Dengue Virus

To elucidate the functional roles of highly conserved residues in the stem region of the E protein in the replication cycle of DENV and other re-emerging flaviviruses (WNV and JEV), and to identify potential targets for the development of antiviral compounds against these medically important mosquito-borne viruses.

Specific Aim 1

Investigate the role of highly conserved residues in the stem region of the E protein on the entry and assembly of DENV serotype 4 (DENV4).

• Hypothesis: Highly conserved residues in the stem are involved in entry and assembly.
• Rationale: Mutational studies of TBEV E protein suggest the stem is involved in the assembly of VLPs. Peptide derived from the stem can block entry of DENV. Highly conserved residues at the stem are likely to be involved in such processes.
• Experimental Plan: Site-directed mutagenesis will be carried out to replace the highly conserved stem residues in DENV4 prM/E expressing construct. Assembly will be assessed by examining VLPs production, and incorporation into VLPs containing replicon (VLPs-R). Entry will be examined by infectivity assays of VLPs-R and DENV4 infectious clone containing E mutations.

Specific Aim 2

Investigate the mechanisms of impairment in entry and assembly of DENV4 E mutants.

• Hypothesis: Highly conserved residues in the stem are involved in the entry by affecting post-receptor binding step, or in the assembly by affecting prM-E heterodimerization or intracellular localization.
• Rationale: The plausible mechanisms involved will be examined.
• Experimental Plan: VLPs containing E mutants will be examined by binding to target cells and membrane fusion assay, and cells transfected with E mutants will be subjected to co-immunoprecipitation assay, subcellular fractionation assay, double-label immunofluorescence assay and digestion with endo H to examine intracellular localization.

Specific Aim 3

Investigate the role of highly conserved residues in the stem on the entry and assembly of other DENV serotypes and other flaviviruses, including WNV and JEV.

• Hypothesis: Highly conserved residues in the stem are also involved in the entry and assembly of other flaviviruses.
• Rationale: Since the residues identified in aim 1 are also conserved by other DENV serotypes and flaviviruses including WNV and JEV, they are likely to be involved in such processes.
• Experimental Plan: Site-directed mutagenesis will be carried out for the residues in the prM/E expressing constructs of other DENV serotypes, WNV and JEV. Entry and assembly will be examined.

Experimental Approach

Specific Aim 1: 

• Proline substitutions introduced to residues at the EH1 or EH2 domain greatly affect the production of VLPS.
• The overall conformation of E protein was not affected by any mutations
• It is significant to have a hydrophobic residue at position 398 to maintain the assembly function of VLPS
• EH1 and EH2 mutations greatly affect the assembly of replicon particles and release (Figure 5).
• Mutations in both EH1 and EH2 affect the assembly and release of virions (Figure 6). 
• A single substitution from proline to leucine at position 398 of E protein can restore viral replication of mutant I398P and at position 429 of E protein can restore viral replication of mutant F429P. 

Specific Aim 2 & 3:

• Proline substitutions introduced into the EH1 or EH2 domain result in an impairment with regard to the release of VLPs from the membrane to the lumen of the ER; thus, reducing the production of VLPs in the culture supernatants (Figure 3). 
• The residues introduced at the center of the EH1 or EH2 domain greatly affect the prM-E heterodimeric interaction (Figure 2).