Abstract
The entry of DENV into the host cell appears to be a very complex process which has been started to be studied in detail. In this report, the route of functional intracellular trafficking after endocytic uptake of dengue virus serotype 1 (DENV-1) strain HW, DENV-2 strain NGC and DENV-2 strain 16681 into Vero cells was studied by using a susceptibility to ammonium chloride assay, dominant negative mutants of several members of the family of cellular Rab GTPases that participate in regulation of transport through endosome vesicles and immunofluorescence colocalization. Together, the results presented demonstrate that in spite of the different internalization route among viral serotypes in Vero cells and regardless of the viral strain, DENV particles are first transported to early endosomes in a Rab5-dependent manner. Then a Rab7-dependent pathway guides DENV-2 16681 to late endosomes, whereas a yet unknown sorting event controls the transport of DENV-2 NGC, and most probably DENV-1 HW, to the perinuclear recycling compartments where fusion membrane would take place releasing nucleocapsid into the cytoplasm. Besides the demonstration of a different intracellular trafficking for two DENV-2 strains that shared the initial clathrin-independent internalization route, these studies proved for the first time the involvement of the slow recycling pathway for DENV-2 productive infection.
Highlights
Dengue virus (DENV) is a member of the family Flaviviridae transmitted to humans by mosquitoes of the genus Aedes
We corroborated that the differential properties between dengue virus serotype 1 (DENV-1) and DENV-2 for endocytic pathway of entry into Vero cells was not privative of these two strains but it was preserved in other reference strains and recent Argentinian clinical isolates of both serotypes: DENV-1 infection was inhibited in the presence of chlorpromazine, a pharmacological inhibitor of clathrin-mediated endocytosis, whereas no effect of this compound was observed against DENV-2 infection, independently of the strain (Fig. 1A)
The lack of participation of the clathrin pathway in the infective entry of DENV-2 was assessed by the overexpression of a dominant negative mutant of the clathrin coat-associated protein Eps15, which interferes with clathrin-coated pit assembly at the plasma membrane without affecting clathrinindependent endocytic pathways [17]
Summary
Dengue virus (DENV) is a member of the family Flaviviridae transmitted to humans by mosquitoes of the genus Aedes. In Vero cells a differential route of entry was demonstrated according to virus serotype: DENV-1 enters by clathrin-mediated endocytosis whereas DENV-2 is internalized through a non classical clathrin- and caveolin- independent process [9]. This variable behavior among DENV serotypes for entry into Vero cells was of particular interest considering that Vero cells are widely used to make vaccines, including flavivirus vaccines, and represent the usual system to test antiviral candidates against DENV. Differences were reported in the intracellular location of viral fusion for different strains of DENV-2: Krishnan et al [5] reported that DENV-2 strain NGC virions fused predominantly from early endosomes in HeLa cells, but other studies demonstrated fusion from late endosomes for DENV-2 strains 16681 and PR159 S1 in C6/36 HT and BSC1 cells, respectively [8,13]
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