(703e) Adherens Junctions Formation Prevents Lentiviral Entry

Adherens junctions (AJs) in epithelial cells involve the inter-cellular interactions and are well known to play a role in number of processes such as proliferation, polarity, differentiation, inflammation etc. Here we explore the role of AJs in mediating lentivirus entry into epithelial cells by controlling junctional stability using calcium (Ca2+) switch, shRNA knockdown or dominant negative overexpression of AJ complex proteins. Epithelial cells form colonies with mature AJs in presence of high Ca2+ (1~1.8mM) and grow as single cells under low Ca2+ conditions (0.06 mM). AJ formation upon increasing Ca2+ concentrations resulted in significant reduction of gene transfer in these cells. Interestingly, cell colonies growing in high Ca2+ conditions exhibited higher transduction preferentially towards the periphery which can be attributed to AJ-free edges of peripheral cells. By shRNA knockdown of junction complex protein α-catenin and overexpression of dominant negative mutant E-Cadherin we were able to prevent AJs formation. Owing to loss of AJs, lentiviral entry lost dependence on Ca2+ and transduction efficiency remained unchanged at varying Ca2+ concentrations. With a tet-ON E-Cadherin dominant negative expression system we weakened AJs in a doxycycline dose dependent manner even in the presence of high Ca2+ concentration. Once again weaker AJ led to higher levels of virus entry and transduction efficiency. Interestingly, we also noticed rapid movement of cells upon lentivirus exposure, eventually weakening AJs and cells spreading out. This suggests a role of actin cytoskeleton in mediating this response to virus entry. To confirm this we disrupted cellular stress fibers using the ROCK inhibitor Y27632 and thereby weakened AJs. Gene delivery was significantly increased upon Y27632 treatment and showed dose dependence. With the use of shRNA and dominant negative forms of ROCK we were able to further delineate the role of ROCK in AJ controlled infection. Our work strongly suggests that AJ formation prevents lentiviral entry and disruption of AJs significantly enhances gene transfer to epithelial cells.