Evading Innate and Adaptive Immunity During Adenovirus Cell Entry

Adenovirus (Ad), a non-enveloped, dsDNA virus, enters cells via clathrin-mediated endocytosis. For viral genome delivery to the nucleus, Ad must penetrate endosomal membranes to create defects sufficient for the passage of the 90 nm diameter capsid across cell membranes. Recent observations suggest that adenovirus type 5 (Ad5) capsid uncoating occurs at the cell surface upon binding to both the coxsackievirus and adenovirus receptor and αv integrins. This uncoating event leads to the exposure of the capsid membrane lytic protein VI. Using the cytosolic protein galectin-3 (gal3) as a marker of membrane rupture, we demonstrate that Ad5 membrane rupture occurs at or near the cell surface upon exposure of protein VI. However, translocation of the virus across ruptured membranes occurs more frequently from perinuclear endosomal locations. Additionally, while Ad5 rupture of cell membranes is unaffected by microtubule depolymerization, egress of Ad5 virions from ruptured endosomes is dependent upon microtubules. Ad pVI encodes a highly conserved PPxY motif, capable of recruiting Nedd4-family E3 ubiquitin ligases. Mutation of the pVI-PPxY motif (Ad-WT) to PGAA (Ad-M1) does not impair viral endosomal membrane rupture but results in reduced specific infectivity. We show that the PPxY motif mediates microtubule-dependent egress of Ad5 capsids from ruptured membranes. Further, the defect in Ad-M1 nuclear trafficking is due to targeting the virus to lysosomes via autophagy. Although both Ad-WT and Ad-M1 infection induce autophagosome formation, a greater percentage of these autophagosomes contain Ad-M1 compared to Ad-WT. Further, significantly more Ad-M1 virus is found in lysosomes than Ad-WT several hours after infection. Inhibiting autophagy using RNAi silencing of Atg5 restores Ad-M1 infectivity to Ad-WT levels. Additionally, RNAi silencing of galectin-8 (gal8), which is also recruited to sites of Ad5 membrane rupture, restored Ad5-M1 infectivity to Ad5-WT levels and decreased Ad5-M1 accumulation in autophagosomes and lysosomes. Further, activation of T cells to Ad5 capsid antigens increased with Ad5-M1 transduction compared to Ad5-WT. These data demonstrate that a conserved PPxY motif in Adenovirus pVI is important for Ad5 endosome escape to evade autophagy during entry