Alanine scanning of poliovirus 2CATPase reveals new genetic evidence that capsid protein/2CATPase interactions are essential for morphogenesis

Abstract

Polypeptide 2C(ATPase) is one of the most thoroughly studied but least understood proteins in the life cycle of poliovirus. Within the protein, multiple functional domains important for uncoating, host cell membrane alterations, and RNA replication and encapsidation have previously been identified. In this study, charged to alanine-scanning mutagenesis was used to generate conditional-lethal mutations in hitherto-uncharacterized domains of the 2C(ATPase) polypeptide, particularly those involved in morphogenesis. Adjacent or clustered charged amino acids (2 to 4), scattered along the 2C(ATPase) coding sequence, were replaced with alanines. RNA transcripts of mutant poliovirus cDNA clones were transfected into HeLa cells. Subsequently, 10 lethal, 1 severely temperature-sensitive, 2 quasi-infectious, and 3 wild type-like mutants were identified. Using a luciferase-containing reporter virus, we demonstrated RNA replication defects in all lethal and quasi-infectious mutants. Temperature-sensitive mutants were defective in RNA replication only at the restricted temperatures. Furthermore, we characterized a quasi-infectious mutant (K(6)A/K(7)A) that produced a suppressor mutation (G(1)R) and a novel 2B^2C(ATPase) cleavage site (Q^R). Surprisingly, this cleavage site mutation did not interfere with normal processing of the polyprotein. These mutants have led to the identification of several new sites within the 2C(ATPase) polypeptide that are required for RNA replication. In addition, analysis of the suppressor mutants has revealed a new domain near the C terminus of 2C(ATPase) that is involved in encapsidation, possibly achieved through interaction with an amino acid sequence between NTP binding motifs A and B of 2C(ATPase). Most importantly, the identification of suppressor mutations in both 2C(ATPase) and the capsid domains (VP1 and VP3) of poliovirus has confirmed that an interaction between 2C(ATPase) and capsid proteins is involved in viral morphogenesis.

Fig 1

Genomic structure of poliovirus, functional motifs, and grouping of the 16 alanine-scanning mutants of protein 2C^ATPase. (A) The PV RNA contains a long 5′ nontranslated region (5′ NTR), a single open reading frame, a short 3′ nontranslated region (3′ NTR), and a poly(A) tail. The proteinase cleavage sites and the P1, P2, and P3 domains of the polyprotein are shown. (B) Functional domains of the 2C^ATPase protein. (C) Locations of previously identified mutations in 2C^ATPase involved in encapsidation or uncoating. H₁₁₈Y and V₁₉₄I are newly discovered hydantoin-resistant mutations (Paul et al., unpublished). (D) Locations and grouping of the 16 alanine-scanning mutants of 2C^ATPase.

Fig 2

In vitro translation of Wt and mutant RNA transcripts. RNA transcripts (200 ng) derived from the Wt and 16 alanine-scanning mutant constructs were translated in vitro in HeLa cell extracts at 34°C for 8 h, as described in Materials and Methods. Positions of the precursor and mature proteins are indicated, and the 2C^ATPase-related proteins are boxed. Those mutants that exhibited a shift in the migration of their 2C^ATPase-related proteins (P2, 2BC, and 2C^ATPase) are bolded and underlined.

Fig 3

Mutant 7 is severely temperature sensitive and exhibits a RNA replication defect at the nonpermissive temperatures. (A) Growth phenotypes of mutant 7 (E₁₄₈A/R₁₄₉A/E₁₅₀A) and its partial revertant variant 7r (E₁₄₈A/R₁₄₉A). The titers of lysates of the Wt and mutant 7 from the 33°C transfections (Tf) and of plaque-purified variant 7r were determined at 33°C, 37°C, and 39.5°C by plaque assay. Virus titers are shown on the left and plaque sizes on the right of the panel. For mutant 7, plaque pictures were taken from different dilutions (10⁻⁵, 10⁻², and 10⁻²) at 33°C, 37°C, and 39.5°C, respectively. (B) Analysis of RNA replication phenotype of mutant 7. The genome structure of the R-Luc reporter virus (R-PPP) is shown on top of the panel. RNA replication and encapsidation by the Wt and mutant 7 were assayed at different temperatures using the luciferase reporter virus. Transfection of RNA transcripts and luciferase assays were performed as described in Materials and Methods. R-PPP, Renilla luciferase gene fused to three domains of the PV polyprotein; Mock, no viral RNA added.

Fig 4

Quasi-infectious mutants 1 and 11 are defective in RNA replication. (A) Growth phenotypes of mutants 1 and 11. At the time of full CPE (passage 2 with mutant 11; passage 1 with mutant 1), the transfection lysates at 33°C were collected and the virus titers were determined by plaque assay at 33°C, 37°C, and 39.5°C. Virus titers are illustrated on the left side and plaque sizes on the right side of the panel. For mutants, plaque pictures are taken from different dilutions at different temperatures as follows: for mutant 1, 10⁻⁵, 10⁻⁶, and 10⁻⁵ at 33°C, 37°C, and 39.5°C, respectively; and for mutant 11, 10⁻⁵, 10⁻³, and 10⁻³ at 33°C, 37°C, and 39.5°C, respectively. (B) HeLa R19 cells were transfected with 5 μg of Wt or mutant Renilla luciferase reporter virus RNA transcripts in both the presence and absence of 2 mM GnHCl, and then they were subjected to passage once at 33°C on HeLa cells. Luciferase activities were determined as described in Materials and Methods. Mock, no viral RNA added.

Fig 5

Quasi-infectious mutant 1 yielded suppressor mutants harboring a novel Q∧R3CD^pro protease cleavage site at 2B∧2C^ATPase. (A) Location of suppressor mutations G₁R and S₃R of 2C^ATPase alanine-scanning mutant 1. The amino acid sequence around the 2B∧2C^ATPase cleavage site of the Wt, mutant 1, R₁A₆A₇ (G₁R), and R₃A₆A₇ (S₃R) suppressor mutants is shown. The G₁R mutation was introduced into the Wt PV background (R₁K₆K₇), which is also included in the panel. A vertical arrow shows the 3CD^pro cleavage site at 2B∧2C^ATPase. The last amino acid in 2B is indicated by −1 and the amino acids of 2C^ATPase by 1 to 7. (B) In vitro translation assays of mutant 1 (Wt-like) and of two reconstructed suppressor mutants, R₁A₆A₇ and R₁K₆K₇. Translations were carried out in HeLa cell extracts in vitro at 34°C for 6 h, 8 h, and 16 h (see Materials and Methods). Only the upper portion of the gel (VP1 and up) is shown. (C) Growth phenotypes of the Wt and of all three derivatives of mutant 1. RNA transcripts were transfected into HeLa cells, and the lysates were collected at the time of full CPE. At 33°C, the R₁A₆A₇ mutant yielded full CPE only at passage 1. The titers of the lysates were determined by plaque assay at 33°C, 37°C, and 39.5°C. The virus titers are shown at the top and the plaque phenotypes at the bottom of the panel.

Fig 6

Growth phenotypes of four suppressor variants of mutant 11. (A) Four suppressor variants of mutant 11, all of which retain the original mutant 11 mutation at K₂₇₉A/R₂₈₀A, as indicated by the diamond in the schematic genomes. The genomic structures of mutant 11 and its four suppressor variants are shown on the top. The locations of suppressor mutations in 2C^ATPase (either C₃₂₃R or E₁₄₈K) and/or capsid proteins VP3 (K₄₁R) and VP1 (T₃₆I or N₂₀₃S) are indicated. The genotypes and the selection temperatures and phenotypes of the four suppressor variants are summarized at the bottom. (B) Growth phenotypes of the four suppressor variants of mutant 11. Plaques were picked and amplified at the temperatures indicated in panel A, and the titers of the lysates were determined by plaque assay at 33°C, 37°C, and 39.5°C and incubated for 72 h. Note that plaque pictures of variant 11 at 37°C and 39.5°C were taken from a different dilution (10⁻⁴) instead of 10⁻⁷ for the Wt and other variants. (C) The genomic structures of F-Luc replicons (FPP) of the Wt and mutant 11 and its suppressor variants, FPP (mutant 11 plus C₃₂₃R) and FPP (mutant 11 plus E₁₄₈K), which carry only single suppressor mutations in 2C^ATPase (either C₃₂₃R or E₁₄₈K) in addition to the original mutant 11 mutation at K₂₇₉A/R₂₈₀A, are shown at the top. RNA replication of these constructs was assayed at 33°C, 37°C, and 39.5°C in the absence and presence of GnHCl (see Materials and Methods). The ratio of F-Luc levels observed in the absence and presence of GnHCl is plotted; at each temperature, the ratio of the Wt is taken as 100%. (D) Capsid suppressor mutations do not rescue the RNA replication defect of mutant 11. HeLa R19 cells were transfected with 5 μg of Renilla luciferase reporter virus RNA transcripts of the Wt or mutant 11 and its variants with capsid suppressor mutations at 33°C or 39.5°C in both the presence and absence of 2 mM GnHCl. Luciferase activities were determined as described in Materials and Methods. The ratio of R-Luc levels observed in the absence and presence of GnHCl is plotted. Mock, no viral RNA added.