Junín virus DC-SIGN-mediated entry

FORLENZA, MB; ROLDÁN, J; MARTINEZ MG, CORDO, SM; CANDURRA, N.

Philadelphia, Pennsylvania

Congreso; ASCB/IFCB meeting, Philadelphia, Pennsylvania, USA. December 6-10, 2014.; 2014

American Society for Cell Biology

Virus entry and disemination may be mediated by calcium dependant c-type lectines as DC-Specific ICAM-3 grabbing non- integrin (DC-SIGN), member of a family of receptor which recognizes carbohidrates structures present in patogens. In previuos work we provided evidence that hDC-SIGN recognizes and internalizes the New World arenavirus Junín (JUNV), etiologic agent of hemorragic argentinian fever, which presents highly glicosilated proteins in its envelope. In this work we aim to support more evidence of the specificity of the interaction and characterize the early stages of DC-SIGN-mediated entry. To show this, we infected NHI3T3 and NIH3T3-hDC-SIGN cultures in presence of EGTA and the carbohydrate bynding agent Griffithsin (GRFT). In both treatments we observed inhibition of multiplication only in cultures which express the human lectin. To analize the molecular components of the lectin involved in virus internalization, NIH3T3 cells were transfected with expression plasmids encoding wild type or mutants for conserved cytoplasmatic motifs of the lectine: dileucine (LL), and triacid cluster (EEE) which are believed to regular ligans uptake, and trafficking respectively. Our results showed that the presence of the LL and EEE mutations interfere with the enhancment of infection. In orden to gain knowledge of the way of entry used after the interaction virus- lectin, we used compounds that specifically blocked different entry pathways. Our results showed significated inhibition of infection with dynasore (DYN); which inhibits the dynamin II-dependent endocytosis, Chlorpromazine (CZ), that inhibit the clathin- dependent endocytosis and methyl-B-cyclodextrin (MBCD) which inhibit the colholesterol- dependent endocytosis. To determine the cellular molecules involved, cells were transitory transfected with plasmids encoding dominat negative proteins involved in endocitosis. Our results showed that JUNV infection depends on EPS15 and Dynamin-2, both involved in clatrhin mediated endocitosis. As many entry pathways are dependant of cell citoesqueleton, we studied the rol of microfilaments and microtubules during early virus infection. Only inhibition of infection was obtained during treatments with LatruculinA, a compound that disrupts cortical and citoplasmatic microfilament whereas Citochalasin D, that only affects citoplasmatic microfilaments did not show inhibitory effect on JUNV multiplication. In contrast, drugs that alters microtubules did not show to affect JUNV multiplication. Taken togheter our results demonstrates that JUNV specifically interacts with hDC-SIGN and that the prefential entry pathway involved is clathrin mediated endocitosis and it dependens of cholesterol and intact cortical actin filaments.