Enterovirus type 71 2A protease functions as a transcriptional activator in yeast
© Yang et al; licensee BioMed Central Ltd. 2010
Received: 7 April 2010
Accepted: 4 August 2010
Published: 4 August 2010
Enterovirus type 71 (EV71) 2A protease exhibited strong transcriptional activity in yeast cells. The transcriptional activity of 2A protease was independent of its protease activity. EV71 2A protease retained its transcriptional activity after truncation of 40 amino acids at the N-terminus but lost this activity after truncation of 60 amino acids at the N-terminus or deletion of 20 amino acids at the C-terminus. Thus, the acidic domain at the C-terminus of this protein is essential for its transcriptional activity. Indeed, deletion of amino acids from 146 to 149 (EAME) in this acidic domain lost the transcriptional activity of EV71 2A protein though still retained its protease activity. EV71 2A protease was detected both in the cytoplasm and nucleus using confocal microscopy analysis. Coxsackie virus B3 2A protease also exhibited transcriptional activity in yeast cells. As expected, an acidic domain in the C-terminus of Coxsackie virus B3 2A protease was also identified. Truncation of this acidic domain resulted in the loss of transcriptional activity. Interestingly, this acidic region of poliovirus 2A protease is critical for viral RNA replication. The transcriptional activity of the EV71 or Coxsackie virus B3 2A protease should play a role in viral replication and/or pathogenesis.
Enterovirus type 71 (EV71) is the causative agent of several human diseases, including hand-foot-and-mouth disease, encephalitis, and meningitis. EV71 is a single-stranded, positive-sense RNA virus, which belongs to the Picornaviridae family . Genomic RNA of picornaviruses (e.g. polioviruses) encodes a polyprotein precursor, which is processed by three proteases (the maturation protease, 2A protease, and the 3C protease) into at least 11 different proteins, which are arranged in the order of NH2-VP4-VP2-VP3-VP1-2A-2B-2C-3A-VPg-3C-3D-COOH . The 2A protease of poliovirus, a representative member of the Picornaviridae, is a cysteine protease with multiple functions . Similar to poliovirus 2A protease, expression of EV71 2A protease led to cleavage of the eukaryotic initiation factor 4GI, a key factor for host protein synthesis [3, 4]. Moreover, transient expression of EV71 2A protease alone also resulted in the induction of apoptotic change [5, 6]. However, the function of EV71 2A protease is not well characterized. The biologic function of EV71 2A protease was investigated by fusing it with the DNA-binding domain of Gal4 and examining its possible interaction with cellular factors .
Materials and Methods
PCR primers used in this study
(5'-CCGCTCGAG TTA CTGCTCCATGGCTTC-3')
(5'-CCATCGAT ATG ATGGGTACGTTC-3')
(5'-GGAATTC ATG GGGAAATTTGGACAGCAG-3')
(5'-GCTCTAGA CTA CTGCTCCATGGCTTCATCATC-3')
(5'-GGAATTC ATG GGACAACAATCAGGGGC-3')
To clone the DNA fragment encoding the full-length Coxsackie virus B3 2A protease for yeast two-hybrid screening, mRNA extracted from a patient infected with Coxsackie virus B3 was converted into cDNA and oligonucleotide primers (CoxB2AY-S and CoxB2AY-AS) were used to perform PCR (the sequence is the same as nucleotides from 3304 to 3744 of GI:323419). PCR was performed using primer pairs (CoxB2AY-61 S and CoxB2AY-AS) to clone the DNA fragments encoding Coxsackie virus 2A protease with the N-terminal truncation of 60 amino acids, while another PCR was performed with primer pairs (CoxB2AY-S and CoxB2AY-127AS) to clone the DNA fragments encoding Coxsackie virus 2A protease with the C-terminal deletion of 20 amino acids. Again, after the PCR, the DNA fragments were treated with T4 polynucleotide kinase, digested by the restriction enzyme EcoRI, and cloned into the pBDGal4 Cam (Stratagene, USA) expression vector which had been linearized with EcoRI and SmaI.
To clone the DNA fragment encoding the C-terminus of EV71 VP1 and the full-length 2A protease (nucleotides from 3124 to 3781 of strain pinf7-54A) for transient expression in mammalian cells, PCR was performed using oligonucleotide primers (VP1/2A-S and 2AY-AS2). After the PCR, the DNA fragment was digested by restriction enzymes (ClaI/XbaI), together with the EMCV IRES sequence (digested with EcoRI/ClaI), and cloned into the expression vector pcDNA3 (Invitrogen, USA) which had been linearized with EcoRI/XbaI. To mutate amino acid 110 of EV71 2A protease from Cys to Ala, primers (VP1/2A-S and C110A-AS) were used to amplify the 5'-end of the gene fragment while primers (C110A-S and 2AY-AS2) were used to amplify the 3'-end fragment. These two DNA fragments were linked together by PCR using primers (VP1/2A-S and 2AY-AS2). After the PCR, the DNA fragment was digested by restriction enzymes (ClaI/XbaI), together with the EMCV IRES sequence (digested with EcoRI/ClaI), and cloned into the expression vector pcDNA3 (Invitrogen, USA) which had been linearized with EcoRI/XbaI.
To clone the DNA fragment encoding the C-terminus of EV71 VP1 and full-length 2A protease with the V5 tag in the C-terminus for confocal microscopy analysis in mammalian cells, PCR was performed using oligonucleotide primers (VP1/2A-S and 2AY-AS3). After the PCR, the DNA fragment was digested by restriction enzymes (ClaI/XbaI), together with the EMCV IRES DNA sequence (digested with EcoRI/ClaI), and cloned into the expression vector pcDNA3.1-V5-His A (Invitrogen, USA) which had been linearized with EcoRI/XbaI. To clone the EV71 2A protease with mutation of amino acid 110 from Cys to Ala for confocal microscopy analysis, the DNA template containing this mutation and primers (2A-S10 and 2A-AS3) was used to amplify the DNA fragment of full-length EV71 2A protein with mutation of amino acid 110 from Cys to Ala. After the PCR, the DNA fragment was digested by the restriction enzymes (EcoRI/XbaI), and cloned into the expression vector pcDNA3.1-V5-His A (Invitrogen, USA) which had been linearized with EcoRI/XbaI. To clone the EV71 2A protease without potential NES (amino acid 31 to 42) for confocal microscopy analysis, the DNA fragment containing the mutation of amino acid 110 from Cys to Ala was used as the PCR template. Primers (2A-S10 and L30/43-AS) were used to amplify the 5'-end of the gene fragment while primers (L30/43-S and 2A-AS3) were used to amplify the 3'-end fragment. These two DNA fragments were linked together by PCR using primers (2A-S10 and 2A-AS3). After the PCR, the DNA fragment was digested by restriction enzymes (EcoRI/XbaI), and cloned into the expression vector pcDNA3.1-V5-His A (Invitrogen, USA) which had been linearized with EcoRI/XbaI. The same approach was used to clone the DNA fragment encoding the C-terminus of EV71 VP1 and 2A protease deleting the amino acids 146-149 with the V5 tag in the C-terminus using primers (VP1/2A-S and 2A-AS301) to perform PCR.
All of the expression plasmids were verified by sequencing.
Yeast two-hybrid screening
The yeast two-hybrid system used for screening was purchased from Clontech Laboratories (USA). The experimental procedures were conducted according to the manufacturer's instructions.
Protein expression and Western blot analysis
HeLa cells were maintained in RPMI (Chemicom, USA) medium containing 10% fetal bovine serum, 1% glutamine (200 mM, Gibco, USA), and 100 ug/ml penicillin/streptomycin (Gibco BRL, USA). Cultured cells were maintained at 37°C with 5% CO2. Cells were seeded at a density of approximately 4 × 105 cells per 60-mm culture dish. After overnight incubation, cells were transfected with plasmids (1-4 ug) using the ExGen 500 in vitro transfection reagent (Fermentas, USA) or Arrest-In™transfection reagent (Open Biosystems, USA). At 48 hours after transfection, recombinant proteins expressed in cells were analyzed by Western blot.
Our previous procedures were followed for Western blot analysis [7, 10]. Rabbit polyclonal antibodies against ERK-2 and eIF4G were purchased from Santa Cruz Biotechnology (USA). Monoclonal antibodies against PARP were purchased from SEROTEC (UK). Monoclonal antibodies against V5 tag were purchased from Invitrogen (USA). Rabbit antibodies against EV71 2A protease were generated in the lab.
Confocal microscopy analysis
HeLa cells were seeded at a density of about 2.5 × 105 cells per 35 mm culture dish. After overnight incubation, cells were transfected with plasmids (0.5 - 2 ug) using the ExGen 500 in vitro transfection reagent (Fermentas, USA) or Arrest-In™transfection reagent (Open Biosystems, USA). At 48 hours after transfection, recombinant proteins expressed in cells were analyzed by confocal microscopy.
Cells with recombinant proteins were fixed with 1% methanol/acetone at 0°C for 10 minutes, washed with incubation buffer (0.05% NaN3, 0.02% saponin, 1% skim milk in PBS) twice for 2 minutes each, and then incubated with the anti-V5 antibody (1:200 dilution) at 37°C for 30 minutes. Cells were washed with PBS at room temperature for five minutes three times, and then incubated with Cy3-conjugated goat anti-mouse IgG antibody (1:20 dilution) at 37°C for 30 minutes. Cells were washed three more times with PBS. DAPI (Merck, Germany) was used to stain the nucleus.
Real-time reverse transcriptase-polymerase chain reaction (RT-PCR)
HeLa cells were transfected with plasmids of vector alone or pcDNA3.1-IRES-2A using Arrest-In™transfection reagent (Open Biosystems, USA). At 24 hours after transfection, G418 was used to select the cells with transfected plasmid. After 72 hours, cellular mRNAs were extracted and our previous procedures were followed for real-time RT-PCR .
EV71 2A protease exhibited strong transcriptional activity in yeast cells
Transcriptional activity of EV71 2A protease is independent of its protease activity
Sub-cellular localization of EV71 2A protease
Deletion of amino acids from 146 to 149 of EV71 2A protease lost its transcriptional activity but retained its protease activity
EV71 2A protease did not transactivate cellular genes reported to enhance the replication of poliovirus or EV 71
Some cellular genes were reported previously to enhance the replication of poliovirus or EV71: poly(rC) binding proteins [15–17], cellular COPII proteins , the polypyrimidine tract binding proteins , Reticulon 3 , and GBF1 . Real-time RT-PCR was performed to determine whether EV71 2A protease could transactivate PCBP2, PTBP1, RTN3, GBF1, CD55, or SAM68 gene. However, EV71 2A protease repressed rather than transactivated all of these cellular genes (data not shown).
Coxsackie virus B3 2A protease exhibited transcriptional activity in yeast cells
EV71 2A protease is expected to enter the nucleus by passive diffusion since it is a small protein with no potential NLS. This protein would not be actively exported from the nucleus since no functional NES was detected (Figure 4). These findings explain why only small portion of EV71 2A protease localized in the nucleus and the majority of this protein was retained in the cytoplasm (Figure 4). Interestingly, 2A proteins of poliovirus and EMCV were reported to localize in the nucleus [22, 23].
As a transcriptional activator, EV71 2A protease did not contain a glutamine-rich domain, a leucine zipper domain, or a proline-rich domain as are found in some other eukaryotic transcriptional activators such as CTF/NF-1 or the amino terminal deletion mutants of HCV NS5A protein [24–27]. The PXXXP motif necessary for full transactivation of HIV Tat protein was also not found in EV71 2A protease (Figure 2A) . However, one acidic domain (rich in Glu (E) or Asp (D), Figure 2B), functioning universally in eukaryotic transcriptional activators from yeast to human [29, 30], was found in the C-terminus of EV71 2A protease (6 amino acids within the last 15 amino acids are acidic, Figure 2A). Moreover, 9aa TAD possessing an autonomous transactivation activity in yeast and mammalian cells was also found at the N-terminus of EV71 2A protease (from aa 27 to 35) (Figure 2A) . Deletion analysis revealed the acidic domain in the C-terminus but not 9 aa TAD in the N-terminus of EV71 2A protease is essential for the transcriptional activation activity of this protein (Figure 1).
The C-terminal 15 amino acid residues of picornaviral 2A protease sequences
Enterovirus type 71
Coxsackie virus B3
Coxsackie virus B5
Coxsackie virus A17
Human rhinovirus 24
Human rhinovirus 52
Human rhinovirus 94
Human rhinovirus C
In summary, 2Apro of enterovirus type 71 and Coxsackie virus B3 possesses transcriptional activity. The transcriptional activity of 2A protease was independent of its protease activity. Furthermore, the acidic domain at the C-terminus of 2Apro is essential for its transcriptional activity. Enteroviral 2A proteases may transactivate some cellular genes to benefit virus replication.
We would like to thank Ms. Chingyn Chang and Dr. Shin-Ru Shih for providing viral DNA fragments of EV71 and Coxsackie virus B3. This work was supported by grants from the National Science Council of Taiwan (NSC 97-3112-B-320-001) and from the Tzu Chi University (TCIRP96004-05) to Dr. Shih-Yen Lo.
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