Enterovirus 71 3C protease cleaves a novel target CstF-64 and inhibits cellular polyadenylation

Abstract

Identification of novel cellular proteins as substrates to viral proteases would provide a new insight into the mechanism of cell-virus interplay. Eight nuclear proteins as potential targets for enterovirus 71 (EV71) 3C protease (3C(pro)) cleavages were identified by 2D electrophoresis and MALDI-TOF analysis. Of these proteins, CstF-64, which is a critical factor for 3' pre-mRNA processing in a cell nucleus, was selected for further study. A time-course study to monitor the expression levels of CstF-64 in EV71-infected cells also revealed that the reduction of CstF-64 during virus infection was correlated with the production of viral 3C(pro). CstF-64 was cleaved in vitro by 3C(pro) but neither by mutant 3C(pro) (in which the catalytic site was inactivated) nor by another EV71 protease 2A(pro). Serial mutagenesis was performed in CstF-64, revealing that the 3C(pro) cleavage sites are located at position 251 in the N-terminal P/G-rich domain and at multiple positions close to the C-terminus of CstF-64 (around position 500). An accumulation of unprocessed pre-mRNA and the depression of mature mRNA were observed in EV71-infected cells. An in vitro assay revealed the inhibition of the 3'-end pre-mRNA processing and polyadenylation in 3C(pro)-treated nuclear extract, and this impairment was rescued by adding purified recombinant CstF-64 protein. In summing up the above results, we suggest that 3C(pro) cleavage inactivates CstF-64 and impairs the host cell polyadenylation in vitro, as well as in virus-infected cells. This finding is, to our knowledge, the first to demonstrate that a picornavirus protein affects the polyadenylation of host mRNA.

Figure 1. Identification of potential EV71 3C^pro substrates by 2D gels.

(A) A [³⁵S]-labeled peptide which contains part of EV71 viral polyprotein was treated with wild-type 3C^pro(WT) or mutant 3C^pro (C147S). The uncleaved peptide precursor and 3C^pro cleavage product was indicated. (B) Full scale of 2D gels for one of the six in vitro cleavage experiments; left panel shows results obtained using the mutant 3C^pro-treated nuclear extract (3C_mut); right panel shows the results obtained using wild-type 3C^pro-treated nuclear extract (3C_WT). Arrows indicate location of CstF-64. (C) Six in vitro cleavage experiments on 2D gels. Arrows indicate location of CstF-64 protein on mutant 3C^pro-treated nuclear extracts gels; circles indicate the corresponding regions on wild-type 3C^pro-treated nuclear extract gels.

Figure 2. CstF-64 in 3C^pro-treated nuclear extract.

(A) CstF-64 proteins in nuclear extracts from SF268, RD and HeLa cells were detected using anti-CstF-64 antibodies. Untreated nuclear extracts (-) were loaded as controls. The amount of CstF64 in wild-type 3C^pro treated nuclear extracts (WT) and in mutant 3C^pro-treated nuclear extracts (C147S) were showen. A cleavage product of 55 kDa was indicated (*). PCNA (proliferatory cell nuclear antigen) detection was employed as the loading control. (B) CstF-64 protein in RD nuclear extract treated with various quantities of 3C^pro - 0.5 µg , 1 µg, 2 µg , 4 µg and 5 µg (lanes 3 to 7) and in the mutant 3C-treated nuclear extract (C147S) or untreated nuclear extract (-) were shown. (C) eIF4-GI in wild-type (WT) and mutant 2A^pro (C110S) treated RD cell extracts were detected using specific antibody (lanes 1–3). CstF-64 in 2A^pro or mutant 2A^pro-treated nuclear extract was also detected (lanes 4–6).

Figure 3. CstF-64 in EV71-infected cells.

(A) After RD cells have been infected with EV71 (m.o.i. = 40), 3C^pro and CstF-64 protein in total cellular proteins of infected RD cells (+) or mock infected cells (-) were detected at various hours post-infection (h.p.i.) using specific antibodies. A potential cleavage product of CstF-64 was also indicated (*). The detection of β-actin was used as a loading control (B) RD cells with transient FLAG-CstF-64 overexpression were also infected with EV71 at an m.o.i. of 40 and the total cellular proteins from mock infected (-) and infected cells (+) were harvested at 6 and 8 h.p.i.. FLAG-CstF-64 was detected using FLAG-specific antibody. The other cleaved FLAG-peptides were indicated as CP1 and CP2 (C) Cytoplasmic (C) and nuclear (N) fractions from EV71-infected RD cells at 8 h.p.i. were extracted and CstF-64 or 3C^pro in each of the fractions were detected using specific antibodies. Detections of β-actin and histon deacetylase (HDAC) were used as cytoplasmic and nucleus protein controls. (D) The locations of CstF-64 in uninfected (Mock) or EV71-infected cells at 2, 4, 6, 8 and 10 h.p.i. were detected using specific antibody. The detection of viral 2B protein was applied as an infection-positive marker. The nuclei of cells were stained using Hoechst dye.

Figure 4. 3C^pro cleaves recombinant CstF-64 protein in vitro.

(A) To locate the 3C^pro cleavage sites on CstF-64, [³⁵S]-labeled CstF-64 and numerous partial CstF-64 peptides which contain 1^st–220^th, 221^st–409^th, 410^th–577^th, 110^th–409^th, 221^st–469^th amino acids of CstF-64 were generated based on the functional domains of CstF-64, including RNA recognition motif (RRM), hinge domain, Pro/Gly rich domains (P/G), MEARA sequence, and C-terminal domain (C-ter) as displayed in (B). These untreated [³⁵S]-labeled peptides (-) and those were treated with wild-type 3C^pro (WT) and mutant 3C^pro (C147S) were analyzed in SDS-PAGE. The mass of cleavage products and suspected cleavage sites (arrows) are summarized in (B), indicating that the predicted 3C^pro cleavage sites of full-length CstF-64 are around the amino acid positions 250 and 500 of CstF-64. (C) The untreated [³⁵S]-labeled CstF-64 proteins (-) and those incubated with catalytic mutant 3C protein (C147S) or wild-type 3C^pro (WT) with various incubation times (15, 30, 60, 90, 120, 150 and 180 minutes) were analyzed in SDS-PAGE. The cleavage products of 55 kDa (p55), 35 kDa (p35), 30 kDa (p30) and 25 kDa (p25) were also indicated.

Figure 5. Multiple 3C^pro cleavage sites around the amino acid positions 250 and 500 of CstF-64 in vitro.

(A) Gln/Gly (Q/G) junctions located around amino acid positions 250 and 500 of CstF-64, including Gln251, 483, 496, 505, 510 and 515. (B) Predicted size of 3C^pro cleavage products on CstF-64 with mutations at the cleavage site close to amino acid position 250 of CstF-64 (250) or at both the cleavage sites at positions 250 and 500 (250+500). (C) Wild-type 3C^pro (WT) or mutant 3C^pro (C147S)-treated [³⁵S]-labeled CstF-64(Q251A) with a single Gln483, 496, 505, 510 or 515 (Q483A, Q496A, Q505A, Q510A, Q515A) mutation (D) Wild-type 3C^pro (WT) or mutant 3C^pro (C147S)-treated [³⁵S]-labeled CstF-64(Q251A) or CstF-64(Q251A) with multiple mutations of Q483A, Q496A, Q505A, Q510A and Q515A.

Figure 6. Pre-mRNA and poly(A)-mRNA in EV71-infected cells.

(A) Primers that target the region downstream of the polyadenylation site and poly(A) tail sequence, were designed to detect pre-mRNA and poly(A) mRNA of GFP. (B) After plasmid pEGFP was transfected into the RD cells, cells were infected with EV71 and total RNA of infected cells was harvested at 6 and 8 h.p.i. for RT-PCR assay. RT-PCR results demonstrate the accumulation of pre-mRNA and the reduction of poly(A)-mRNA in EV71-infected cells at six and eight hours post-infection (h.p.i.). Following calibration to the total amount of GFP RNA, the fold changes from the amount of pre-mRNA and poly(A)-RNA in mock-infected cells (Mock) to that of EV71-infected cells (EV71) were calculated. (C) The endogenous pre-mRNA and poly(A)-mRNA of Interleukin-10 receptor beta (IL-10RB) in mock or EV71 infected cells at 8 h.p.i. were estimated based on real-time PCR. The relative amounts of pre-mRNA and poly(A)-mRNA were normalized by total IL-10RB RNA.

Figure 7. In vitro 3′-end pre-mRNA processing and polyadenylation of 3C^pro-treated HeLa nuclear extract.

(A) Cleavage and (B) polyadenylation of pre-RNA substrates by untreated nuclear extract (-) or nuclear extracts treated with 3C^pro buffer indicated as B, mutant 3C^pro (C147S) and wild-type 3C^pro (WT) were analyzed. The recruitment of recombinant CstF-64 protein after 3C^pro treatment (WT + CstF-64) was also tested. The un-treated RNA substrate with buffer only was used as a control. The cleavaged producted (clev) and polyadenylated RNA [poly(A)] from nuclear extract-treated pre-mRNA substrate (pre) were indicated.