Regulation of poliovirus 3C protease by the 2C polypeptide

Abstract

Poliovirus-encoded nonstructural polypeptide 2C is a multifunctional protein that plays an important role in viral RNA replication. 2C interacts with both intracellular membranes and virus-specific RNAs and has ATPase and GTPase activities. Extensive computer analysis of the 2C sequence revealed that in addition to the known ATPase-, GTPase-, membrane-, and RNA-binding domains it also contains several "serpin" (serine protease inhibitor) motifs. We provide experimental evidence suggesting that 2C is indeed capable of regulating virus-encoded proteases. The purified 2C protein inhibits 3C(pro)-catalyzed cleavage of cellular transcription factors at Q-G sites in vitro. It also inhibits cleavage of a viral precursor by the other viral protease, 2A(pro). However, at least three cellular proteases appear not to be inhibited by 2C in vitro. The 2C-associated protease inhibitory activity can be depleted by anti-2C antibody. A physical interaction between 2C and His-tagged 3C(pro) can be demonstrated in vitro by coimmunoprecipitation of 2C with anti-His antibody. Deletion analysis suggests that the 2C central and C-terminal domains that include several serpin motifs are important for 3C(pro)-inhibitory activity. To examine the 2C protease inhibitory activity in vivo, stable HeLa cell lines were made that express 2C in an inducible fashion. Infection of 2C-expressing cells with poliovirus led to incomplete (or inefficient) processing of viral precursor polypeptides compared to control cell lines containing the vector alone. These results suggest that 2C can negatively regulate the viral protease 3C(pro). The possible role of the 2C protease inhibitory activity in viral RNA replication is discussed.

FIG. 1.

Schematic diagram of domain structures of 2C. A linear diagram of 2C shows the locations of putative domains as determined by computer analyses and prior studies from various laboratories. The numbers at the top indicate the amino acids from N (1) to C (329) termini of the 2C polypeptide.

FIG. 2.

Poliovirus 3C^pro activity is inhibited by the 2C polypeptide. (A) Totals of 300 ng of 2C (lane 1) and 2A (lane 2) and 800 ng of 3C (lane 3) were analyzed by SDS-PAGE, followed by silver staining as described in Materials and Methods. (B) Totals of 500 ng (lanes 3 to 8) and 2 μg (lanes 9 to 14) of 3C^pro was preincubated with buffer (lane 1), 500 ng of 2C (lane 2), or various amounts of purified 2C prior to labeled substrate addition (1 μl of in vitro-translated, [³⁵S]methionine-labeled CREB) as described in Materials and Methods. Reactions were terminated by the addition of 2× SDS sample buffer (1:1 [vol/vol]), and proteins were resolved by SDS-14% PAGE, followed by autoradiography. The numbers on the left show the position and approximate molecular mass (in kilodaltons) of the marker proteins. The arrowheads on the right indicate the migration of full-length and the proteolyzed CREB products (∼28 and ∼20 kDa). Lanes 4 through 8 contain 50, 125, 250, 375, or 500 ng of 2C, respectively. Lanes 10 through 14 contain 50, 125, 250, 375, or 500 ng of 2C, respectively.

FIG. 3.

2BC inhibits 3C^pro activity. (A) In vitro-translated, [³⁵S]methionine-labeled CREB was incubated with buffer (lane 1) or 500 ng each of bacterially expressed, affinity-purified 2B (lanes 2 and 6), 3D^pol (lanes 3 and 7), 2BC (lanes 4 and 9), and 2C (lane 8) in the absence (lanes 1 to 4) or presence (lanes 5 to 9) of 500 ng of 3C^pro. Lane 10 is the same as lane 5 except that 500 ng of an extract from bacteria harboring the plasmid without the 2C insert was used. (B) 2A^pro activity is inhibited by 2C. Various amounts of 2C were preincubated with 20 ng of purified 2A^pro before addition to the reactions containing [³⁵S]methionine-labeled 3CD. Reactions were terminated by the addition of 2x SDS sample buffer and proteins resolved on a 14% SDS polyacrylamide gel. All reactions contain labeled [³⁵S] methionine-labeled 3CD. Lane 1, control reaction with labeled 3CD as substrate plus buffer; lane 2, 500 ng of 2C; lane 3, 20 ng of 2A^pro; lanes 4 to 7, reactions containing 2A^pro (20 ng) preincubated with 125 ng (lane 4), 250 ng (lane 5), 375 ng (lane 6), 500 ng (lane 7) of 2C, or 500 ng of heat-inactivated 2C (lane 8) before the addition of labeled 3CD substrate. The arrowheads indicate full-length 3CD and proteolyzed products 3C′ and 3D′, respectively. Positions of the migration of molecular markers are indicated on the right (in kilodaltons).

FIG. 4.

Effect of 2C protein on cellular proteases. Two eukaryotic proteases, thrombin, and enterokinase were used in this experiment. The activity of the proteases was monitored by Western blot analysis after cleavage of a control protein of 48 kDa supplied by the manufacturer (Novagen, Inc.) in the presence or absence of 2C polypeptide as detailed in Materials and Methods. Lane 1, control protein plus buffer; lane 2, control protein in the presence of 0.06 U of enterokinase; lane 3, same as lane 2 but also containing 500 ng of purified 2C; lane 4, control protein and 0.005 U of thrombin; lane 5, same as lane 4 but also containing 500 ng of purified 2C. The numbers on the right indicate the positions of the prestained molecular weight markers (lane M). The migrations of the 32- and 16-kDa cleaved products and the full-length protein are marked by arrows.

FIG. 5.

The protease inhibitory activity of 2C is depleted specifically by 2C antibody. (A) A 1.5-μg portion of 2C was incubated with 3 (lane 4) or 4.5 (lane 5) μg of anti-2C IgG or 4.5 μg (lane 6) of preimmune IgG for 2 h at 4°C. The antigen-antibody complex was removed after incubation with protein A-Sepharose, and the supernatants were assayed for 2C-mediated inhibition of 3C^pro activity as described in Materials and Methods. [³⁵S]methionine-labeled CREB was incubated with 2C (lane 1), 3C (lane 2), or both 2C and 3C (lane 3) and analyzed directly. Anti-2C depleted supernatants from reactions containing 3 μg of anti-2C IgG (lane 4), 4.5 μg of anti-2C IgG (lane 5), and 4.5 μg of preimmune IgG (lane 7) were assayed for inhibition of 3C^pro activity. (B) Analysis of physical interaction between 3C^pro and 2C. A fixed amount (200,000 cpm) of in vitro-translated [³⁵S]methionine-labeled 2C was incubated with various amounts of unlabeled, purified His-tagged 3C^pro as described in Materials and Methods. An antibody directed against the His epitope was used to immunoprecipitate the 2C-3C complex. Lane 1, [³⁵S]methionine-labeled 2C protein after immunoprecipitation with anti-2C IgG; lanes 2 to 5, [³⁵S]methionine-labeled 2C was preincubated in the presence of 5 μg of BSA (lane 2) or 1.25 (lane 3), 2.5 (lane 4), or 5 (lane 5) μg of unlabeled His-3C^pro, and the 2C-3C complex was immunoprecipitated with anti-His. [³⁵S]methionine-labeled CAT was preincubated with 5 μg of unlabeled His-3C^pro and immunoprecipitated with anti-His (lane 6) or anti-CAT (lane 7) antibodies. The arrows indicate positions of full-length 2C and CAT, and the numbers to the left correspond to the position and approximate molecular masses (in kilodaltons) of marker proteins.

FIG. 6.

Expression and purification of recombinant wt 2C and deletion mutants. (A) Diagram of wt and various 2C deletion mutants; (B) V5-tagged wt 2C or various 2C mutants were expressed in E. coli and purified by affinity chromatography. The figure shows a Western blot of wt 2C (lane 2) and deletion mutants NΔ35 (lane 3), NΔ70 (lane 4), NΔ105 (lane 5), Δ101-270 (lane 6), CΔ35 (lane 7), CΔ70 (lane 8), or CΔ105 (lane 9) with anti-V5 tag antibody. In lane 1, proteins purified thorough an affinity column from bacterial extracts without the 2C plasmid were analyzed.

FIG. 7.

Effect of 2C mutations on its 3C inhibitory activity. First, 2 μg of 3C^pro was preincubated with various amounts of purified wt or mutant 2C polypeptides as indicated. The preincubated proteins were then added to reactions containing [³⁵S]methionine-labeled CREB. Reactions were terminated by the addition of 2× SDS sample buffer (1:1 [vol/vol]), and proteins were resolved on 14% SDS-acrylamide gels. The arrow and arrowheads on the left show the migration of full-length CREB and the proteolyzed CREB products (28 and 20 kDa), respectively. All lanes contain [³⁵S]methionine-labeled CREB, and all lanes except lanes 1 and 2 contain 3C^pro. Lanes 1 and 2, CREB alone and CREB incubated with buffer, respectively; lane 3, same as lane 2 except 3C^pro was added instead of buffer; lanes 4 and 5, same as lane 3 except that 0.25 and 1.0 μg of wt 2C was included; lanes 6, 9, 12, 15, 18, 21, and 24, same as lane 3 except 0.25 μg of various mutant 2C proteins (as indicated at the top) was added; lanes 7, 10, 13, 16, 19, 22, and 25, same as lane 3 except 0.5 μg of various 2C mutants were added; lanes 8, 11, 14, 17, 20, 23, and 26, same as lane 3 except 1.0 μg of various 2C mutants was added.

FIG. 8.

Reversal of 3C^pro-mediated transcription inhibition by 2C in vitro in HeLa cell extract. In vitro RNA polymerase II-catalyzed transcription was performed in HeLa nuclear extracts as described in Materials and Methods. RNA polymerase II transcription was carried out in the presence of 100 μg of HeLa nuclear extract plus 2C buffer (lane 1), 2.5 μg of 2C (lane 2), 1 μg of 3C^pro (lane 3), 2.5 μg of wt 2C and 1 μg of 3C^pro (lane 4), 2.5 μg of mutant (Δ101-270) 2C and 1 μg of 3C^pro (lane 5), or 2.5 μg of mutant (Δ101-270) 2C (lane 6). The arrow indicates the appropriate transcript.

FIG. 9.

Inducible expression of 2C in HeLa cells. A HeLa cell line that expresses 2C when induced by doxycycline was prepared as described in Materials and Methods. (A) Rate of cell growth for the 2C cell line (E2 [▪]) versus that of the control line with vector alone (9E [♦]) for up to 48 h. (B) Genomic DNA was used to detect 2C sequence from various antibiotic-resistant colonies transformed with a vector containing the 2C sequence by PCR with appropriate primers. The arrowhead indicates the position of the appropriate PCR product. (C) Genomic DNA from various colonies transformed with the vector alone was analyzed by PCR. (D) Total RNA isolated from E2 (lane 2) and 9E (lane 3) cells after 10 h of induction with doxycycline and from HeLa cells (lane 1) was analyzed to detect 2C RNA with appropriate primers. The arrowhead indicates the position of the RT-PCR product. (E) Fifty micrograms of total protein from the E2 cell line (lanes 2 and 3, duplicate) or 9E (lane 1) were used for Western blot with a polyclonal antibody to 2C.

FIG. 10.

Proteolytic processing of viral precursor polypeptides in stable HeLa cell line that express 2C in an inducible fashion. (A) Pulse-chase analysis of protein processing in poliovirus-infected HeLa cells expressing the 2C polypeptide. HeLa cells lines 9E (with vector alone, lanes 1 to 5) and E2 (2C expressing cells, lanes 6 to 10) were infected at a multiplicity of infection of 10 with type 1 poliovirus as described in Materials and Methods. Cells were labeled with [³⁵S]methionine at 3 h postinfection. The numbers above the lanes indicate times in minutes after the chase with unlabeled methionine. The lane marked PV shows the migration of poliovirus proteins from HeLa cells infected with poliovirus for 5 h. The migration of molecular markers is indicated on the right (in kilodaltons). The arrowheads pointing right indicate precursor and mature viral proteins that are either present exclusively or in higher amounts in the E2 cell line compared to the control 9E cell line. The arrowhead pointing left indicates the position of migration of 3D. (B) A lower exposure of lanes 1 to 3 and 6 to 8 from Fig. 10A is shown. (C) Quantification of viral precursor proteins from E2 and 9E cells. The viral precursor polypeptides P0, P1, P3, 3CD, 2BC, 60 kDa, and 50 kDa were quantified by densitometric scanning as described in Materials and Methods. The numerical values were normalized with respect to total cellular proteins from virus-infected E2 and 9E cells. The ratio of the numerical values for E2 to 9E cells for each polypeptide at 0, 20, and 40 min of chase are shown.