A Pepscan strategy was employed to screen 95-overlapping synthetic peptides corresponding to the VP1 capsid protein for antiviral activity against EV-71. 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-71 genotypes and sub-genotypes. Our results demonstrated that the SP40 peptide inhibited EV-71 infection in a dose-dependent manner corresponding to the reduction of viral RNA, VP1 protein and plaque formation. The IC50 values reported in our studies ranged from 6–9.3 µM against all representative strains of EV-71 genotypes A, B and C. Interestingly, the SP40 peptide also inhibited CV-A16 and poliovirus type 1 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 poliovirus type 1 infection. This could be due to the high degree of dissimilarity of the amino acid sequence present in EV-71 and poliovirus.
The possible mechanism of action of the SP40 peptide could be either through direct viral inactivation or it could block viral attachment and entry. Our data confirmed that the SP40 peptide was not virucidal, but it blocked viral attachment to the cell-surface and hence prevented EV-71 infection. Our immunofluorescence assay and Cellomics HCS ArrayScan 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. The results indicated that the SP40 peptide probably first interacted with a cell-surface receptor and subsequently prevented EV-71-cellular receptor interactions. However, the SP40 peptide lost its antiviral activity when the peptide was added 1 hour after EV-71 infection.
Previous studies have shown that peptides could play a significant role in surface protein-protein interactions and could exert inhibitory activities against viruses like influenza virus 
, Herpes Simplex virus-1 
, Hepatitis B virus 
, Hepatitis C virus 
, HIV-1 
, Dengue virus and West Nile virus 
. Although the EV-71 capsid protein VP1 has been reported to be responsible in mediating viral adsorption and uncoating process, little information is available about the molecular interactions of EV-71 and cell receptors 
. Recently, Chen et al.
had identified several amino acid residues present in the EV-71 capsid protein VP1 that were critical for the molecular interaction between EV-71 and the SCARB2 receptor. These amino acid residues were found within the residues 152–236 of the VP1 protein. None of the amino acids identified was mapped within the SP40 peptide amino acid sequence. This finding suggested that the SP40 peptide probably did not interact with the SCARB2 receptor.
We have demonstrated the importance of the SP40 amino acid sequence for antiviral activities by comparison with a peptide carrying scrambled sequence. The inhibitory effect of the scrambled peptide, SP40X, was significantly lower (at 42.5%) than the effect observed with the SP40 peptide. Since the amino acid sequence of the SP40 peptide was highly conserved across all EV-71 genotypes and was exposed on the surface, this sequence might carry important motif/domain that interacted with an unidentified cell-surface receptor. The SP40 peptide could prevent viral attachment by interacting with cell receptors present on the surfaces of the RD cells, thereby blocking the availability of the receptor for attachment of the EV-71 viral particles. The observation of a significantly reduced IC50 value when RD cells were pre-treated with SP40 before EV-71 infection strongly supports this view. The cellular receptor that the SP40 peptide interacted with remained unknown. However, the SP40 peptide could also inhibit CV-A16 and poliovirus type 1 infections in vitro, indicating that the SP40 peptide could interact with a common receptor that was probably 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-71 infection in different cell lines. This indicated that the SP40 peptide was probably interacting with the receptor that was commonly expressed in most cell types. Interestingly, cell surface glycosaminoglycans are present ubiquitously on the surface of most animal cells and in the extracellular matrix 
. The presence of several arginine residues in SP40 draws similarity to the antiviral peptide displaying positively charged poly-arginine residues against herpes simplex virus-I (HSV-I) 
. The antiviral property of the poly-arginine peptide against HSV-I infection in mice was due to an interaction with heparan sulfate. Sequence analysis of the SP40 peptide revealed that it consisted of heparan sulfate glycosaminoglycan specific binding domains (G1
) present in bovine and human lactoferrins 
. Several studies have reported that lactoferrin was able to bind to ligands such as heparan sulfate and chondroitin sulfate 
. It is possible that through this interaction lactoferrin was able to inhibit EV-71 infection 
. These findings suggested that the SP40 peptide could have interacted with cell surface glycosaminoglycans and prevented EV-71 attachment.
This is the first time that a small novel viral-based peptide (15-mer) derived from VP1 is reported to exhibit antiviral activities against all genotypes and sub-genotypes of EV-71 infection in vitro
. The development and use of antivirals like enviroxime 
, pleconaril 
, nucleoside analog ribavirin 
and 3C protease inhibitors 
for treating enteroviral infection showed variable efficacies against the neurotrophic EV-71 virus 
. Our results showed that some EV-71 strains were even resistant to ribavirin at 800 µM (unpublished data), this contradicts with the finding of an IC50
of 266 µM reported by Li et al.
. This indicated that ribavirin might not serve as an effective antiviral agent against all EV-71 strains. Other antiviral agents like the viral capsid-binding pyridyl imidazolidinones were found to be ineffective when a single amino acid mutation occurred at position 192 of the hydrophobic pocket of the VP1 capsid protein 
. The SP40 peptide was found to exhibit very similar antiviral properties with bovine and human lactoferrins which were predicted to prevent viral attachment, possibly by blocking an unknown cellular receptor 
. However, the exact antiviral mechanism of lactoferrin remains to be determined and the SP40 peptide reported in our study has an even lower IC50
value at 15 µg/ml when compared to the IC50
value of bovine lactoferrin at 34.5 µg/ml 
or human lactoferrin at 103.3–185.0 µg/ml 
. Thus, SP40 is a good antiviral candidate.
The peptides that blocked the SCARB2 receptor could also be developed as antiviral agents. Chen et al.
have discovered the amino acid residues in the VP1 capsid protein that are critical for SCARB2 receptor binding. Interestingly, the amino acids in VP1 that are critical for SCARB2 binding were found in the SP45, SP55, and SP81 peptides. The amino acid residues that are important for SCARB2 binding in the SP45, SP55 and SP81 peptides were illustrated in Fig. S1
. These peptides were able to inhibit EV-71 infections in a dose-dependent manner with no cytotoxicity to the RD cells (unpublished data). Strong synergistic antiviral activities were observed between the SP40 and the SP81 peptides in RD cells. Since the amino acids critical for binding to SCARB2 were not present in the SP40 peptide, the data suggested that the SP40 peptide could have interacted with a different receptor compared with the SP81 peptide. The additive effects of these two peptides could have significantly reduced the availability of receptors for viral attachment.
Inhibition of viruses at the stage of viral attachment provides a target for therapeutic intervention. Therapeutic peptides have become an attractive tool in drug discovery due to their active regulatory role in the biological system and their extreme high specificity of recognition. The best characterized therapeutic peptide inhibitor is Enfuvirtide (fusion inhibitor) which mimics the N terminal sequence in HIV fusion protein, gp41 
. Using peptides as therapeutic agents offer some significant advantages over small chemical molecules or large therapeutic antibodies. A major advantage of peptides is their small size and their high activity and specificity when compared to the antibodies. Peptides are better candidates to inhibit protein-protein interactions that comprise a surface area often too large to be inhibited by small chemical molecules. Peptides accumulate in lesser quantity in tissues, and have very low cell toxicity when compared to small synthetic molecules 
. Antimicrobial peptides such as lactoferrin, human β-defensin-2 and dermaseptins have been reported to exhibit antiviral properties against various viruses 
. Therefore, therapeutic peptides have some advantages over the smaller chemical compounds as antiviral agents.
Since the SP40 peptide works at a very low micromolar concentration and is non-cytotoxic to RD cells, it is potentially an excellent candidate for further development as an antiviral agent. The SP40 peptide was effective when administered before EV-71 infection and could be considered as an excellent candidate for prophylactic intervention. The exact cellular receptor(s) that the SP40 peptide interacted with still remained unknown. Further in vivo
studies are needed for development of the 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 to circumvent these disadvantages