Dengue-3 virus (DENV-3) entry into the host cell: mechanism and antiviral target for heparan sulfate-mimicking agents

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

Belem

Congreso; IV Pan-American Dengue Research Network Meeting; 2014

Dengue virus (DENV) entry into the host cell represents an attractive antiviral strategy for chemotherapy to suppress the beginning of infection. However, it appears to be a very complex process regulated by several cell- and virus-dependent factors that may affect antiviral susceptibility. Here, we studied first the mode of entry of DENV-3 strain H87 into the host cell by analyzing the effect of pharmacological and molecular inhibitors of different endocytic pathways on viral infectivity and antigen expression. Together, the results obtained demonstrated that DENV-3 entered mosquito C6/36 cells by clathrin-mediated endocytosis whereas the entry into monkey Vero and human A549 cells occurred via a clathrin-independent, dynamin and acid pH-mediated route with a partial involvement of caveolae. The kinetics of viral entry, analyzed by using a resistance to ammonium chloride assay, indicated that virion penetration started at 5 min post-binding and the time for viral escape from endosomes was about 15 min. The antiviral susceptibility of DENV-3 to entry inhibitors was next analyzed in both Vero and A549 cells. Diverse classes of sulfated polysaccharides (SP), compounds mimicking the cellular heparan sulfate residues, exhibited a potent and selective antiviral activity against DENV-3. Carrageenans were the most active inhibitors with effective concentration 50% (EC50) values around 1 µg/ml  and selectivity indices (ratio cytotoxicity/antiviral activity) higher than 1000. The target of these compounds was DENV-3 entry by dual blockade of virus adsorption and internalization of the nucleocapsid from endosomes. The comparison with our previous studies on Vero cell entry with DENV-1 (clathrin-mediated endocytosis and resistance to SP) and DENV-2 (non classical clathrin- and caveola-independent pathway and susceptibility to SP) confirms that diverse viral and cellular factors affect DENV entry and should be considered for evaluation of safe antiviral agents reactive against all DENV serotypes.