Picornavirus genome replication. Identification of the surface of the poliovirus (PV) 3C dimer that interacts with PV 3Dpol during VPg uridylylation and construction of a structural model for the PV 3C2-3Dpol complex

Abstract

Picornaviruses have a peptide termed VPg covalently linked to the 5'-end of the genome. Attachment of VPg to the genome occurs in at least two steps. First, Tyr-3 of VPg, or some precursor thereof, is used as a primer by the viral RNA-dependent RNA polymerase, 3Dpol, to produce VPg-pUpU. Second, VPg-pUpU is used as a primer to produce full-length genomic RNA. Production of VPg-pUpU is templated by a single adenylate residue located in the loop of an RNA stem-loop structure termed oriI by using a slide-back mechanism. Recruitment of 3Dpol to and its stability on oriI have been suggested to require an interaction between the back of the thumb subdomain of 3Dpol and an undefined region of the 3C domain of viral protein 3CD. We have performed surface acidic-to-alanine-scanning mutagenesis of 3C to identify the surface of 3C with which 3Dpol interacts. This analysis identified numerous viable poliovirus mutants with reduced growth kinetics that correlated to reduced kinetics of RNA synthesis that was attributable to a change in VPg-pUpU production. Importantly, these 3C derivatives were all capable of binding to oriI as well as wild-type 3C. Synthetic lethality was observed for these mutants when placed in the context of a poliovirus mutant containing 3Dpol-R455A, a residue on the back of the thumb required for VPg uridylylation. These data were used to guide molecular docking of the structures for a poliovirus 3C dimer and 3Dpol, leading to a structural model for the 3C(2)-3Dpol complex that extrapolates well to all picornaviruses.

Figure 1. Assembly and organization of the picornavirus VPg ribonucleoprotein complex

Step 1: Two 3C(D) molecules bind to oriI with the 3C domains contacting the upper stem (solid lines) and the 3D domains contacting the lower stem (dashed lines). Step 2: The 3C dimer opens the RNA stem by forming a more stable interaction with single strands forming the stem. Step 3: 3Dpol is recruited to and retained in this complex by a physical interaction between the back of the thumb sub-domain of 3Dpol and a surface of one or both 3C subunits of the dimer. Taken from ref. (13).

Figure 2. Solvent accessible acidic amino acid residues of PV 3C

The structure of PV 3C (1L1N) is shown. The residues targeted for mutational analysis are labeled and shown as ball-and-sticks. The N- and C-termini are displayed as light and dark spheres, respectively.

Figure 3. Kinetics of virus growth for selected PV 3C mutants

HeLa cells were infected at a multiplicity of infection of 10 with PV encoding WT or the indicated mutant 3C protein. The cells were incubated at 37 °C for 0, 1, 2, 3, 4, 6 and 8 h post-infection. Virus was harvested by 3 repeated freeze-thaw cycles and virus titers were performed by plaque assays on HeLa cells. Viral titer (pfu/mL) was plotted as a function of time post-infection.

Figure 4. Kinetics of RNA synthesis using a luciferase-expressing, subgenomic replicon

HeLa cells were transfected with the indicated replicon RNA (5 μg) and incubated for 4 h (A) or 8 h (B) post-transfection. Extracts were prepared and analyzed for luciferase activity. Luciferase specific activity is reported in relative light units (RLU) per microgram of total protein in the extract. WT + GuHCl represents a control for translation of input RNA without replication. The dashed line indicates luciferase activity observed for WT at 4 h. The results are the average of two independent transfections of replicon RNA transcribed from two independent clones. Error bars indicate the standard deviation.

Figure 5. Proteolytic processing activity of PV 3C mutants

HeLa extracts containing [³⁵S]-methionine were programmed with subgenomic replicon RNAs encoding the indicated 3C protein. After 2 h, samples were mixed with SDS-PAGE sample buffer and resolved by SDS-PAGE on a 12.5% acrylamide gel. Gels were fixed, dried and the radiolabeled proteins were detected by phosphorimaging.

Figure 6. VPg uridylylation activity of selected PV 3C mutants

A. Sypro stained gel of spin-column-purified proteins (0.1 μg) employed in the VPg uridylylation experiment. The sypro-stained proteins were detected by using a Typhoon-8600 Variable Mode Imager. B. VPg uridylylation reactions were performed by using the indicated purified 3C derivative. Uridylylation products were resolved from [α-³²P]UTP by using a 15% polyacrylamide gel and a Tris-Tricine buffer system. The “-3C” lane indicates a reaction performed in the absence of 3C.

Figure 7. OriI binding by selected PV 3C derivatives

A. OriI binding by WT 3C. 3′-fluorescein-labeled oriI RNA (29-nt) was titrated with 3C. Binding was measured by monitoring the change in fluorescence polarization (Δ mP). The data were fit to a hyperbola. The K_d value is 260 ± 40 nM. B. OriI binding by 3C derivatives. 3C protein (150 nM) was mixed with 3′-fluorescein labeled oriI RNA and the change in polarization (Δ mP) was measured. The results are the average of three independent binding experiments with error bars indicating the standard deviation.

Figure 8. PV mutants encoding substitutions in 3C residues proposed to interact with 3Dpol exhibit synthetic lethality in a PV background producing 3Dpol R455A

A. Conceptual framework for this experiment. (i) WT 3C₂ and 3Dpol interact via specific surface residues. (ii) R455A mutation in 3Dpol weakens 3C₂-3Dpol interaction and reduced replication occurs. (iii) R455A 3Dpol mutation combined with 3C₂ interaction surface mutation disrupts 3C₂-3Dpol interaction and prevents replication (synthetic lethality). (iv) R455A 3Dpol mutation combined with 3C₂ remote site (non-interaction surface) mutation results in 3C₂-3Dpol interaction and replication as in (ii). B. Replication of WT, 3Dpol-R455A or 3C mutations as indicated in either 3Dpol-WT or 3Dpol R455A background. Luciferase activity (RLU/μg) was determined 2, 4, 6, 8 and 10 h post-transfection into HeLa cells. Dashed line indicates luciferase activity attributable to translation only. The 3C E45A and E96A/D99A mutants exhibit synthetic lethality in the 3Dpol R455A background, further confirming an interaction of these residues with 3Dpol.

Figure 9. Model of 3C₂-3Dpol complex

A. The surfaces of the 3C dimer and 3Dpol are shown in cyan and purple, respectively. The 3Dpol sub-domains are abbreviated as follows: F, fingers; T, thumb; P, palm. B. Same as in (A) with 90° rotation around y-axis. C. Same as in (A) with 180° rotation around the y-axis D. The model of 3C₂-3Dpol is shown rotated 180° around the Y-axis relative to the view in (A); 3Dpol residues known to be required for VPg uridylylation are shown. These are: Asp406 (14,32), Arg455 (38,41) and Phe461 (69). E. Residues of the 3C dimer that contribute to interaction with 3Dpol are shown in yellow. Selected 3C residues are shown for orientation. The complementary residues from 3Dpol are displayed as sticks. These interactions are listed in Table 4.

Figure 10

A. Previously isolated 3Dpol mutants map to the 3C₂-interaction surface defined in this study. The 3Dpol surface is colored purple, and regions of 3Dpol involved in 3C₂-3Dpol interactions are shown in raspberry. 3Dpol mutants constructed in ref. (42) are shown in orange. Those that killed the virus include: Arg128, Lys383, Lys405, Asp406, Asp412, Arg455 and Arg456. Those that caused a temperature-sensitive phenotype include: Glu98, Asp99, Asp105, Glu108, Arg136, Asp137, Glu426, Glu427 and Glu428. Residues that overlap directly with those for which biochemical data exist are underlined above and shown in green on the structure. B. Diversifying residues on 3C and 3Dpol map to the 3C₂-3Dpol interface. [i] The 3Dpol surface is colored purple and regions that interact with the 3C dimer are shown in raspberry. Diversifying residues of 3Dpol identified in ref. (75) are shown in orange including: Met86, Glu93, Thr138, Lys200, Glu254, Gly259, Asp260, Glu369, Pro385, Lys431, and Ala434. Residues identified in both studies are shown in green. [ii] The 3C surface is colored in cyan and regions that interact with 3Dpol are shown in yellow. Diversifying residues of 3C identified in ref. (75) are shown in red including: Ser21, Gly44, Glu45, Ser46, Thr68, Tyr109, and Thr120. Residues identified in both studies are shown in green.