Biochemical and genetic studies of the VPg uridylylation reaction catalyzed by the RNA polymerase of poliovirus

Abstract

The first step in poliovirus (PV) RNA synthesis is the covalent linkage of UMP to the terminal protein VPg. This reaction can be studied in vitro with two different assays. The simpler assay is based on a poly(A) template and requires synthetic VPg, purified RNA polymerase 3D(pol), UTP, and a divalent cation. The other assay uses specific viral sequences [cre(2C)] as a template for VPg uridylylation and requires the addition of proteinase 3CD(pro). Using one or both of these assays, we analyzed the VPg specificities and metal requirements of the uridylylation reactions. We determined the effects of single and double amino acid substitutions in VPg on the abilities of the peptides to serve as substrates for 3D(pol). Mutations in VPg, which interfered with uridylylation in vitro, were found to abolish viral growth. A chimeric PV containing the VPg of human rhinovirus 14 (HRV14) was viable, but substitutions of HRV2 and HRV89 VPgs for PV VPg were lethal. Of the three rhinoviral VPgs tested, only the HRV14 peptide was found to function as a substrate for PV1(M) 3D(pol) in vitro. We also examined the metal specificity of the VPg uridylylation reaction on a poly(A) template. Our results show a strong preference of the RNA polymerase for Mn(2+) as a cofactor compared to Mg(2+) or other divalent cations.

FIG. 1.

Structure of PV genomic RNA and processing of the P3 domain of the polyprotein. The single-stranded RNA genome of PV is shown with the terminal protein VPg (3B) at the 5′ end of the 5′ NTR and the 3′ NTR with the poly(A) tail (20). The 5′NTR consists of a cloverleaf and a large internal ribosomal entry site element. The attachment site of the 5′-terminal UMP of the RNA to the tyrosine of VPg (2, 45) is shown enlarged. Also shown enlarged is the predicted secondary structure of a cis replicating RNA element located in the coding sequence of 2C^ATPase [cre(2C)] (15, 39, 44). The nucleotides required for PV cre function are shown in boldface letters (44, 58). The polyprotein contains structural (P1) and nonstructural (P2 and P3) domains. The vertical lines within the polyprotein box represent the proteinase cleavage sites. Processing of the P3 domain of the polyprotein is shown enlarged.

FIG. 2.

Effects of mutations in VPg on the viability of PV. (A) Enteroviral and rhinoviral VPg sequences. Conserved amino acids are indicated with uppercase letters. The fully conserved tyrosine, which provides the VPg-linking site, is shown in boldface letters. (B) Growth properties of PV VPg mutants. The table summarizes the growth properties of VPg mutants made in this study (Y3T T4Y, T4A, G5P, P7A, N8A, and K10A) and those previously described (Y3F, K9A K10A, R17E, R17Q, and R17K) (7, 21, 22, 43, 54). Also shown are two mutants in which either the N- or the C-terminal 3C^pro-specific cleavage site is eliminated (A−4E Q−1H in 3A and A19E Q22H in VPg). Mutations in the wt PV1(M) VPg sequence are indicated with boldface letters; the dashes represent conserved residues. *, quasi-infectious (q.i); **, Y3F and T3Y4 mutants were also made by Reuer et al. (43) and Kuhn et al. (21), respectively, but they contained an additional K10R mutation; ***, virus contained an additional K10R mutation in VPg, which had no effect on viability (22). The numbers in parentheses are references. ++++, wt-type growth; +++, slower than wt growth; ++, much slower than wt growth; −, no growth. (C) Plaque assay of viruses containing mutant VPgs. The plaque sizes of the three viable VPg mutants are compared to that of wt PV.

FIG. 3.

Effects of mutations in PV1(M) VPg on uridylylation in vitro. (A) Assay 1 (see Materials and Methods) was used with poly(A) as a template, except that the synthetic wt VPg peptide was replaced with a mutant peptide, as indicated. (B) Assay 2 (see Materials and Methods) was used with cre(2C) RNA as a template in the presence of 3CD^pro, except that wt VPg was replaced by a mutant peptide, as indicated.

FIG. 4.

Yeast two-hybrid analysis of the interaction between wt PV 3D^pol and wt or mutant VPgs. Yeast strain EGY48 was transformed with the indicated DNA binding domain (DB)- and transcriptional activation domain (AD)-viral protein fusion plasmids (see Materials and Methods). Protein-protein interaction was quantitated by analysis of β-galactosidase activity.

FIG. 5.

Growth properties of wt and chimeric PVs containing rhinoviral VPgs. (A) The sequences of HRV14, HRV2, and HRV89 VPgs are compared to that of PV. Amino acids conserved among all four peptides are shown with boldface letters. Transfections with transcripts derived from pT7PVM HRV14 VPg, pT7PVM HRV2 VPg, and pT7PVM HRV89 VPg were carried out as described in Materials and Methods. Virus derived from two transfections with pT7PVM HRV14 VPg was passaged four times; several large-plaque variants were isolated, and their VPg sequences were determined. All revertants contained an amino acid change (L12P) in HRV14 VPg (underlined). (B) Plaque size of wt PV1(M) compared to those of PVM HRV14 VPg and its revertant. ++++, wt-type growth; +++, slower than wt growth; ++, much slower than wt growth; −, no growth. Dots indicate missing amino acids relative to the HRV14 VPg sequence.

FIG. 6.

Comparison of PV and rhinovirus VPgs as substrates for uridylylation. (A) Assay 1 (see Materials and Methods) was used with poly(A) as a template, except that PV VPg was replaced with those of HRV14, HRV2, and HRV89, as indicated. (B) Assay 2 (see Materials and Methods) with cre(2C) RNA as a template was used in the presence of 3CD^pro to test uridylylation with the viral VPgs, as indicated.

FIG. 7.

Effect of divalent cation concentration on the uridylylation of VPg. Assay 1 (see Materials and Methods) with a poly(A) template was used to determine the optimal divalent concentration required for VPg uridylylation. (A) Mg²⁺; (B) Mn²⁺; (C) Zn²⁺; (D) Co²⁺.

FIG. 8.

Metal specificity of VPg uridylylation. Assay 1 (see Materials and Methods) was used for VPg uridylylation, except that Mn²⁺ (0.5 mM) was replaced with Co²⁺ (0.5 mM), Zn²⁺ (0.25 mM), or Mg²⁺ (3.5 mM), as indicated. The amount of product obtained with Mn²⁺ was taken as 100%.