To identify proteins that interact with the IRES of EV71, biotinylated EV71 IRES probes were used to capture the interacting proteins. As shown in Fig. 1A, compared with nonbiotinylated RNA (lane 2), the biotinylated EV71 IRES RNA enriched 6 differential bands (lane 1). Using MALDI-TOF MS analysis, 12 proteins were identified, of which PTB, poly(rC)-binding protein 2 (PCBP2), and proliferation-associated protein 2G4 (ITAF45) have been found to interact with the IRES of picornavirus (Table 1). PTB has been found to be an important protein during the life cycle of enterovirus. However, the details of PTB's interaction with the EV71 IRES remain to be determined.
Figure 1. Interaction of PTB with the EV71 IRES region. A Pull down of proteins with the EV71 IRES in T98G cell lysate. The specific bands interacting with EV71 IRES are shown by silver staining. Lane 1: biotinylated IRES RNA, Lane 2: nonbiotinylated EV71 IRES RNA. B A specific association between PTB and the EV71 IRES region was confirmed by Western blotting and a competition assay. Increasing amounts of unlabelled RNA were added to compete with the biotin-labeled EV71 IRES RNA interacting with PTB. The eluted proteins were separated by 12% SDS-PAGE. Lanes are as follows: lanes 1-4, unlabeled EV71 IRES RNA; lanes 5-8, unlabeled yeast tRNA. C Extracts of RD cells, SH-SY5Y cells, and HA cells were prepared and then incubated without RNA (lane 2), with biotinylated actin RNA (lane 3), non-biotinylated EV71 IRES RNA (lane 4), or biotinylated EV71 IRES RNA (lane 5). After pull-down assay, the bound proteins were eluted, boiled, and subjected to 12% SDS-PAGE. PTB protein was detected by western blot with a rabbit anti-PTB antibody. The inputs were cell extracts of RD, SH-SY5Y and HA (lane 1). D EV71 IRES RNA was pulled down with PTB from EV71-infected T98G cell lysate. T98G cells were infected with EV71 at an MOI of 20 for 6 h and then cell extracts were incubated with rabbit anti-PTB antibody (lanes 2 and 6), normal rabbit IgG (lanes 3 and 7), or without antibody (- Ab) (lanes 4 and 8). Following washing and dissociation, the RNA extract was prepared and subjected to RT-PCR analysis with primers specific for the ribosomal protein S16 (PRS16) RNA (lanes1-4) or for EV71 IRES region RNA (lanes 5-8). Lane 1, cell lysate without immunoprecipitation as a positive RT-PCR control; Lane 2, anti-PTB antibody incubated with 200 mg infected-cell lysate; Lane 3, negative control with rabbit IgG; Lane 4, negative control with no antibody.
Table 1. MALDI-TOF MS results of cellular proteins associated with the EV71 IRES.
To elucidate the specific interaction of PTB and the EV71 IRES region, a competition assay and Western blotting were carried out. The specific interaction was outcompeted by non-biotinylated EV71 IRES (Fig. 1B, lanes 1-4), but not by non-biotinylated yeast tRNA (Fig. 1B, lanes 5-8). To confirm this interaction, additional cell lines were assayed. RNA pull-down and Western blot results showed that PTB interacted with EV71 IRES in all of the cell lines tested, including the RD, SH-SY5Y, and HA cell lines (Fig. 1C). Non-biotinylated IRES RNA or biotinylated β-actin probe were used as negative control.
To further confirm the results in infected cells, PTB antibody was used to pull-down the EV71 IRES. EV71 IRES was detected in total RNA extract and in the assay with PTB antibody, but not with rabbit IgG or in the absence of antibody (Fig. 1D). Primers specific to ribosomal protein S16 (RPF16) were used as negative control. These results illustrated that PTB interacts with EV71 IRES region.
To identify the binding region of EV71 IRES with PTB, the secondary structure of EV71 5′UTR was predicted, and found to be conserved in picornavirus (Fig. 2A). Three functional regions including cloverleaf-like (CL), IRES, and linker regions, were identified based on a previous report (Lin et al. 2008). Stem-loops Ⅱ-Ⅵ, which are important for initiation of virus translation, were contained in the EV71 5′UTR. To map the regions of interaction between PTB and EV71 IRES, six different stem-loop RNA probes were generated, including SL Ⅱ (nt 121-180), SL Ⅲ (nt 190-230), SL Ⅳ (nt 241-450), SL Ⅴ (nt 451-563), SL Ⅵ (nt 564-742), and the entire IRES (nt 90-742) (Fig. 2B). After RNA pull-down assay, Western blot results showed that PTB specially binds to the SL Ⅵ (stem-loop Ⅵ and linker region) and whole IRES, but not other SLs (Fig. 2C).
Figure 2. Determination of the EV71 IRES sequences required for the binding of PTB. A Prediction of the RNA secondary structure of the EV71 5′UTR by the mfold web serve. The modified schematic representation of the secondary structure of poliovirus (PV) 5′UTR (modified from Hellen et al. 1994) is shown in the top right corner. B Plasmids carrying different deletions in the five stem-loops of EV71 IRES: pGEM-3zf-(SL Ⅱ, 121-181), (SL Ⅲ, 190-230), (SL Ⅳ, 241-450), (SL Ⅴ, 451-463), (SL Ⅵ, 564-742). C Analysis of the regions responsible for the interaction in the EV71 IRES region using various truncated RNA forms, transcribed in vitro and biotinylated. T98G cell lysate were incubated with these biotin-labeled RNAs and the non-biotinylated IRES RNA probes were used as controls. After being pulled down by streptavidin, the protein complex was separated by SDS-PAGE and Western blot was carried out to detect PTB in the pulled-down complex (lanes 1-7).
PTB protein contains four RRMs (RRM1, RRM2, RRM3, and RRM4), which are important for RNA binding (Clery et al. 2008). The amino acids constituting these four RRM domains are underlined in Fig. 3A. To identify the domains that account for interaction with the EV71 IRES region, three plasmids expressing the one full-length and two truncated PTB proteins (RRM1-2, RRM3-4) were constructed (Fig. 3B). These plasmids were individually transfected into 293ET cells, and the resulting cell lysates were used in pull-down assays with biotinylated EV71 IRES. The results showed that all the three PTB proteins were expressed in transfected cells, and that they were detected in the input lanes. PTB RRM1-2 protein, but not RRM3-4 protein, was detected in the presence of biotinylated EV71 IRES (Fig. 3C). These results indicated that RRM1-2 of PTB is responsible for the binding of PTB to the EV71 IRES.
Figure 3. Analysis of the expression of PTB in neuro-related cells and RRMs required for its binding to EV71 IRES. A The RRM domains of full-length PTB protein as described. The amino acids of the four RRM domains, RRM1, RRM2, RRM3, and RRM4, are underlined. B Three constructed plasmids expressing the full-length PTB, or truncated PTB proteins in which RRM3-4 was deleted (RRM1-2), or RRM1-2 was deleted (RRM3-4), respectively. C RNA-protein binding assay showing PTB protein as determined by Western blot analysis using anti-His antibody, from 293ET cells transfected with plasmids coding the His-Tagged proteins, pcDNA4.0-PTB, RRM1-2, RRM3-4, respectively.
PTB is localized mainly in the nucleus, but can shuttle between nucleus and cytoplasm to complete different functions (Xie et al. 2003). To further illustrate whether EV71 infection induces changes in PTB localization, T98G cells were infected with EV71, and the localization of PTB was monitored. Confocal microscopy demonstrated that PTB was mainly expressed in the nucleus in mock cells; however, after EV71 infection, a portion of the PTB proteins were shuttled from the nucleus to the cytoplasm (Fig. 4A). To further confirm this changed expression pattern, the PTB expression pattern during EV71 infection was examined. The results showed that partial PTB protein was translocated into the cytoplasm in EV71-infected cells, while PTB was expressed exclusively in the nuclei in non-infected cells (Fig. 4B). Consistent with the confocal results, EV71 VP1 protein was exclusively located in the cytoplasm. The positive controls, β-actin and CREB, were exclusively expressed in the cytoplasm and nucleus, respectively. These results illustrated that, upon EV71 infection, PTB translocated from the nucleus to the cytoplasm, implying that PTB might be recruited by EV71 to participate the viral life cycle.
Figure 4. Subcellular distribution of PTB protein during EV71 infection. T98G cells mock-infected (upper panels) or infected with EV71 at an MOI of 20 (lower panels). At 6 h post infection, cells were fixed with formaldehyde, washed, and detected with antibody against PTB or EV71 3C protein. FITC-conjugated goat anti-rabbit IgG or TRITC-conjugated goat anti-mouse IgG was used as secondary antibody and stained with DAPI. Images were captured by confocal laser scanning microscopy. PTB (red), EV71 3C (green), DAPI (blue). Bar = 10 µm. B T98G cells infected with or without EV71 at an MOI of 20 for 6 h. Whole cell lysate, cytoplasm, and nucleus extractions were prepared from mock infected and EV71-infected cells, respectively. Endogenous proteins as detected by Western blot analysis using antibodies to CREB, EV71 VP1, PTB, or β-actin. The blot is representative of three independent experiments with similar results.
Cap-independent initiation of translation is a major pathway for picornavirus translation, and IRES provides an alternative means of viral polyprotein translation initiation. To evaluate the effects of PTB on EV71 IRES activity, a dicistronic reporter plasmid was used (Fig. 5A). The first cistronic (Renilla luciferase, Rluc) is cap-dependent, while the translation of the second cistronic (Firefly luciferase, Fluc) is dependent on EV71 IRES activity. One stem loop was added upstream of Rluc to prevent a high Rluc background signal arising from false initiation of translation. The Ratio of Fluc to Rluc expression could be considered as the relative IRES activity. Compared with the siNC negative control, knockdown of PTB decreased EV71 IRES activity to 70% (P < 0.05) (Fig. 5B), while overexpression of PTB increased EV71 IRES activity to 160% (P < 0.05). These results suggest that PTB can positively regulate EV71 IRES activities related to initiation of translation.
Figure 5. Analysis of PTB in the regulation of EV71 viral translation and virus production. A Schematic diagram of dicistronic reporter plasmids pRF-EV71 IRES. B T98G cells were transfected with either PTB siRNA or pcDNA4.0-PTB. 48 h later, dicistronic construct pRF or pRF-EV71 IRES and siRNA duplexes were co-transfected into T98G cells. After 48 h, the relative ISRE activity were analyzed by monitoring the luciferase activity. Western blot were used to analyze the expression level of PTB and β-actin. C-F T98G cells were transfected with siRNA targeting PTB (siPTB) and control siRNA (siNC), respectively. 48 h later, T98G cells were transfected with plasmids containing either full-length PTB cloned into pcDNA4.0 vector (pcDNA-PTB), or vector without PTB (pcDNA). After 24 h, cells were infected with EV71 at an MOI of 20 for 4, 8 and 12 h. Cell culture supernatants from EV71-infected cells were prepared and subjected to plaque assays. Western blotting was used to determine the expression level of EV71 VP1 protein, PTB or β-actin protein. E, F The effects of PTB on EV71 growth. Cell culture supernatants from C and D were determined by plaque assay. Each bar represents the average of triplicate data points with the standard deviation represented as the error bar. *P < 0.05 and **P < 0.01 versus negative control.
To study the roles of PTB in virus translation, the expression level of EV71 VP1 was examined. The expression level of EV71 VP1 was decreased in PTB knockdown cells at 4 h, 8 h, and 12 h post-EV71 infection, respectively (Fig. 5C), while the expression level of EV71 VP1 was increased in PTB-overexpression cells during infection (Fig. 5D), indicating that PTB promotes the expression of viral protein.
The effects of PTB on EV71 growth were also evaluated. As illustrated in Fig. 5E, 5F, virus production and viral titer in EV71-infected cells were enhanced by overexpression of PTB, but reduced by knockdown of PTB, suggesting that PTB positively regulates viral replication.
PTB Interacts with EV71 Viral RNA in IRES Region
PTB Binds to the Stem-loop Ⅵ within the EV71 IRES Region
RRM1-2 of PTB Interacts with EV71 IRES
PTB Transfers to the Cytoplasm during EV71 Infection
PTB Promotes IRES-Dependent Translation
PTB Promotes EV71 Viral Protein Expression and Virus Production
Table S1. Primers used in the study.