The C-terminal 50 amino acid residues of Dengue NS3 protein are important for NS3-NS5 interaction and viral replication — ASN Events
  1. Schedule
  2. Session Five - Poster Session A including Refreshments
  3. The C-terminal 50 amino acid residues of Dengue NS3 protein are important for NS3-NS5 interaction and viral replication

The C-terminal 50 amino acid residues of Dengue NS3 protein are important for NS3-NS5 interaction and viral replication (#170)

Moon Y.F Tay 1 , Wuan Geok Saw 2 , Yongqian Zhao 1 3 , Kitti W.K. Chan 1 , Daljit Singh 1 , Yuwen Chong 1 , Jade K. Forwood 4 , Eng Eong Ooi 1 , Gerhard Grüber 2 , Julien Lescar 5 , Dahai Luo 6 , Subhash G. Vasudevan 1 3
  1. Program in Emerging Infectious Diseases, Duke-NUS Graduate Medical School, Singapore
  2. School of Biological Sciences, Nanyang Technological University, Singapore
  3. NUS Graduate School for Integrative Sciences and Engineering, National University of Singapore, Singapore
  4. School of Biomedical Sciences, Charles Sturt University, Wagga Wagga, NSW, Australia
  5. Division of Structural Biology and Biochemistry, School of Biological Sciences, , Nanyang Technological University, Singapore
  6. Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore

Background: Dengue virus (DENV), a re-emerging mosquito-borne flavivirus that exists as four closely related serotypes yet antigenically distinct, continues to be a threat to >40% of the world’s population. Despite tremendous efforts, no clinically approved drug or licensed vaccine is available to combat dengue disease.

Objective: The interaction between the two major replicative enzymes, DENV nonstructural protein 3 and 5 (NS3 and NS5) has been proposed to be an attractive target for antiviral drug development. In order to facilitate the target validation and druggability, this study investigated the details of the molecular interaction between NS3 and NS5.

Methods: Peptide-phage display, surface plasmon reasonance (SPR), competitive NS3-NS5 interaction enzyme-linked immunosorbant assay (ELISA) and small-angle X-ray scattering (SAXS) were used to narrow down the region involved in NS3-NS5 interaction. The importance of the interaction interface was examined by site directed mutagenesis, in vitro enzyme activity assay and reverse genetics with DENV2 cDNA clone. A strand-specific real-time quantitative reverse transcription-PCR (qRT-PCR) was developed to examine the dynamics of positive- and negative-strand RNA synthesis in recombinant viruses carrying mutations that disrupted NS3-NS5 interaction. Synthetic peptide inhibitors were designed to target the interaction interface.

Results: NS3 residues 566-585 was identified to be involved in NS3-NS5 interaction and within this region, a highly conserved amino acid, N570 was found to be critical in the interaction; mutation of NS3 N570 to alanine reduced NS3-NS5 interaction but did not affect its in vitro ATPase activity. Through reverse genetics, comparison of NS3:N570A mutant with a published NS3-NS5 interaction defective mutant, NS5:K330A revealed that the NS3-NS5 interaction was indeed important for replication. However, unlike NS5:K330A mutant which was completely replication defective, NS3:N570A mutant transfected cells stained positive for dsRNA and also the viral proteins NS3 and NS5. Strand-specific qRT-PCR studies indicate defective RNA replication in NS3:N570A mutant could be attributed to its reduced positive-strand RNA synthesis.

Conclusion: NS3-NS5 interaction may be required for coordinated positive- and negative-strand RNA synthesis; different NS3-NS5 interaction defective mutants can impair viral replication to a varying extent, which is likely to be dependent on the importance of the amino acids that are involved in the interaction.