genome replication, inadvertently deleted one complete VPg sequence. Using two genetically marked viral genomes with two VPg sequences, we now provide evidence that this deletion occurs via homologous recombination. The mechanism was abrogated when the second VPg was engineered such that its nucleotide sequence differed from that of the first VPg sequence by 36%. Such genomes also expressed a qi phenotype, but progeny viruses resulted from (i) random deletions yielding single VPg coding sequences of varying length lacking the Q*G cleavage site between the VPgs and (ii) mutations in the AKVQ*G cleavage sites between the VPgs at either the P4, P1 or P1' position. These variants present a unique genetic system defining the cleavage signals recognized in 3Cpro-catalyzed proteolysis. We propose a recognition event in the cis cleavages of the polyprotein P2-P3 region, and we present a hypothesis why the poliovirus genome does not tolerate two tandemly arranged VPg sequences.