File(s) under permanent embargo
Reason: Restricted by author. A copy can be supplied under Section 51(2) of the Australian Copyright Act 1968 by submitting a document delivery request through your library or by emailing firstname.lastname@example.org
Identification of a novel anti-dengue virus 2 peptide using phage display technology and its interaction with the virus surface proteins
thesisposted on 26.02.2017, 22:34 by Chew, Miaw Fang
Dengue is a mosquito-borne disease caused by the infection of dengue viruses (DENV-1, 2, 3, and 4). Despite the importance of dengue being a public health problem worldwide, there is no specific therapeutic treatment available. Treatment for DENV remains limited to supportive care. Therefore, there is an urgent need to discover and develop effective anti-DENV drugs. In this study, we screened for novel antiviral peptides against DENV-2 using peptide phage display technology. Three potential peptide-bearing phages were identified by biopanning of a randomised 12-mer phage display peptide library against purified DENV-2. Binding abilities of these peptide-bearing phages were assessed via ELISA. The most promising peptide-bearing phage which demonstrated the highest frequency of occurrence in biopanning and strongest binding affinity in ELISA was selected and its peptide sequence (Pgg-ww) was synthesized for further studies. The toxicity and antiviral property of Pgg-ww were evaluated via WST-1 cell proliferation assay and plaque reduction assay respectively. Antiviral properties of Pgg-ww were further validated using RT-qPCR and immunofluorescence assays. Approximately 100% inhibition was successfully achieved at 250µM with an IC50 value of approximately 77µM. Further analyses on its mode of action indicated that Pgg-ww inhibited DENV-2 infection by binding to the virus, thereby, blocking viral entry into host cells. To further elucidate its interaction with DENV-2 surface proteins (prM and E proteins), recombinant prM and E(III) proteins were produced for subsequent peptide-protein interaction studies. The recombinant prM and E(III) proteins were expressed and purified using Ni2+ resins. The antigenicity and authenticity of the recombinant proteins were then validated using Western blot, dot blot, immunoprecipitation assays and liquid chromatography–mass spectrometry (LCMS) assays. In co-localization assays, the Pgg-ww showed a stronger co-localization fluorescence signals with the E protein as compared to the prM protein, indicating that Pgg-ww interacted and bound with E protein. This observation was further validated via fluorescence polarization assay and confirmed via co-immunoprecipitation assay using recombinant E(III) and prM proteins. The interaction was also demonstrated using in silico docking study. The in silico docking analyses had predicted the binding of Pgg-ww to 6 amino acid residues of the E domain II/III (Glu195, Arg350, Glu370, Pro371, Phe373 and Lys394). The binding of Pgg-ww to these highly conserved amino acid residues have indicated the potential of Pgg-ww to be used as inhibitor for other DENV serotypes and flaviviruses. On the other hand, in silico alanine substitution of each of the 12 amino acid residues in Pgg-ww have revealed six `hot spots’ that can potentially destabilize the Pgg-ww:E protein interface. These hot spots may be used as important leads for downstream peptide optimizations. It was shown that Pgg-ww had a half-life of less than 0.5 h in serum, nonetheless, its stability could be improved using numerous modifications including cyclization, amidation and acetylation. In summary, we have identified a novel antiviral peptide (Pgg-ww) against DENV-2. We postulated that Pgg-ww binds to domain II/III region of DENV envelope protein which hinder the structure rearrangement of E protein leading to viral entry blockage. Pgg-ww may represent a new therapeutic candidate for the treatment of DENV infections and is a potential candidate to be developed as a peptide drug.