Identification of regions of poliovirus 2BC protein that are involved in cytotoxicity

Abstract

The expression of poliovirus 2BC protein in yeast and mammalian cells leads to a number of metabolic and morphological alterations, such as growth inhibition, intracellular membrane proliferation, blockade of the exocytic pathway, and enhanced membrane permeability. Yeast cells that express poliovirus 2BC in an inducible manner were used to identify the regions of 2BC implicated in the modifications of these cellular functions. Several 2BC deletion mutants were generated to define the minimal portion of 2BC required to alter these activities. Additional deletion mutants that were obtained by random mutagenesis followed by selection in yeast cells provided new insights into the structure and mechanism of action of 2BC. The activity responsible for membrane proliferation is located in 2C, while the activities responsible for membrane permeabilization and inhibition of the exocytic pathway are located in 2B. Several regions of 2B and 2C required for the different functions of 2BC were identified. Thus, the integrity of the N termini of both 2B and 2C is necessary for 2BC-induced cytotoxicity. It is also possible to separate the different cellular alterations provoked by 2BC by the use of several 2BC variants. Deletion of amino acids 52 to 65 in 2B generates a 2BC deletion variant, 2bC deltaAvrII, that still blocks yeast growth but is unable to enhance membrane permeability or to inhibit the exocytic pathway. On the other hand, 2Bcl28*.32b and 2Bcl28*.3c, which contain only 73 and 77 amino acids of 2B, interfere with yeast division and enhance membrane permeability but affect the exocytic pathway only weakly and do not induce membrane proliferation. Our findings indicate that Saccharomyces cerevisiae represents a useful model system to analyze the functions of poliovirus 2BC and show the feasibility of separating the activities assigned to this protein.

FIG. 1

Schematic representation of 2BC mutants and summary of the effects of their expression in yeast cells. The amino acids of each protein are indicated. Black bars indicate sequences unrelated to 2BC. Growth agar (−HB/+HB) indicates growth on YNB.Gal plates supplied (+HB) or not supplied (−HB) with 50 μg of HB per ml; +++, growth rate similar to that of control cells; ++ growth at a rate lower than that of control cells; +, growth resulting in individual colony formation; −, no growth. The third value shown for 2B(1-97) corresponds to cells streaked for a second time on YNB.Gal plates supplemented with HB. Growth liquid indicates that exponentially growing yeast cultures were diluted in YNB.Lac medium, and galactose (2%) was added (initial A₆₆₀, 0.150). Cells were harvested at 2-h intervals, and the A₆₆₀ was measured to quantitate cell density: the optical density of the culture after 24 h of incubation increased more than 10-fold (+++), between 7- and 10-fold (++), between 5- and 7-fold (+), between 3- and 5-fold (−), or less than 3-fold (−−). pCPY arrest indicates that the ratio of precursor form of CPY (pCPY) to mature CPY in wild-type 2BC-expressing cells was calculated by densitometric analysis, and this value was taken as 100%. The relative proportion of pCPY was calculated for the rest of the mutants: +++, 100 to 70% wild-type 2BC activity; ++, 70 to 40% wild-type 2BC activity; +, 40 to 10% wild-type 2BC activity; −, no pCPY accumulation. HB sensitivity indicates that the ratio of protein synthesis in yeast cells expressing 2BC in the absence or in the presence of HB (1 mM) was calculated by densitometric analysis, and this value was taken as 100%. Therefore, 100% represents full permeabilization to HB. The relative inhibition of protein synthesis by HB was calculated for the rest of the mutants: +++, 100 to 70% wild-type 2BC activity; ++, 70 to 40% wild-type 2BC activity; +, 40 to 10% wild-type 2BC activity; −, no HB permeabilization.

FIG. 2

Growth of yeast cells expressing different 2BC mutants. (A) Yeast cells expressing different 2BC variants were streaked on agar plates with the indicated composition and incubated for 3 days at 30°C. HigB, hygromycin B. (B) Extracts of yeast cells expressing the different 2BC variants indicated or bearing plasmid pEMBLyex4 (V) were obtained after 6 h of induction and assayed with antibodies against 2B or 2C proteins. Immunoblot analysis of these samples was performed as described in Materials and Methods.

FIG. 3

Action of 2BC variants on HB permeabilization. (A) 2BC induces permeability changes. Protein synthesis in yeast cells bearing plasmids pEMBLyex4 (V) and pEMBL.2BC (2BC) in the presence (+) or absence (−) of HB (1 mM) was assayed. The samples were obtained as described in Materials and Methods at the postinduction times indicated in hours. (B) Effects of 2BC variants on membrane permeability. Protein synthesis in yeast cells expressing the 2BC variants indicated or bearing plasmid pEMBLyex4 (V) in the presence (+) or absence (−) of HB (1 mM) was assayed. The samples were obtained as described in Materials and Methods at 5 h postinduction. This experiment was performed with all of the mutants obtained, and the results of the densitometric analysis of the corresponding sodium dodecyl sulfate-polyacrylamide gel electrophoresis autoradiograms are included in Fig. 1.

FIG. 4

Action of 2BC variants on the exocytic pathway. (A) Extracts of yeast cells expressing the different 2BC variants indicated or bearing plasmid pEMBLyex4 (V) were obtained after 6 h of induction and assayed with antibodies against CPY. mCPY, mature CPY; pCPY, precursor form of CPY. (B) Yeast cells at 4 h postinduction in YNB.LGal medium were labeled for 10 with [³⁵S]methionine and chased for 0 or 30 min. Invertase immunoprecipitation of control cells (bearing pEMBLyex4 [V]) or cells expressing the different 2BC variants indicated was performed as described in Materials and Methods. This pulse-chase experiment was carried out three times for mutant 2Bc128. In all cases, the amount of invertase precipitated was much smaller than that obtained with any other 2BC variant. mInv, mature invertase; pInv, precursor forms of invertase; MW, molecular weight (in thousands).

FIG. 5

Ultrastructure of yeast cells expressing different 2BC variants. Thin-section electron microscopy was carried out for yeast cells expressing 2bCΔ30N (A), 2BC (B), 2Bc258 (C), 2bC-D (D), 2Bc128 (E and F), 2Bc108 (G), 2B (H), 2B-2cΔ40N (I), or 2Bc128*3c (J) or transformed with plasmid pEMBLyex4 (K). Cells were chemically fixed at 20 h postinduction and were processed for electron microscopy as described in Materials and Methods. N, nucleus; V, vacuole; M, mitochondria. Black arrows indicate ER swelling. Bars, 500 nm.

FIG. 6

Sequences of 2B deletion mutants. Amino acid sequences from position 51 to the C terminus of the indicated mutants (Mut) are shown. Shaded sequences correspond to the wild-type 2B (2Bwt) sequence.