A protein linkage map of the P2 nonstructural proteins of poliovirus

Abstract

The yeast two-hybrid system was used to catalog all detectable interactions among the P2 nonstructural cleavage products of poliovirus type 1 (Mahoney). Evidence has been obtained for specific associations among 2A(pro), 2BC, 2C, and 2B. Specifically, 2A(pro) can interact with itself and 2BC and its cleavage products (2B and 2C) interact in all possible combinations, with the exception of 2C/2C. Detected interactions were confirmed in vitro by a glutathione S-transferase pulldown assay, which allowed us to detect 2C/2C association. transdominant-negative mutants of 2B (K. Johnson and P. J. Sarnow, J. Virol. 65:4341-4349, 1991) were examined and were found to retain interaction with wild-type 2B, perhaps reflecting a need for 2B multimerization in viral RNA replication. The multimerization of 2B was examined further by screening a mutagenized library for 2B variants that have lost the ability to bind wild-type 2B. The screen identified two nonconservative missense mutations within a central hydrophobic region, as well as truncations and frameshifts that implicate the C terminus in homointeraction. Introduction of the missense mutations into the genome of the virus conferred a quasi-infectious phenotype, an observation strongly suggesting that the 2B/2B interaction is required for replication of the viral genome.

FIG. 1

Gene organization and polyprotein processing of poliovirus. (A) Structure of the genomic RNA of poliovirus and its coding region. The 5′ terminus is covalently linked to the VPg peptide (3B). The highly structured 5′ nontranslated region (NTR) regulates translation and positive-sense RNA synthesis, while the 3′ NTR regulates negative-sense RNA synthesis. The product of the single open reading frame, the polyprotein, is divided into three regions, as shown. Proteolytic processing of the P2 nonstructural precursor yields 2A^pro, 2BC, 2B, and 2C. (B) Representation of the 2B polypeptide and the map positions of 2B mutants. LB21, W6, W36, W10, LB5, and LB21 were isolated from a randomly mutagenized library of GAD-2B assayed for loss of multimerization with the wild-type protein (see Results). Italicized residue numbers indicate codons that have been altered by mutation. Linker insertion mutant 2B(201) was generated by Bernstein et al. (8). Linker insertion mutants 2B(204) and 2B(205) were generated by Johnson and Sarnow (29). A computer prediction of the 2B secondary structure was carried out with PeptideStructure (Genetics Computer Group software package, Biotechnology Center, University of Wisconsin). Features depicted constitute a general consensus between the predictions of Chou and Fasman and those of Garnier, Osguthorpe, and Robson.

FIG. 2

Two-hybrid assays for interactions among 2BC and its cleavage products in strain L40-ura3 transformed with LexA and GAD fusions of 2B, 2C, and 2BC. GAD-retinoblastoma (Rb), GAD empty vector (Vector), and LexA-lamin were used as negative controls. (A) Filter lift assay for lacZ expression with X-Gal as the substrate. (B) Colorimetric quantitation in Miller units of positive pairings from panel A with chlorophenol red-β-d-galactopyranoside as the substrate for β-galactosidase. Bars represent the means of duplicate samples; error bars represent maximum measured values. (C) Assay for expression of the HIS3 reporter gene by selection of transformants on minimal media lacking His.

FIG. 2

Two-hybrid assays for interactions among 2BC and its cleavage products in strain L40-ura3 transformed with LexA and GAD fusions of 2B, 2C, and 2BC. GAD-retinoblastoma (Rb), GAD empty vector (Vector), and LexA-lamin were used as negative controls. (A) Filter lift assay for lacZ expression with X-Gal as the substrate. (B) Colorimetric quantitation in Miller units of positive pairings from panel A with chlorophenol red-β-d-galactopyranoside as the substrate for β-galactosidase. Bars represent the means of duplicate samples; error bars represent maximum measured values. (C) Assay for expression of the HIS3 reporter gene by selection of transformants on minimal media lacking His.

FIG. 2

Two-hybrid assays for interactions among 2BC and its cleavage products in strain L40-ura3 transformed with LexA and GAD fusions of 2B, 2C, and 2BC. GAD-retinoblastoma (Rb), GAD empty vector (Vector), and LexA-lamin were used as negative controls. (A) Filter lift assay for lacZ expression with X-Gal as the substrate. (B) Colorimetric quantitation in Miller units of positive pairings from panel A with chlorophenol red-β-d-galactopyranoside as the substrate for β-galactosidase. Bars represent the means of duplicate samples; error bars represent maximum measured values. (C) Assay for expression of the HIS3 reporter gene by selection of transformants on minimal media lacking His.

FIG. 3

Two-hybrid assays for interaction of 2A^pro with cleavage products of P2 in strain Y153 transformed with GBD and GAD fusions of 2A^pro, 2B, 2C, and 2BC. (A) Filter lift assay for lacZ expression. (B) Colorimetric quantitation of the 2A^pro multimerization seen in panel A. Bars and error bars are as defined for Fig. 2B.

FIG. 4

GST pulldown assay of putative interacting pairs, performed as described in Materials and Methods. Lanes marked “translation products” were loaded with 0.5 μl of in vitro translation reaction mixture with the indicated contents. (A) Assays for the interaction of ³⁵S-labeled 2B, 2C, and 2BC with GST, GST-2B, GST-2C, and GST-2BC. (B) Assays for the interaction of ³⁵S-labeled 2A^pro with GST and GST-2A^pro.

FIG. 5

Two-hybrid tests for the interaction of 2B with trans-dominant-negative mutants in strain L40-ura3 transformed with LexA and GAD fusions of 2B, 2B(201), 2B(204), and 2B(205) (Fig. 1B). (A) Filter lift assays for lacZ expression. (B) Colorimetric quantitation of interactions seen in panel A. Bars and error bars are as defined for Fig. 2B. (C) Assay for expression of the HIS3 reporter gene.

FIG. 5

Two-hybrid tests for the interaction of 2B with trans-dominant-negative mutants in strain L40-ura3 transformed with LexA and GAD fusions of 2B, 2B(201), 2B(204), and 2B(205) (Fig. 1B). (A) Filter lift assays for lacZ expression. (B) Colorimetric quantitation of interactions seen in panel A. Bars and error bars are as defined for Fig. 2B. (C) Assay for expression of the HIS3 reporter gene.

FIG. 5

Two-hybrid tests for the interaction of 2B with trans-dominant-negative mutants in strain L40-ura3 transformed with LexA and GAD fusions of 2B, 2B(201), 2B(204), and 2B(205) (Fig. 1B). (A) Filter lift assays for lacZ expression. (B) Colorimetric quantitation of interactions seen in panel A. Bars and error bars are as defined for Fig. 2B. (C) Assay for expression of the HIS3 reporter gene.

FIG. 6

Two-hybrid assays for the interaction of wild-type 2B with mutagenized library isolates that have been reconstructed into pGAD.GH by using strain L40-ura3. The expression of lacZ and HIS3 reporter genes was assayed by filter lift and selection on media lacking HIS. The double mutations in W10 were reconstructed into separate constructs (W10f and W10s). An asterisk denotes the position of missense mutations outlined in Fig. 1B.

FIG. 7

Two-hybrid assays for the interaction with mutant 2B and 2BC. (A) Filter lift assays for the interaction of 2B constructs carrying the W36 mutation (I53N) with 2B, 2C, and 2BC, and for the multimerization of 2B(W36). The multimerization of wild-type 2B was assayed in parallel as a positive control. (B) Two-hybrid assays for the interaction of 2BC mutants carrying the W36 mutation (I53N) with 2B, 2C, 2BC, and for the multimerization of 2BC(W36). (C) Schematic summary of interactions involving 2B(W36) and 2BC(W36). Double arrows indicate positive reciprocal two-hybrid interactions, while single arrows denote unidirectional (polar) interactions.

FIG. 8

Luciferase activity in cell cultures transfected with luciferase-expressing viral replicons encoding either the wild-type protein, LB21, W10s, or 2B(W36). Following transfection, cell cultures were maintained either in the absence or presence of 2 mM guanidine hydrochloride. Light units are arbitrary.

FIG. 9

SDS–15% PAGE analysis of in vitro-translated full-length transcript RNAs harboring the LB21, W10s, and W36 mutations, as compared to the wild type. The “Marker” lane consists of the (³⁵S-Translabel) pulse-labeled products from PVM-infected HeLa R-19 cells.

FIG. 10

Protein linkage map of the cleavage products of the P2 protein precursor, suggested by data obtained by two-hybrid and GST pulldown assays. See the text for an explanation of the two classes of interaction. Double arrows indicate positive reciprocal interactions, whereas single arrows denote unidirectional (polar) interactions.