4.2 Inhibition of enterovirus A71 infections by a novel antiviral peptide derived
Figure 4.7: Identification of antiviral peptides. A library containing 95 overlapping synthetic peptides (15-mers) covering the entire EV-A71 capsid protein VP1 was synthesized. Each peptide was screened at a concentration of 100 µM for its ability to inhibit EV-A71 infection in a plaque reduction assay. A schematic representation of the EV-A71 genome is shown; and the percentage of EV-A71 infectivity following treatment with each peptide is shown in the bar graph. Arrows denote the four peptides that inhibited > 80% of plaques.
4.2.2 Antiviral analysis of the SP40 peptide
To further investigate the antiviral activities of SP40 peptide, the peptide was re-synthesized at a higher purity of > 95% by HPLC. A scrambled peptide, SP40X (Ac-REFTMKRMVLFRQDY-NH2) was synthesized and used as a control throughout the experiment. The inhibitory effect of SP40 peptide against EV-A71 at a MOI of 0.1 was determined using a comprehensive inhibition assay. SP40 peptide significantly reduced EV-A71-induced CPE (Figure 4.8A), plaque formation (Figure 4.8B) and viral protein expression (Figure 4.8C). The results also confirmed that SP40 peptide inhibited EV-A71 infection in RD cells, with reduction of viral plaques by 96.3% ± 0.8 (Figure 4.9A) and viral RNA level by 92.0% ± 9.3 (Figure 4.9B). The inhibition concentration 50%
(IC50) for SP40 peptide was 7.9 µM ± 3.5. The scramble SP40X did not inhibit EV-A71 infection, except at higher concentration. This is likely due to the accumulation of positively-charged amino acids. Taken together, these data suggests that the amino acid sequence of SP40 is critical for its antiviral activity.
To verify whether the antiviral activity of SP40 peptide was cell-specific, the SP40 peptide was tested against EV-A71 infection in HeLa, Vero and HT-29 cell lines. As depicted in Figure 4.10A and B, SP40 peptide greatly inhibited EV-A71 infection in HeLa, Vero and HT-29 cells in a dose-dependent manner, as determined by CPE observation and TaqMan real-time PCR. However, SP40 peptide exhibited reduced efficacy in HT-29 cells as compared to the HeLa or Vero cells.
This study also further investigated whether SP40 peptide inhibited different EV-A71 genotypes as well as other enteroviruses. As shown in Table 4.1, SP40 peptide also exhibited antiviral activity against EV-A71 BrCr (genotype A), EV-A71 SHA66/97 (genotype B3), EV-A71 SHA52/97 (genotype C2), CV-A16 strain 22159 and PV
vaccine strain. However, reduced antiviral activity was observed against the PV vaccine strain (IC50 of 18.2 µM ± 10.4).
4.2.3 Cytotoxicity analysis of the SP40 peptide
To evaluate whether SP40 peptide was cytotoxic to cells, RD cells were treated with increasing concentrations of SP40 peptide from 0 µM to 280 µM. The cell viability was then determined. As depicted in Figure 4.11, SP40 peptide was not toxic to cells when tested up to 280 µM.
Figure 4.8: Inhibitory effects of SP40 and SP40X peptides on CPE, plaque formation and protein synthesis. (A) For CPE, EV-A71 at a MOI of 0.1 was pre-incubated with peptides for 1 hour before infection of peptide-treated RD cells. The images were taken at 24 hours post-infection. CPE was seen as round and shrunken cells, which eventually dislodged from the surface.
(B) For the plaque reduction assay, approximately 100 PFU of EV-A71 were pre-incubated with peptides for 1 hour before infection of the peptide-treated RD cells. The cells were fixed with 4% formaldehyde and stained with 0.5% crystal violet at 48 hours post-infection. (C) Western blot analysis of total protein isolated from virus-infected cells using the anti-EV-A71 monoclonal antibodies and monoclonal anti-actin antibodies. The molecular weights of the EV-A71 protein and β-actin are 36 kDa and 42 kDa, respectively.
Figure 4.9: Antiviral activities of the SP40 and SP40X peptides. Both RD cells and EV-A71 were separately pre-incubated with increasing concentrations of each peptide for 1 hour before viral inoculation. The inhibitory levels of the peptide were evaluated at 24 hours post-infection by (A) plaque assay and (B) TaqMan real-time PCR. At each peptide concentration, titers are the average of two biological replicates; error bars represent the standard deviation of the mean.
Figure 4.10: The antiviral activities of the SP40 peptide in various cell lines. Vero, HeLa and HT-29 cell lines were pre-treated with the SP40 peptide at various concentrations for 1 hour at room temperature before infection with EV-A71 at a MOI of 0.1. (A) The viral-induced CPE in various cell lines were observed 24 hours post-infection. CPE was seen as round and shrunken cells, which eventually dislodged from the surface. (B) Viral RNA inhibition was quantitated by TaqMan real-time PCR. At each peptide concentration, percentages of inhibition are the average of two biological replicates; error bars represent the standard deviation of the mean.
Table 4.1: Inhibition concentration 50% (IC50) of the SP40 peptide against various enteroviruses
Enterovirus Genotype Clinical manifestations IC50 (µM)a
EV-A71 BrCr A Aseptic meningitis 9.3 ± 2.5
EV-A71 SHA66/97 B3 HFMD 6 ± 0.7
EV-A71 41 B4 Fatal 7.9 ± 3.5
EV-A71 SHA52/97 C2 HFMD 8.5 ± 2.8
CV-A16 - HFMD 6 ± 0.8
PV vaccine strain - - 18.22 ± 10.4
aThe IC50s are presented as mean ± standard deviation determined from at least two independent experiments
Figure 4.11: Cytotoxicity assay. RD cell monolayers in a 96-well plate were treated with increased concentrations of SP40 peptide. Cell viability was assayed as an absorbance reading at 490 nm. At each peptide concentration, the absorbance readings are averages of two biological replicates; error bars represent the standard deviation of the mean.
4.2.4 Mechanism of action of the SP40 peptide
Since the amino acid sequence of SP40 peptide was critical for its antiviral activity, further understanding of the mechanism of action is required. The data from the comprehensive assay suggests that the SP40 peptide could exert its antiviral activity through inhibition of the viral binding step or inactivation of the virus. To further elucidate the mechanism, either cells or viruses were pre-treated with SP40 peptide before infection. When the viruses at a MOI of 10 were pre-treated with the SP40 peptide, followed by 200-fold dilution prior to infection, the SP40 peptide lost its antiviral activity against EV-A71, and this implied that SP40 peptide did not inactivate the virus (Figure 4.12).
When only the cells were pre-treated with SP40 peptide, significant inhibition of EV-A71 infection was observed with an IC50 value of 15 µM. This result suggests that the SP40 peptide could either inhibit EV-A71 at the pre-binding step or post-binding step.
To address this, RD cells were pre-treated with various concentrations of the SP40 peptide at 4°C for 1 hour, followed by EV-A71 at a MOI of 100 at 4°C for 1 hour. The EV-A71 viral particles that attached to the cells surface were then determined by immunofluorescence assay and further quantitated by high content screening analysis.
As shown in Figure 4.13A, the number of EV-A71 viral particles (with Alexa Fluor 488 green fluorescence) attached to the SP40 peptide-treated cell surface was significantly lower compared to the untreated cells. This result was further verified by Cellomics HCS ArrayScan Spot Detector Bio-Application and TaqMan real-time PCR. As shown in Figure 4.13B, the number of spots per field for SP40 peptide-treated RD cells (68 ± 20 spots/field) was lower compared to the untreated cells (210 ± 39 spots/fields). The results correlated well with TaqMan real-time PCR (Figure 4.13C). To investigate if SP40 peptide inhibited the EV-A71 post-binding step, RD cells were pre-treated with
EV-A71 at 4°C for 1 hour to allow the virus to bind to the cells, followed by addition of SP40 peptide. The cells were washed and then immediately shifted to 37°C for 1 hour to allow the virus to enter the cells. However, SP40 peptide lost its antiviral activity, which implies that SP40 peptide blocked EV-A71 infection at the pre-binding step. These findings suggest that SP40 peptide could interact with the attachment receptor targeted by EV-A71 and hence block the attachment event.
To further verify whether the SP40 peptide still inhibits EV-A71 infection in the post-infection event, SP40 peptide was administered 1 hour after EV-A71 post-infection. The SP40 peptide was found to be non-inhibitory when added 1 hour after infection, with an IC50 value of 200 µM (Figure 4.12).
Figure 4.12: Mechanism of action studies of SP40 peptide. The SP40 peptide was administered at different time points relative to viral inoculation, and plaque assay was performed 24 hours post-infection. In the comprehensive assay, both RD cells and EV-A71 were pre-treated with the SP40 peptide for an hour before infection. In the cell protection assay, RD cells were pre-treated with the peptide for an hour before virus inoculation. In the post-infection assay, RD cells were infected with EV-A71 for an hour before addition of the peptide-containing media. In the virus inactivation assay, EV-A71 was pre-treated with the peptide for an hour, and diluted 200-fold before infection of RD cells. At each peptide concentration, percentages of plaque inhibition are the average of two biological replicates; error bars represent the standard deviation of the mean
Figure 4.13: Effect of SP40 peptide on EV-A71 attachment. (A) Immunofluorescence analysis of A71 viral particles attached on the RD cell surface. EV-A71 viral particles were probed with anti-EV-EV-A71 antibodies and Alexa Fluor 488; and cell nuclei were stained with DAPI. EV-A71 viral particles and nuclei are shown in green and blue fluorescence, respectively. The number of virus particles attached to the cell surface were quantified by (B) TaqMan real-time PCR assay and (C) Cellomics HCS ArrayScan Spot Detector Bio-Application. At each peptide concentration, titers are the average of two biological replicates; error bars represent the standard deviation of the mean.
4.2.5 Alanine scanning analysis
Identification of the amino acids critical for the antiviral activity of SP40 peptide can determine the mechanism of action, and identify the potential receptor binding site.
Alanine scanning is the most common method used to identify the amino acid residues critical for activities. Thirteen peptides with alanine substitutions in each amino acid position of the 15-mer SP40 peptide were synthesized. The inhibitory effects in RD cells at 200 µM of all peptides were evaluated. Reduction of viral RNA levels by each of the peptides were evaluated and are summarized in Figure 4.14. Substitution of an arginine residue at position 3 (P3) with alanine significantly reduced the antiviral activity from 95.9% to 60.8%. Substitution of other positively-charged amino acids (lysine and arginine) at positions 4, 5 and 13 (P4, P5 and P11) of the SP40 peptide with alanine caused a moderate reduction of antiviral activity, from 95.9% to 74.3%, 70.9%
and 70.6%, respectively. Substitution of a polar amino acid, methionine, at position 12 (P10) with alanine also reduced the antiviral activity moderately to 74.7%. Alanine substitution of other amino acids at other positions of the SP40 peptide did not alter its antiviral activity. These results indicated that the positively-charged amino acids of SP40 peptide were critical for its antiviral properties. Substitution of a polar methionine also reduced the antiviral properties.
Figure 4.14: Alanine scanning analysis of SP40 peptide. Thirteen different peptides (P1-P13) were synthesized by replacing one residue at a time with an alanine and their inhibitory effect was determined, compared with the original SP40 represented by the red line. SP40X, a scrambled peptide, was used as a negative control. Percentages higher than the red line showed a gain of antiviral activity whereas a lower number represented a loss of antiviral activity. Data presented are mean of two biological replicates; error bars represent the standard deviation of the mean.
4.2.6 Three-dimensional structure analysis
With the recent available crystal structure of EV-A71, the position of the SP40 peptide was located and characterized (Figure 4.15). The SP40 peptide was not positioned on the surface, but the lysine residues of SP81 and SP82, which also exhibited antiviral activity (see section 4.2.1) were highly exposed on the surface.
4.2.7 Synergistic antiviral activities of the SP40 peptide with SP81
To investigate whether the SP40 peptide exhibited synergistic antiviral activities with other peptides, the SP40 peptide was combined with SP45 and SP81 (> 70% purity by HPLC) to a final concentration of 200 µM. The SP40 peptide exhibited synergistic antiviral activity with SP81 peptide to achieve a total viral RNA inhibition of 99.7%
when compared to 92.0% inhibition by the SP40 peptide alone. However, the SP40 peptide had less synergistic effect when combined with the SP45 peptide, with viral RNA inhibition of 94.3%. When all the three peptides were combined, viral RNA inhibition achieved was 98.8%. There were only few amino acids difference between SP81 and SP82 peptides, implying that these two peptides could share similar mechanism in action. Therefore, synergistic antiviral activities of SP40 and SP82 peptides were not included in this study. These data suggest that all these peptides could potentially target different host factors that are important for EV-A71 infection.
Figure 4.15: Proposed location of the SP40 peptide based on the recently determined EV-A71 crystal structure (PDB accession number 4AED). The molecular structures of EV-A71 VP1, VP2, VP3, and VP4 are represented by red, blue, green and grey, respectively. The SP40 peptide sequence is indicated in yellow.
4.3 Enterovirus A71 uses cell surface heparan sulfate glycosaminoglycan as an