Dengue Virus (DENV), a flavivirus spread by mosquito vectors, can cause vascular leakage and hemorrhaging. However, the processes that underlie increased vascular permeability and pathological plasma leakage during viral hemorrhagic fevers are largely unknown. Mast cells (MCs) are activated in vivo during DENV infection, and we show that this elevates systemic levels of their vasoactive products, including chymase, and promotes vascular leakage. Treatment of infected animals with MC-stabilizing drugs or a leukotriene receptor antagonist restores vascular integrity during experimental DENV infection. Validation of these findings using human clinical samples revealed a direct correlation between MC activation and DENV disease severity. In humans, the MC-specific product, chymase, is a predictive biomarker distinguishing dengue fever (DF) and dengue hemorrhagic fever (DHF). Additionally, our findings reveal MCs as potential therapeutic targets to prevent DENV-induced vasculopathy, suggesting MC-stabilizing drugs should be evaluated for their effectiveness in improving disease outcomes during viral hemorrhagic fevers.DOI: http://dx.doi.org/10.7554/eLife.00481.001

The envelope glycoprotein located at the outermost surface of the flavivirus particle mediates entry of virus into host cells. In this study, the involvement of domain III of West Nile virus (WNV-DIII) envelope protein in binding to host cell surface was investigated. WNV-DIII was first expressed as a recombinant protein and purified after a solubilization and refolding procedure. The refolded WNV-DIII protein displays a content of b-sheets consistent with known homologous structures of other flavivirus envelope DIII, shown by using circular dichroism analysis. Purified recombinant WNV-DIII protein was able to inhibit WNV entry into Vero cells and C6/36 mosquito cells. Recombinant WNV-DIII only partially blocked the entry of dengue-2 (Den 2) virus into Vero cells. However, entry of Den 2 virus into C6/36 was blocked effectively by recombinant WNV-DIII. Murine polyclonal serum produced against recombinant WNV-DIII protein inhibited infection with WNV and to a much lesser extent with Den 2 virus, as demonstrated by plaque neutralization assays. Together these results provided strong evidence that immunoglobulin-like DIII of WNV envelope protein is responsible for binding to receptor on the surface of host cells. The data also suggest that similar attachment molecule(s) or receptor(s) were used by WNV and Den 2 virus for entry into C6/36 mosquito cells.

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The complete genome of West Nile (Sarafend) virus [WN(S)V] was sequenced. Phylogenetic trees utilizing the complete genomic sequence, capsid gene, envelope gene and NS5 gene/39 untranslated region of WN(S)V classified WN(S)V as a lineage II virus. A full-length infectious clone of WN(S)V with a point mutation in the glycosylation site of the envelope protein (pWNS-S154A) was constructed. Both growth kinetics and the mode of maturation were affected by this mutation. The titre of the pWNS-S154A virus was lower than the wild-type virus. This defect was corrected by the expression of wild-type envelope protein in trans. The pWNS-S154A virus matured intracellularly instead of at the plasma membrane as shown for the parental WN(S)V.

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SummaryThe Flaviviridae family consists of several medically important pathogens such as West Nile virus (WNV) and Dengue virus (DENV). Flavivirus capsid (C) protein is a key structural component of virus particles. However, the role of C protein in the pathogenesis of arthropod-borne flaviviruses is poorly understood. To examine whether flavivirus C protein can associate with cellular proteins, and contribute to viral pathogenesis, WNV/DENV C protein was screened against a human brain/liver cDNA yeast two-hybrid library. This study identified human Sec3 exocyst protein (hSec3p) as a novel interacting partner of WNV and DENV C protein. Mutagenesis studies showed that the SH2 domain-binding motif of hSec3p binds to the first 15 amino acids of C protein. We report for the first time that hSec3p can modulate virus production by affecting viral RNA transcription and translation through the sequestration of elongation factor 1a (EF1a). This molecular discovery shed light on the protective role of hSec3p during flavivirus infection. This study also highlighted the antagonistic mechanism adopted by flavivirus C protein that can negatively regulate the formation of hSec3p-EF1a complex by sequestering hSec3p to establish successful infection.

Flavivirus premembrane (prM) protein plays an important role in conformational folding of the envelope (E) protein and protects it against premature fusion in acidic vesicles of the Golgi network. Currently, molecular determinants on the prM protein ectodomain which mediate critical steps during the flavivirus assembly process are poorly characterized. In this study, bioinformatics analysis and alanine scanning mutagenesis showed that the amino acid triplet valine 76, tyrosine 78 and glycine 79 is absolutely conserved among flavivirus prM ectodomains. Triple mutations engineered at these residues in prM ectodomain of West Nile virus (WNV) completely abrogated virus infectivity. Site-directed mutagenesis of prM protein revealed that tyrosine 78 of the amino acid triplet was required for virus infectivity and secretion. The mutation did not affect folding, post-translational modifications and trafficking of the prM and E proteins. Ultrastructural studies using transmission electron microscopy confirmed that virus particle formation was blocked by tyrosine 78 mutation. Specificity of assembly defect conferred by tyrosine 78 mutation was demonstrated by positive and negative trans complementation studies. Collectively, these results defined tyrosine 78 as a novel critical determinant present on prM protein ectodomain that is required for flavivirus assembly. Molecular dissection of prM protein function provides the crucial knowledge much needed in the elucidation of flavivirus particle formation.