A stable HeLa cell line that inducibly expresses poliovirus 2A(pro): effects on cellular and viral gene expression

Abstract

A HeLa cell clone (2A7d) that inducibly expresses the gene for poliovirus protease 2A (2A(pro)) under the control of tetracycline has been obtained. Synthesis of 2A(pro) induces severe morphological changes in 2A7d cells. One day after tetracycline removal, cells round up and a few hours later die. Poliovirus 2A(pro) cleaves both forms of initiation factor eIF4G, causing extensive inhibition of capped-mRNA translation a few hours after protease induction. Methoxysuccinyl-Ala-Ala-Pro-Val-chloromethylketone, a selective inhibitor of 2A(pro), prevents both eIF4G cleavage and inhibition of translation but not cellular death. Expression of 2A(pro) still allows both the replication of poliovirus and the translation of mRNAs containing a picornavirus leader sequence, while vaccinia virus replication is drastically inhibited. Translation of transfected capped mRNA is blocked in 2A7d-On cells, while luciferase synthesis from a mRNA bearing a picornavirus internal ribosome entry site (IRES) sequence is enhanced by the presence of 2A(pro). Moreover, synthesis of 2A(pro) in 2A7d cells complements the translational defect of a poliovirus 2A(pro)-defective variant. These results show that poliovirus 2A(pro) expression mimics some phenotypical characteristics of poliovirus-infected cells, such as cell rounding, inhibition of protein synthesis and enhancement of IRES-driven translation. This cell line constitutes a useful tool to further analyze 2A(pro) functions, to complement poliovirus 2A(pro) mutants, and to test antiviral compounds.

FIG. 1

tet regulates poliovirus 2A^pro expression in a stable HeLa cell clone: morphological alteration. (A) Phase-contrast microscopy of HeLa 2A7d cells at different times after induction of 2A^pro. OFF, uninduced 2A7d cells; ON28, 2A7d-On cells 28 h postinduction; ON46, 2A7d-On cells 46 h postinduction. (B) Immunofluorescence microscopy of 2A^pro-expressing cells. 2A7d cells were fixed and doubly stained with anti-2A antibodies (α-2A; rabbit polyclonal antiserum) and with a mouse monoclonal antibody against tubulin (α-tub) after 25 h in the presence (off) or absence (on) of tet. Immunofluorescence microscopy of HeLa cells was carried out as described previously (1). (C) Northern blot of mRNAs isolated at 24 h postinduction from 2A7d cells that express (on) or do not express (off) poliovirus 2A^pro hybridized with a 2A ³²P-labeled probe. As a loading control, the same samples were hybridized to a human β-actin mRNA probe. (D) HeLa 2A7d cells grown in 24-well dishes were treated with the indicated concentrations of tet. tet was present (+) or removed (−) from the medium by extensive washing to induce 2A^pro expression as described in Materials and Methods. Cells were harvested at 24 h postinduction, extracts were analyzed by SDS-PAGE, and the gel was immunoblotted with anti-eIF4GI antiserum. CP, cleavage products. Extracts from Hela cells infected with poliovirus (p) or not infected (m) were used as controls for eIF4G cleavage.

FIG. 2

Kinetics of protein synthesis and eIF4G cleavage at different times after induction of poliovirus 2A^pro in HeLa 2A7d cells. (Top) Kinetics of protein synthesis. HeLa 2A7d cells grown in 24-well dishes were extensively washed to remove tet to induce 2A^pro expression. Cell monolayers were then labeled with 25 μCi of [³⁵S]methionine/ml for 1 h at the indicated times (hours) postinduction (h.p.id.). Cell extracts were analyzed by SDS–15% PAGE. Aliquots of the same samples were also analyzed by immunoblotting. The blots were incubated with anti-eIF4GI antiserum (eIF4GI) (middle) or with an antiserum against the C-terminal domain of eIF4GII (eIF4GII; bottom). CP, cleavage products.

FIG. 3

Effect of MPCMK on the shutoff of protein synthesis and eIF4G cleavage in poliovirus-infected cells and in 2A7d-On cells. (A) HeLa 2A7d-Off cells grown in 24-well dishes were infected with poliovirus at a MOI of 10 PFU/cell and treated with 1 mM MPCMK after virus adsorption (+) or not treated (−). At 5.5 h postinfection cells were labeled with 25 μCi of [³⁵S]methionine/ml for 1 h and extracts were analyzed by SDS–15% PAGE (top). Aliquots of these samples were also assayed by immunoblotting with anti-eIF4GI antiserum (bottom). CP, cleavage products. (B) Poliovirus 2A^pro was induced in 2A7d cells grown in 24-well dishes by tet removal. MPCMK was added (+) or not added (−) to the culture medium from the beginning of 2A^pro induction. At the indicated times (hours) postinduction (h.p.id.), cells were labeled with 25 μCi of [³⁵S]methionine/ml for 1 h. Cell extracts were analyzed by SDS–15% PAGE (top). Aliquots were also assayed by immunoblotting using anti-eIF4GI antiserum (middle) or anti-eIF4GII antiserum (bottom). (C) Phase-contrast microscopy at 50 h postinduction of 2A7d-Off and 2A7d-On cells treated (+) or not treated (−) with 0.2 mM MPCMK during the time of induction.

FIG. 4

Kinetics of protein synthesis in 2A7d-Off and 2A7d-On cells infected with poliovirus, EMCV, or vaccinia virus. HeLa 2A7d cells grown in 24-well dishes were induced (on) or not induced (off) to synthesize poliovirus 2A^pro by tet removal. After 15 h, cells were infected with poliovirus (10 PFU/cell), with EMCV (8 PFU/cell), or with vaccinia virus (5 PFU/cell). Sixteen hours postinduction coincides with 1 h postinfection. Protein synthesis was estimated by pulse-labeling the cell monolayers with 25 μCi of [³⁵S]Met-Cys/ml as described in Materials and Methods. The same extracts were analyzed in parallel by immunoblotting using anti-eIF4GI antiserum as indicated for Fig. 1 (bottom). CP, eIF4GI cleavage products.

FIG. 5

Complementation of a poliovirus 2A^pro variant (M3) in 2A7d-On cells. HeLa 2A7d cells grown in 24-well dishes were induced to synthesize poliovirus 2A^pro (on) or not induced (off). After 13.5 h of induction, 2A7d cells were infected with wt virus or with poliovirus mutant M3 (10 PFU/cell). At the indicated times postinfection (hours), cells were labeled for 1 h with [³⁵S]Met-Cys and samples were analyzed by SDS-PAGE (A) or by immunoblotting using the corresponding specific antisera for eIF4GI (B) and poliovirus 3C (C) as described in Materials and Methods. m, mock-infected cells. The poliovirus obtained by transfection of HeLa cells with pT7XLD was used (8). The positions of 3C′, 3D′, 3CD, 3C, and eIF4GI and its cleavage products (CP) are indicated.

FIG. 6

Effect of eIF4G cleavage on the translation of mRNAs bearing different leader sequences. (A) After 8 h of induction of 2A expression, HeLa 2A7d cells were infected with recombinant vaccinia virus VT7 (5 PFU/cell). Cells were transfected after 45 min of VT7 adsorption with plasmid pTM1.2C, pAR.2C, or pT7.2C. AraC (40 μg/ml) was present (+) or not present (−) during transfection. At 16 h postinfection cells were labeled with [³⁵S]Met-Cys for 1 h as described in Materials and Methods. Proteins were analyzed by SDS-PAGE (top) or by immunoblotting. Blots were incubated with anti-eIF4GI antiserum (middle) or with poliovirus anti-2C antiserum (bottom). Positions of poliovirus 2C and eIF4GI and its cleavage products (CP) are indicated. (B) 2A7d cells grown in 35-mm-diameter dishes were induced to express 2A^pro for 15 h, and then cells were transfected with 1-μg portions of different mRNAs encoding luciferase under different leader sequences: uncapped (luc), capped (cap-luc), or uncapped luciferase mRNA bearing the 5′ leader sequence of poliovirus (Lluc). After 3 and 6 h posttransfection, cells were harvested and analyzed for luciferase activity as described previously (66). rlu, relative luciferase units.