Shutoff of RNA polymerase II transcription by poliovirus involves 3C protease-mediated cleavage of the TATA-binding protein at an alternative site: incomplete shutoff of transcription interferes with efficient viral replication

Abstract

The TATA-binding protein (TBP) plays a crucial role in cellular transcription catalyzed by all three DNA-dependent RNA polymerases. Previous studies have shown that TBP is targeted by the poliovirus (PV)-encoded protease 3C(pro) to bring about shutoff of cellular RNA polymerase II-mediated transcription in PV-infected cells. The processing of the majority of viral precursor proteins by 3C(pro) involves cleavages at glutamine-glycine (Q-G) sites. We present evidence that suggests that the transcriptional inactivation of TBP by 3C(pro) involves cleavage at the glutamine 104-serine 105 (Q104-S105) site of TBP and not at the Q18-G19 site as previously thought. The TBP Q104-S105 cleavage by 3C(pro) is greatly influenced by the presence of an aliphatic amino acid at the P4 position, a hallmark of 3C(pro)-mediated proteolysis. To examine the importance of host cell transcription shutoff in the PV life cycle, stable HeLa cell lines were created that express recombinant TBP resistant to cleavage by the viral proteases, called GG rTBP. Transcription shutoff was significantly impaired and delayed in GG rTBP cells upon infection with poliovirus compared with the cells that express wild-type recombinant TBP (wt rTBP). Infection of GG rTBP cells with poliovirus resulted in small plaques, significantly reduced viral RNA synthesis, and lower viral yields compared to the wt rTBP cell line. These results suggest that a defect in transcription shutoff can lead to inefficient replication of poliovirus in cultured cells.

FIG. 1.

Shutoff of RNA polymerase II-mediated transcription does not correlate with 3C^pro-induced cleavage at the 18th glutamine-glycine site in TBP. (A) The domain structure of TBP consisting of the core, N-terminal with the glutamine stretch (Q), and acidic domain (IV) is shown. (B) Transcription shutoff in vitro does not correlate with TBP cleavage at the 18th Q-G site. Kinetics of transcription shutoff was examined in HeLa transcription extracts (80 μg) using a template DNA containing the TATA element (37) in the presence of 1 μg of purified 3C^pro, and the reactions were stopped at indicated times. The transcribed RNA was analyzed by primer extension using a ³²P-labeled Sp6 promoter primer (37). The arrowhead indicates the size (79 nucleotides) of the correctly initiated transcription product. (C) The same reactions as shown in panel B were examined by Western blotting using a polyclonal antibody to human TBP. The positions of migration of wt TBP and ΔN18 TBP are indicated. (D) The primary sequence of TBP is shown. The Q-G and Y-G sites in TBP are underlined. The arrow and the arrowhead indicate the known 2A^pro and 3C^pro cleavage sites within TBP, respectively.

FIG. 2.

TBP cleavage by 3C^pro generates other proteolytic products in addition to ΔN18 TBP. (A) In vitro 3C^pro induced cleavage of TBP at sites other than the 18th Q-G site. One microliter of in vitro translated [³⁵S]methionine-labeled TBP was incubated with buffer (lane 1) or 1 μg of purified 3C^pro (lane 2) for 4 h, and products were analyzed by SDS-PAGE. The positions of migration of TBP, Δ18 TBP, and two additional bands at 27 and 24 kDa are indicated. (B) Incubation of TBP with PV-infected extract generates the 27- and 24-kDa products. [³⁵S]methionine-labeled TBP (∼150,000 cpm) was incubated with 40 μg of 4-h mock- or PV-infected (lanes 1 to 3 and lanes 4 to 6, respectively) extracts for 1, 5, and 16 h as indicated, and the reactions were analyzed by SDS-PAGE. (C and D) Detection of the 27-kDa polypeptide in PV-infected cells. In a Western blot using anti-TBP, the cell extracts from 4-h mock- or PV-infected (lanes 3 and 4, respectively) cells were compared with HeLa cell extract treated with buffer (lane 1) or purified 3C^pro (lane 2) in vitro. An overexposure of the blot shown in panel C is shown in panel D. The migrations of TBP, ΔN18TBP, and the 27-kDa polypeptide are indicated. (E) Generation of 27-kDa polypeptide by direct cleavage of TBP. Approximately 60 ng of purified TBP was incubated with buffer alone (lane 1) or with 250 ng of purified 3C^pro (lane 2) for 4 h, and the TBP-related products were visualized by Western blot analysis using a polyclonal anti-TBP antibody.

FIG. 3.

Effects of various TBP terminal and internal deletions on 3C^pro-induced cleavage of TBP. (A) Schematic representation of amino- and carboxy-terminal and internal deletion mutants of TBP. The pluses and minuses in parentheses indicate qualitatively whether these polypeptides are cleaved (+) or not cleaved (−) to generate the 27- and 24-kDa products by 3C^pro. (B) Various N-terminal deletion mutants of TBP were translated in vitro in the presence of [³⁵S]methionine and subjected to digestion with buffer only (lanes 1, 3, and 5) or 1 μg of purified 3C^pro (lanes 2, 4, and 6). (C) The wild-type (lanes 5 and 6) and internal deletion mutants of TBP, Δ101-129 (lanes 1 and 2), and Δ132-167 (lanes 3 and 4) were digested with buffer alone (lanes 1, 3, and 5) or purified 3C^pro (lanes 2, 4, and 6). The arrows indicate the proteolyzed products. (D) The in vitro translated, [³⁵S]methionine-labeled C-terminal deletion mutants ΔC210 (lanes 1 and 2), ΔC139 (lanes 3 and 4), ΔC105 (lanes 5 and 6), and ΔC68 (lanes 7 and 8) of TBP were digested with buffer alone (odd-numbered lanes) or purified 3C^pro (even-numbered lanes).

FIG. 4.

Effects of double and single amino acid substitution mutations of potential 3C^pro cleavage sites between amino acid residues 101 and 126 of TBP. (A) The primary sequence of amino acid residues 101 through 129 of TBP is shown. The potential 3C^pro alternative cleavage sites are underlined. (B) In vitro translated, [³⁵S]methionine-labeled wild type or various point mutants of TBP were digested with buffer alone (odd-numbered lanes) or 1 μg of 3C^pro (even-numbered lanes) prior to SDS-PAGE. (C) wt TBP or various double mutants were digested with buffer (odd-numbered lanes) or 3C^pro (even-numbered lanes). The proteolysis products are indicated by arrowheads to the right.

FIG. 5.

Comparison of complete 3C^pro digestion products of TBP, ΔN100 TBP, and ΔC239 TBP. (A) In vitro translated [³⁵S]methionine-labeled wt TBP (lanes 1 and 2), ΔN100 (lanes 3 and 4), and ΔC239 (lanes 5 and 6) were digested with buffer (lanes 1, 3, and 5) or 3C^pro (lanes 2, 4, and 6) to completion. The radiolabeled products were analyzed by SDS-PAGE. (B) A schematic presentation of the predicted 3C^pro cleavage products generated from TBP and truncated TBP molecules. Arrowheads indicate Q18-G19 and Q104-S105 cleavage sites. The numbers within parentheses indicate the molecular weights of the products, while the numbers marked with asterisks indicate actual molecular weights of these products compared to how they migrate during gel electrophoresis.

FIG. 6.

Amino acid residues preceding the Q104-S105 bond important for 3C^pro-induced cleavage of TBP. (A) The primary sequence of amino residues 95 to 111 of TBP is shown. The Q104-S105 bond is underlined. The upward arrowhead indicates the P4 residue. (B) Single amino acid substitution between residues 96 to 99 does not influence TBP cleavage at the Q104-S105 site. TBP mutants with the single amino acid substitutions A96L, V97L, A98L, and A99L were digested with buffer (lanes 1, 3, 5, and 7) or 3C^pro (lanes 2, 4, 6, and 8). (C) Substitution of A101 (P4 position) with G, but not with V, significantly inhibits 3C^pro-mediated cleavage that generates the 27- and 24-kDa products. Single amino acid substitution mutants A101G, A101V, V102A, Q103A, and wt TBP were digested with buffer (lanes 1, 3, 5, 7, and 9) or 3C^pro (lanes 2, 4, 6, 8, and 10) prior to analysis by SDS-PAGE. (D) Double amino acid substitution at A100-A101 completely blocks the 3C^pro-mediated cleavage that generates 27- and 24-kDa products. TBP double amino acid substitution mutants A100G-A101G, A100L-A101L, A101L-V102L, V102L-Q103L, and wt TBP were digested with buffer alone (lanes 1,3, 5, 7, 9, and 11) or 3C^pro (lanes 2, 4, 6, 8, 10, and 12).

FIG. 7.

ΔN100 TBP is highly defective in restoring RNA Pol II-mediated transcription in PV-infected extracts. (A) TATA box-dependent in vitro transcription from a template DNA containing Ad MLP was performed in poliovirus-infected (4 h postinfection) cell extracts in the absence and presence of purified ΔN100 TBP or wt TBP. The primer-extended product was quantified by densitometric scanning, and the percentage of relative transcription with respect to 4-h mock-infected control was plotted against the concentration of TBP in the reaction. (B) Western blots of various concentrations of wt and ΔN100 TBP used in the transcription assay are shown.

FIG. 8.

Purification and transcriptional activity of GG rTBP and LL rTBP. (A) Recombinant His-tagged wt TBP, GG rTBP, and LL rTBP were expressed in bacteria and purified by Ni⁺⁺ affinity chromatography. Silver-stained gel of the purified proteins is shown. (B) The transcriptional activity at 3 and 5 μM of each of the wt (lanes 2 and 3), GG (lanes 4 and 5), and LL (lanes 6 and 7) rTBP was measured using a template containing the Ad MLP (lanes 2 to 7) in PV-infected cell extract as previously described (37). Bovine serum albumin (5 μM) was used as a negative control (lane 8). Lane 1 shows transcriptional activity of the infected extract in the absence of any added protein. The numbers at the bottom indicate the increase in stimulation (n-fold) over control as determined by densitometric quantification of the transcript.

FIG. 9.

Cell lines that constitutively express wt and mutant TBP resistant to cleavage by 3C^pro. (A) Detection of mutant TBP in various cell lines. Five clonally selected HeLa cell lines that constitutively express His-TBP were isolated as described in Materials and Methods. Each line was examined for expression of recombinant TBP by Western analysis using an antibody to the His tag. Two of these lines (GG1 and GG2) contained His-TBP in which the two alanines at positions 100 and 101 were replaced by glycine. The two alanine residues in three other lines (LL1, LL2, and LL3) were replaced by L. In both mutants the 18Q-19G and the 34Y-35G bonds were mutated to 18A-19A and 34A-35A, respectively. The wild-type line expresses wt His-TBP. In lane 1, HeLa cell extracts containing the endogenous TBP, not detected by anti-His antibody, was used. (B) GG1 rTBP is resistant to 3C^pro-mediated cleavage. Cell extracts from wild-type and GG1 rTBP (GG1) lines were digested with buffer (lanes 1 and 3) or purified 3C^pro (lanes 2 and 4) prior to Western analysis using anti-His antibody. (C) Five cellular mRNA levels as indicated were examined by RT-PCR from wt rTBP (odd-numbered lanes) and GG1 rTBP (even-numbered lanes) cells.

FIG. 10.

Constitutive expression in HeLa cells of recombinant mutant TBP resistant to 3C^pro cleavage yields virus with a small-plaque phenotype. (A to D) HeLa cells expressing wild-type His-rTBP (A and B), GG1 His-rTBP (C), or GG2 His-rTBP (D) were infected with poliovirus type 1 (Mahoney strain) in duplicates and assayed for plaque formation as described in Materials and Methods. (E to H) Transcription shutoff is delayed and viral RNA synthesis is significantly inhibited in GG1 His-rTBP cells. HeLa cell lines expressing either wt rTBP or GG1 rTBP were infected with poliovirus type 1 (Mahoney strain) as described in Materials and Methods. Mock- and PV-infected cells were labeled with 5μCi of [³H]uridine per 0.3 × 10⁶ cells in the absence and presence of 5 μg/ml actinomycin D, and cellular (E) and viral RNA synthesis (G) were measured as described in the text. Transcription shutoff (F) was determined by using the values of acid-insoluble [³H]uridine incorporation according to the following formula: Inf − [(Inf + actD) − (Uninf + actD)]/[(Uninf) − (Uninf + actD)]. The experiments shown in panels E to G were performed in duplicate, and the average values are plotted. (H) Northern analysis of viral RNA was performed from total RNA isolated from wt and GG1 rTBP cells at 0 (lanes 1 and 4), 2 (lanes 2 and 5), and 4 (lanes 3 and 6) h postinfection. The lower panel shows GAPDH RNA used as a loading control.