EV71 suppresses the induction of innate antiviral immunity by poly(I · C) in infected cells.
To study the effect of EV71 on the TLR3 pathway, we measured the induction of antiviral immunity by poly(I · C), a prototype TLR3 agonist. HeLa cells were initially mock infected or infected with EV71 for 4 h. At various time points after incubation with poly(I · C), the cells were examined for the expression of ISG54, ISG56, interleukin-6, and GAPDH by RT-PCR analysis. As shown in A, poly(I · C) induced ISG54 and ISG56 expression in mock-infected cells, which peaked at around 4 h after the treatment (lanes 1, 3, 5, 7, and 9). Such induction by poly(I · C) was sharply reduced in cells infected with EV71 at all time points examined (A, lanes 2, 4, 6, 8, and 10). Under these conditions, most cells were viable (96%) as measured by the trypan blue exclusion assay (data not shown). Interestingly, EV71 inhibited IRF3 phosphorylation by poly(I · C) compared to that in mock-infected cells (B, lanes 1 to 4). Poly(I · C) stimulated interleukin-6 expression in mock-infected cells, with an early kinetics (A, lanes 1, 3, 5, and 7). Intriguingly, EV71 infection enhanced this effect (A, lanes 4, 6, 8, and 10), suggesting that EV71 replication stimulated interleukin-6 expression. As expected, GAPDH expression remained at comparable levels in cells treated or not treated with poly(I · C) (A, lanes 1 to 10). Here, EV71 infection differentially modulates the expression of ISG54, ISG56, and interleukin-6.
Fig. 1. (A) EV71 inhibits the induction of ISG54 and ISG56 by poly(I · C). HeLa cells were mock infected or infected with EV71 at an MOI of 2. At 4 h after infection, cells were incubated with or without poly(I · C). At indicated time points, (more ...) The 3C protein of EV71 impairs the induction of antiviral molecules by poly(I · C).
Among proteins encoded by EV71, 3C inhibits virus-induced immunity by interacting with RIG-I (22
). Since EV71 blocked poly(I · C)-mediated responses, we hypothesized that 3C might contribute to this process. To test this possibility, HeLa cells were transfected with GFP or GFP-3C plasmid, with approximately 60% transfection efficiency. At 24 h after transfection, cells were incubated with poly(I · C) and the expression of ISG54, ISG56, IL-6, and GAPDH was analyzed by RT-PCR assays. As indicated in A, GFP-3C reduced the expression of ISG54, ISG56, and IL-6, whereas GFP displayed no inhibitory effect (lanes 1 to 10). In correlation, GFP-3C inhibited poly(I · C)-induced IRF3 phosphorylation compared to GFP (B, lanes 1 to 4). When expressed in 293/TLR3 cells, the 3C protein also inhibited ISG56 and IFN-β promoter activation induced by poly(I · C) compared to GFP (C and D). These results suggest that EV71 3C is able to inhibit dsRNA-induced innate immunity.
Fig. 2. (A) The 3C protein inhibits the induction of ISG54, ISG56, and IL-6 by poly(I · C). HeLa cells were transfected with GFP or GFP-3C. At 24 h after transfection, cells were treated with or without poly(I · C). At the indicated time points, (more ...) The 3C protein inhibits IFN-β and NF-κB activation by downregulation of TRIF.
When engaged with TLR3, TRIF activates IRF3 and NF-κB, leading to type I IFN production (17
). Therefore, we assessed the impact of 3C on TRIF in reporter assays. As illustrated in A, TRIF stimulated IFN-β promoter activation in 293T cells. Addition of 3C inhibited it in a dose-dependent manner. Likewise, 3C suppressed NF-κB activation by TRIF (B). Under this experimental condition, EV71 3C did not inhibit MyD88-stimulated NF-κB activation (C). It appears that the 3C protein inhibited TRIF but not MyD88 function, suggesting TRIF as a potential cellular target. To evaluate this, we determined whether EV71 3C affected the TRIF expression. As shown in , EV71 3C reduced the level of endogenous TRIF in a dose-dependent manner when expressed in HeLa cells. This reduction was not detectable with TBK1. Thus, EV71 3C reduces TRIF expression and inhibits its activity.
Fig. 3. Effects of 3C on NF-κB and IFN-β promoter activation. (A and B) 293T cells were transfected with TRIF and GFP-3C along with IFN-β-Luc (A) or NF-κB-Luc (B). (C) In parallel, cells were transfected with MyD88, GFP-3C, and (more ...)
Next, we analyzed TRIF expression in EV71-infected cells. Cells were mock infected or infected with EV71. At different time points postinfection, cells were processed and examined by Western blot analysis. A shows that HeLa cells constitutively expressed TRIF, MyD88, TBK1, IRF3, and β-actin (lane 1). EV71 infection had different effects on these proteins (lanes 2 to 5). Strikingly, the level of TRIF decreased as EV71 infection progressed. This coincided with an increase in 3C expression. In contrast, MyD88, IRF3, TBK1, and β-actin exhibited no or little reduction in EV71-infected cells. This was reproducible in several experiments. At 12 h after infection, EV71 reduced TRIF by approximately 70% (B). A similar pattern was also observed in RD cells (C, lanes 1 to 5). These results indicate that EV71 specifically downregulates TRIF in mammalian cells.
Fig. 4. Effects of EV71 infection on protein expression. (A) HeLa cells were mock infected or infected with EV71 at an MOI of 5. At the indicated time points, cell lysates were subjected to Western blot analysis with antibodies against TRIF, MyD88, IRF3, TBK1, (more ...) EV71 3C associates with and induces TRIF cleavage independently of caspases.
To examine the nature of 3C-TRIF interactions, we carried out immunoprecipitation analysis in 293T cells expressing Flag-TRIF, GFP, and GFP-3C. As illustrated in A, GFP-3C but not GFP coprecipitated with Flag-TRIF, indicating an interaction between 3C and TRIF. Notably, 3C reduced the TRIF level in the immunoprecipitates compared to GFP (lanes 3 and 4). This was accompanied by the appearance of a small species (45 kDa) (lane 4). Western blot analysis revealed comparable levels of GFP, GFP-3C, and β-actin in cell lysates (lanes 1 to 4). However, GFP-3C decreased TRIF expression (lane 4). In correlation, a small band appeared (lane 4). This is presumably a TRIF cleavage product. To further address this issue, we carried out a dose-response analysis in 293T cells cotransfected with 3C and TRIF. As shown in B. TRIF expression decreased as the level of GFP-3C increased. As expected, a smaller species appeared (lanes 2 to 6). Similar results were obtained with Flag-3C (data not shown). Hence, EV71 3C has the capacity to induce TRIF cleavage.
Fig. 5. (A) The 3C protein associates with TRIF and induces TRIF cleavage. 293T cells were transfected with plasmids encoding Flag-TRIF (lanes 3 and 4), GFP (lanes 1 and 3), or GFP-3C (lanes 2 and 4). The total amount of DNA was kept constant with an empty vector. (more ...)
Previous studies showed that the 3C protein of EV71 activates caspases (25
). To test whether 3C functioned via caspases, we assessed TRIF cleavage in the presence or absence of Z-VAD-FMK, a pancaspase inhibitor. C shows that GFP-3C induced the cleavage of TRIF, resulting in a 45-kDa protein band (lane 2). Treatment with Z-VAD-FMK did not inhibit this cleavage (lane 3). The enhanced TRIF cleavage by Z-VAD-FMK is attributable to a block of apoptosis. To corroborate this result, we further analyzed a TRIF mutant carrying D281E and D289E substitutions, which is resistant to caspase cleavage (40
). Similarly to wild-type TRIF, the D281E D289E mutant remained susceptible to 3C-mediated cleavage (). These results indicated that 3C-mediated TRIF cleavage does not require caspases.
Glutamine 312 and serine 313 pairs within TRIF are required for 3C-induced cleavage.
As TRIF cleavage produced a 45-kDa product, we inferred that the cleavage site(s) might fall in the central region. To test this, we analyzed a series of TRIF mutants which had deletions from amino acid 311 to 485 (A). These mutants were expressed along with a control GFP or GFP-3C in 293T cells. Cell lysates were subjected to Western blot analysis. As illustrated in B, wild-type TRIF was cleaved when ectopically expressed with GFP-3C, resulting in a 45-kDa species (lane 2). Similarly, the TRIF deletion mutants Δ337-367, Δ368-398, Δ399-438, and Δ439-485 were cleaved in the presence of GFP-3C (lanes 6, 8, 10, and 12). In contrast, Δ311-336 and Δ311-485 were resistant to the 3C cleavage (lanes 4 and 14). Thus, the TRIF cleavage site may sit between amino acids 311 and 336 (C). This region contains three amino acid pairs, Q312-S313, Q331-T332, and Q335-L336, which resemble a signature Q-G sequence of other picornavirus 3C proteolytic sites.
Fig. 6. Mapping of the putative TRIF cleavage site. (A) Schematic representation of wild-type TRIF and its derivatives. The open bar denotes wild-type TRIF, with amino acid numbers shown. Solid lines underneath represent various TRIF variants. Broken lines indicate (more ...)
To define the putative TRIF cleavage site, we analyzed additional TRIF mutants. As shown in , like wild-type TRIF, the Δ322-336 mutant was susceptible to 3C-induced cleavage, whereas the Δ311-325 mutant was resistant (lanes 2, 4, and 6). This implies that the TRIF cleavage site is located between amino acids 311 and 325. Consistently, TRIF substitution mutants Q312A S313A (, lanes 7 and 8) and Q312V S313V (E, lanes 1 and 2) became resistant to 3C. In contrast, Q331A T332A (, lanes 9 and 10), Q335A L336A (, lanes 11 and 12), Q331V T332V (E, lanes 3 and 4), and Q335V L336V (E, lanes 5 and 6) remained susceptible to the 3C cleavage. Collectively, these data indicate that the Q312-S313 junction in TRIF is critical for its cleavage mediated by EV71 3C.
3C protease activity is crucial to suppress IFN-β and NF-κB activation mediated by TRIF.
Besides being a viral protease, EV71 3C also possesses RNA binding activity (45
). To further study 3C, we characterized a panel of 3C mutants (A). H40D has a single amino acid substitution in the catalytic center, which abrogates the 3C protease activity (45
). R84Q and V154S substitution mutants are incapable of binding to RNA. ΔKFRDI has a deletion of amino acids 82 to 86, whereas ΔVGK has a deletion of amino acids 154 to 156. These last two mutants do not exhibit protease and RNA binding activities (45
). As illustrated in B, wild-type 3C inhibited the IFN-β promoter activation mediated by TRIF. However, H40D, ΔKFRDI, and ΔVGK were unable to block the IFN-β promoter activation. Interestingly, R84Q or V154S effectively inhibited the TRIF-mediated activation. Similar phenotypes were observed for NF-κB activation (C). It seems that the region containing H40, KFRDI, and VGK motifs is crucial to overcome TRIF-mediated responses. Nevertheless, this activity does not involve R84 or V154, which are required for RNA binding.
Fig. 7. Mutational analysis of 3C. (A) Schematic diagrams of 3C variants. (B and C) Effects of 3C variants on TRIF-mediated IFN-β promoter activation (B) or NF-κB promoter activation (C). 293T cells were transfected with plasmids encoding TRIF (more ...)
We evaluated TRIF cleavage in cells expressing 3C variants by Western blot analysis. The data in D indicate that wild-type 3C induced a small TRIF species of 45 kDa (lane 2), whereas the control GFP did not. Similarly, R84Q and V154S were able to cause TRIF cleavage (lanes 4 and 5). In stark contrast, H40D, ΔKFRDI, and ΔVGK lost their ability to induce TRIF cleavage and a 45-kDa species (lanes 3, 6, and 7). These phenotypes correlated well with the ability of 3C variants to suppress IFN-β and NF-κB activation by TRIF. To assess protein-protein interactions, we performed an immunoprecipitation experiment. As indicated in D (lower panels), wild-type 3C and all mutants coprecipitated with Flag-TRIF (lanes 2 to 7). Thus, H40D, ΔKFRDI, ΔVGK, R84Q, and V154S mutations had no effect on the 3C-TRIF interaction, suggesting that association of 3C with TRIF is not sufficient to display its inhibitory effect. We conclude that the protease function associated with 3C is to block IFN-β and NF-κB activation by TRIF.