Release of canine parvovirus from endocytic vesicles

AbstractCanine parvovirus (CPV) is a small nonenveloped virus with a single-stranded DNA genome. CPV enters cells by clathrin-mediated endocytosis and requires an acidic endosomal step for productive infection. Virion contains a potential nuclear localization signal as well as a phospholipase A2 like domain in N-terminus of VP1. In this study we characterized the role of PLA2 activity on CPV entry process. PLA2 activity of CPV capsids was triggered in vitro by heat or acidic pH. PLA2 inhibitors inhibited the viral proliferation suggesting that PLA2 activity is needed for productive infection. The N-terminus of VP1 was exposed during the entry, suggesting that PLA2 activity might have a role during endocytic entry. The presence of drugs modifying endocytosis (amiloride, bafilomycin A1, brefeldin A, and monensin) caused viral proteins to remain in endosomal/lysosomal vesicles, even though the drugs were not able to inhibit the exposure of VP1 N-terminal end. These results indicate that the exposure of N-terminus of VP1 alone is not sufficient to allow CPV to proliferate. Some other pH-dependent changes are needed for productive infection. In addition to blocking endocytic entry, amiloride was able to block some postendocytic steps. The ability of CPV to permeabilize endosomal membranes was demonstrated by feeding cells with differently sized rhodamine-conjugated dextrans together with the CPV in the presence or in the absence of amiloride, bafilomycin A1, brefeldin A, or monensin. Dextran with a molecular weight of 3000 was released from vesicles after 8 h of infection, while dextran with a molecular weight of 10,000 was mainly retained in vesicles. The results suggest that CPV infection does not cause disruption of endosomal vesicles. However, the permeability of endosomal membranes apparently changes during CPV infection, probably due to the PLA2 activity of the virus. These results suggest that parvoviral PLA2 activity is essential for productive infection and presumably utilized in membrane penetration process of the virus, but CPV also needs other pH-dependent changes or factors to be released to the cytoplasm from endocytic vesicles