Junin virus DC-SIGN- mediated entry

FORLENZA, M. B.; ROLDAN, J. S.; MARTINEZ, M. G.; CORDO, S. M.; CANDURRA, N. A.

Philadelphia

Congreso; ASCB/IFCB; 2014

Virus entry and disemination can be mediated by calcium dependant c-type lectines as DC-Specific ICAM-3 grabbing non-integrin (DC-SIGN), which is a member of a family of receptor that recognizes carbohidrates structures. In previous studies we provided evidence that hDC-SIGN recognizes and internalizes the New World arenavirus Junín (JUNV), etiologic agent of hemorragic argentinian fever, which presents highly glycosylated proteins in its envelope. In the present study we provide evidence of the interaction between hDC-SIGN and JUNV, and characterize the early stages of hDC-SIGN-mediated entry. NHI3T3 and NIH3T3-hDC-SIGN cell were infected in presence or absence of EGTA or the carbohydrate bynding agent Griffithsin (GRFT). Both compounds inhibited infection in cultures that express the human lectin. To better understand how the lectins are involved in virus internalization, NIH3T3 cells were transfected with expression plasmids encoding hDS-SIGN wild type or mutants of the conserved cytoplasmatic motifs: dileucine (LL), and triacid cluster (EEE) which are believed to be involved in the regulation of ligands uptake, and trafficking respectively. Our results show that mutations in the cytosolic domain of DC-SIGN impair its ability to enhance JUNV infection. We used compounds that specifically blocked different entry pathways to determine if the virus endocytic route is affected when lectins are expressed in the cell surface. Our results show that virus infection is inhibited with dynasore (DYN), which inhibits the dynamin II-dependent endocytosis, Chlorpromazine (CZ), that inhibit the clathrin- dependent endocytosis and methyl-B-cyclodextrin (MBCD) which inhibit the cholesterol-dependent endocytosis. Using plasmids codifying proteins involved in the endocytic pathway, or its dominant negative forms, we determine cellular proteins and compartments involved in virus entry in the presence of hDC-SIGN. Our studies show that JUNV infection depends on EPS15 and Dynamin-2, both involved in clatrhin-mediated endocytosis. As many entry pathways are dependant of cell cytoskeleton, we studied the rol of microfilaments and microtubules during early virus infection.Inhibition of infection was observed when cells were treated with LatrunculinA which disrupts cortical and cytoplasmic microfilament whereas cytochalasin D, that affects cytoplasmic microfilaments did not inhibit JUNV multiplication. In contrast, drugs that depolymerize microtubules did not show to affect JUNV multiplication. Taken togheter our results demonstrate that JUNV interacts with hDC-SIGN and that JUNV internalization is dependent on clathrin-mediated endocytosis. In addition JUNV infection in the presence of the human lectin depends on cholesterol and intact cortical actin filaments.