molecular level (Arita et al., 2005; Arita et al., 2008a; Chua et al., 2008; Kung et al., 2010; Yeh et al., 2011; Huang et al., 2012). In the current study, EV-A71 infectious cDNA clones were used to investigate the degree of tolerance of mismatches of vivo-MO-1 towards its targeted sequence within the EV-A71 RNA genome.
5.2 Development of an antiviral peptide against enterovirus A71 infections
virus (Jones et al., 2006), HSV-1 (Akkarawongsa et al., 2009; Tiwari et al., 2011), HBV (Kim et al., 2008), HCV (Cheng et al., 2008; Liu et al., 2010), HIV-1 (Kilby et al., 1998), DENV and WNV (Hrobowski et al., 2005). Although the EV-A71 capsid protein VP1 has been reported to be responsible in mediating viral adsorption and the uncoating process, little information is available about the molecular interactions of EV-A71 and cell receptors (Li et al., 2007). Recently, Chen et al. (2012) identified several amino acid residues present within position 152-236 in the EV-A71 capsid protein VP1 that were critical for the molecular interaction between EV-A71 and the SCARB2 receptor.
A Pepscan strategy was employed to screen 95-overlapping synthetic peptides corresponding to the VP1 capsid protein for antiviral activity against EV-A71. Four peptides SP40, SP45, SP81 and SP82 were found to exhibit significant antiviral activities. The SP40 peptide was selected for further investigation as the amino acid sequence of SP40 is highly conserved across all EV-A71 genotypes and subgenotypes.
The results demonstrated that the SP40 peptide inhibited EV-A71 infection in a dose-dependent manner corresponding to the reduction of viral RNA, viral protein and plaque formation. The IC50 values ranged from 6-9.3 µM against all representative strains of EV-A71 genotypes A, B and C. Interestingly, the SP40 peptide also inhibited CV-A16 and PV1 infection in vitro, implying that the SP40 peptide could function as a broad-spectrum antiviral agent. However, a higher concentration of SP40 peptide was required to block PV1 infection. This could be due to the high degree of dissimilarity of the amino acid sequences of EV-A71 and PV.
The possible mechanism of action of the SP40 peptide could be either through direct virus inactivation or blocking of viral attachment and entry. This study confirmed that the SP40 peptide was not virucidal, but that it blocked viral attachment to the cell surface and hence prevented EV-A71 infection. Immunofluorescence assay showed the number of viral particles attached to the cell surface was reduced significantly when the
RD cells were pre-treated with the SP40 peptide before addition of virus at 4˚C. This indicated that the SP40 peptide probably first interacted with a cell surface receptor and subsequently prevented EV-A71-cellular receptor interactions. However, the SP40 peptide lost its antiviral activity when the peptide was added 1 hour after EV-A71 infection.
Results also demonstrated the importance of the SP40 amino acid sequence for its antiviral activity. The inhibitory effect of the scrambled peptide SP40X, was significantly lower (42.5%) than the effect observed with the SP40 peptide. The SP40 peptide could prevent viral attachment by interacting with cell receptors present on the surface of RD cells, thereby blocking the availability of receptors for attachment of the EV-A71 viral particles. The observation of a significantly reduced IC50 value when RD cells were pre-treated with SP40 before EV-A71 infection strongly supported this view.
The cellular receptor that the SP40 peptide interacted with remained unknown. None of the amino acids identified that interact with SCARB2 (Chen et al., 2012) was mapped within the SP40 peptide amino acid sequence. This suggested that the SP40 peptide probably did not interact with the SCARB2 receptor. The SP40 peptide could also inhibit CV-A16 and PV1 infections in vitro, indicating that it could interact with a common receptor shared by these viruses.
Since the positively-charged amino acids were critical for antiviral activities, the SP40 peptide could interact with cell surface receptors through electrostatic charge interactions. The antiviral activity of the SP40 peptide was not restricted to a specific cell type, but it could block EV-A71 infection in different cell lines. This indicated that the SP40 peptide was probably interacting with a receptor that was commonly expressed in most cell types. Cell surface GAGs are present ubiquitously on the surface of most animal cells and in the extracellular matrix (Liu and Thorp, 2002). The presence of several arginine residues in SP40 showed similarity to an antiviral peptide against
HSV-1 which bore positively-charged poly-arginine residues, and which was found to interact with heparan sulfate (Tiwari et al., 2011). Sequence analysis of the SP40 peptide revealed that it contained heparan sulfate GAG-specific binding domains (G1RRRRS6 and R28KVR31) present in bovine and human lactoferrins (Mann et al., 1994; Shimazaki et al., 1998; Jenssen et al., 2006). Several studies have reported that lactoferrin was able to bind to ligands such as heparan sulfate and chondroitin sulfate (van der Strate et al., 2001; Marchetti et al., 2004; Jenssen et al., 2006). It is possible that through this interaction, lactoferrin was able to inhibit EV-A71 infection (Weng et al., 2005). These findings suggest that the SP40 peptide could interact with cell surface GAGs to prevent EV-A71 attachment. The SP40 peptide reported in this study had an even lower IC50 value at 15 µg/ml when compared to the IC50 value of bovine lactoferrin at 34.5 µg/ml (Weng et al., 2005) or human lactoferrin at 103.3-185.0 µg/ml (Lin et al., 2002; Wu et al., 2010).
Interestingly, the amino acid domains in VP1 that are critical for SCARB2 binding were found in the SP45, SP55, and SP81 peptides. These peptides were able to inhibit EV-A71 infections in a dose-dependent manner with no cytotoxicity to the RD cells. Strong synergistic antiviral activities were observed between the SP40 and the SP81 peptides in RD cells. The additive effects of these two peptides could have significantly reduced the availability of receptors for viral attachment.
Since the SP40 peptide works at a very low micromolar concentrations, is non-cytotoxic to RD cells, and has activity against multiple EV-A71 genotypes, it is potentially an excellent candidate for further development as an antiviral agent. The SP40 peptide was effective when administered before EV-A71 infection and may be of particular use for prophylactic intervention, for example to contacts of a case of neurological EV-A71 disease. The exact cellular receptor(s) that the SP40 peptide interacts with still remains unknown, but it was postulated to interact with cell surface negatively-charged GAGs
such as heparan sulfate. Further in vivo studies are needed for development of SP40 as an antiviral agent. Although a major disadvantage of peptides is their low bioavailability due to their rapid degradation in the gastrointestinal system, new formulations, such as the D-isomer peptide and other delivery options are being developed (Huther and Dietrich, 2007).
5.3 Cell surface heparan sulfate as an enterovirus A71 attachment receptor