Domain-III FG loop of the dengue virus type 2 envelope protein is important for infection of mammalian cells and Aedes aegypti mosquitoes

Abstract

The FG extended loop in domain III of the dengue virus type 2 (DENV2) envelope protein is postulated to be a molecular determinant for host cell infectivity. To determine the contribution of the FG loop to virus infectivity, an infectious cDNA clone of DENV2 was manipulated by deleting amino acids in the loop (VEPGΔ) to mimic tick-borne flaviviruses or by substituting these AAs with RGD or RGDK/S to mimic motifs present in other mosquito-borne flaviviruses. We found the FG loop to be dispensable for infection of C6/36 cells but critical for infection of Aedes aegypti mosquito midguts and mammalian cells. All the FG loop mutants were able to bind to and enter mammalian cells but replication of VEPGΔ in Vero cells at 37 °C was delayed until acquisition of secondary mutations. Reduced binding of DENV2 type-specific monoclonal antibody 3H5 to mutant viruses confirmed the FG loop motif as its target epitope.

Secretion of dengue virus envelope protein ectodomain from mammalian cells is dependent on domain II serotype and affects the immune response upon DNA vaccination

“…Additionally, DII provides the surface where the main interactions for E dimerization occur (Modis et al, 2005;Rey et al, 1995). The C-terminal DIII domain has an Ig-like b-barrel structure with a hydrophobic inner surface in a pocket that accommodates the fusion loop of the opposing monomer (Allison et al, 2001;Erb et al, 2010). DIII is also believed to contain the receptor-binding sites to the host cell (Erb et al, 2010;Mukhopadhyay et al, 2005) and has been implicated in determining host range, tropism and virulence (Lindenbach et al, 2007).…”

“…The C-terminal DIII domain has an Ig-like b-barrel structure with a hydrophobic inner surface in a pocket that accommodates the fusion loop of the opposing monomer (Allison et al, 2001;Erb et al, 2010). DIII is also believed to contain the receptor-binding sites to the host cell (Erb et al, 2010;Mukhopadhyay et al, 2005) and has been implicated in determining host range, tropism and virulence (Lindenbach et al, 2007). A hinge region formed by the four strands that span between the different DI and DII coding segments provides the flexibility for E conformational changes during virus maturation (Butrapet et al, 2011;Monath et al, 2002), while a linker of 11 aa connects DI to DIII and is fundamental for proper E folding (de Wispelaere & Yang, 2012).…”

Recombinant Dengue Type 2 Viruses with Altered E Protein Domain III Epitopes Are Efficiently Neutralized by Human Immune Sera

“…1C and D, respectively. The recombinant DENV2 (rDENV2) cDNAs were made by site-directed mutagenesis using the parental DENV2 clone, pD2/ IC-30P-NBX, which was originally developed using DENV2 strain 16681, as previously described (7,15). The rDENV2 viruses were derived by transfection of in vitro-transcribed RNA into the C6/36 Aedes albopictus cell line at 28°C, and viral RNAs extracted from resulting viruses were sequence analyzed to verify that the genomes contained the engineered substitutions without other unexpected mutations (15).…”

Structure-Based Mutational Analysis of Several Sites in the E Protein: Implications for Understanding the Entry Mechanism of Japanese Encephalitis Virus

“…Mutational analysis of the E protein of flavivirus has been used to investigate the role of E protein in virus assembly, release, or entry (35,38,(51)(52)(53). In this study, utilizing mutagenesis of an infectious cDNA clone of JEV, we introduced mutations into the receptor-binding motif or the amino acids critical for membrane fusion to systematically study and reveal the JEV entry mechanism.…”