Peptides targeting dengue viral nonstructural protein 1 inhibit dengue virus production

Pucharee Songprakhon,Chunya Puttikhunt,Prida Malasit,Sissades Thongsima,Chatchawan Srisawat,Prasit Luangaram,Thanawat Thaingtamtanha,Thawornchai Limjindaporn,Chairat Uthaipibull,Pa-Thai Yenchitsomanus,Sansanee Noisakran,Thanyaporn Dechtawewat

doi:10.1038/s41598-020-69515-9

Abstract

Viruses manipulate the life cycle in host cells via the use of viral properties and host machineries. Development of antiviral peptides against dengue virus (DENV) infection has previously been concentrated on blocking the actions of viral structural proteins and enzymes in virus entry and viral RNA processing in host cells. In this study, we proposed DENV NS1, which is a multifunctional non-structural protein indispensable for virus production, as a new target for inhibition of DENV infection by specific peptides. We performed biopanning assays using a phage-displayed peptide library and identified 11 different sequences of 12-mer peptides binding to DENV NS1. In silico analyses of peptide-protein interactions revealed 4 peptides most likely to bind to DENV NS1 at specific positions and their association was analysed by surface plasmon resonance. Treatment of Huh7 cells with these 4 peptides conjugated with N-terminal fluorescent tag and C-terminal cell penetrating tag at varying time-of-addition post-DENV infection could inhibit the production of DENV-2 in a time- and dose-dependent manner. The inhibitory effects of the peptides were also observed in other virus serotypes (DENV-1 and DENV-4), but not in DENV-3. These findings indicate the potential application of peptides targeting DENV NS1 as antiviral agents against DENV infection.

Highlights

Viruses manipulate the life cycle in host cells via the use of viral properties and host machineries
It colocalizes with doublestranded viral RNA 21 and interacts with NS4A-2K-NS4B precursor and dengue virus (DENV) structural proteins (C, prM and E) that are essential for viral RNA replication and production of infectious virions, respectively[19,20]
Eighty individual phage clones were randomly selected from biopanning round 3, and the binding activities of displayed peptides towards DENV NS1 were quantitatively confirmed by enzyme-linked immunosorbent assay (ELISA)

Summary

Introduction

Viruses manipulate the life cycle in host cells via the use of viral properties and host machineries. Development of antiviral peptides against dengue virus (DENV) infection has previously been concentrated on blocking the actions of viral structural proteins and enzymes in virus entry and viral RNA processing in host cells. We proposed DENV NS1, which is a multifunctional nonstructural protein indispensable for virus production, as a new target for inhibition of DENV infection by specific peptides. In DENV-infected cells, NS1 protein is required for the formation of virus-induced membrane structures as the sites of DENV replication 20. It colocalizes with doublestranded viral RNA 21 and interacts with NS4A-2K-NS4B precursor and DENV structural proteins (C, prM and E) that are essential for viral RNA replication and production of infectious virions, respectively[19,20]. The soluble form of NS1 contributes to enhancement of DENV production, immune evasion of DENV, induction of proinflammatory cytokine production, disruption of endothelial barrier, and vascular leakage[32,33,34,35,36,37,38,39,40,41]

Methods

Results

Conclusion