Characterization of dengue virus type 3 entry in Vero and A549 cells

PICCINI, L.E.; CASTILLA, V.; DAMONTE, E.B.

Buenos Aires

Workshop; 3rd. ICGEB Workshop on Human RNA Viruses; 2012

Dengue Virus (DENV) is an arthropod-borne virus of the family Flaviviridae that can cause in the man a feverish acute benign disease and self-limited, the dengue fever, or the more severe and potentially lethal forms of dengue hemorrhagic fever/dengue shock syndrome. The knowledge of the viral multiplication cycle is indispensable to the finding of potential target for antiviral agents. In this field, the initial steps of the viral cycle, which determines the viral entry to the host cell, are an interesting alternative as new antiviral strategy. The recent studies about DENV mechanisms of entry shows little and contradictory information. The general objective of this project is the study of dengue virus entry to facilitate the finding of new molecules that has therapeutic potential directed the treatment and prevention of the infection. For it the following partial aims appear: a) Study of the mechanism of entry of DENV-3 to the host cell: It will be analyzed the viral and cellular factors involved in the internalization and fusion and their influence on the above mentioned processes. The characterization of the internalization processes in Vero cells, A549 and mosquito will be done using pharmacological inhibitors and dominant negative mutants of cells proteins implied in the different endocytic pathways. The influence of the viral strains, the kind of cell where the virus was propagated and the polarization state of those will be also evaluated. b) Characterization of the antiviral activity of compounds of natural origin that blocks the viral entry, to evaluate the implied of the entry on the chemotherapy direct to this antiviral target. c) Comparative analysis of the mechanisms and responses of DENV-3, DENV-1 and DENV-2, to assure the therapeutic effectiveness again all the serotypes present in the region. Recently, we have shown that possibly the entry of DENV-3 to Vero cells is by a non classical endocytic pathway. We have done biochemical and microscopic essays. Multiplication of DENV-3 is strongly reduce when Vero cells were pretreated with ammonium chloride, an inhibitor of endocytocis, but no inhibition occurs when we pretreat with chlorpromazine or dansylcadaverine, inhibitors of clathrin mediated endocytosis. Also we have seen that dynasore inhibits the entry of the virus, therefore, DENV-3 requires dynamin to infect Vero cells. Then, DENV-3 apparently enters to Vero cell by clathrin - independent endocytosis route, but dependent on dynamin. More studies are needed to determine if DENV-3 uses alternative pathways, because a slightly inhibition was seen when cells were pretreat with nistatine, an inhibitor of caveolae mediated endocytosis.