Rhinovirus 16 2A protease affects nuclear localization of 3CD during infection

The human rhinovirus (HRV) 3C and 2A proteases (3C^pro and 2A^pro, respectively) are critical in HRV infection, as they are required for viral polyprotein processing as well as proteolysing key host factors to facilitate virus replication. Early in infection, 3C^pro is present as its precursor 3CD, which, although the mechanism of subcellular targeting is unknown, is found in the nucleus as well as the cytoplasm. In this study, we use transfected and infected cell systems to show that 2A^pro activity is required for 3CD nuclear localization. Using green fluorescent protein (GFP)-tagged forms of 3C^pro, 3D, and mutant derivatives thereof, we show that 3C^pro is located in the cytoplasm and the nucleus, whereas 3CD and 3D are localized predominantly in the cytoplasm, implying that 3D lacks nuclear targeting ability and that 3C^pro activity within 3CD is not sufficient to allow the larger protein into the nucleus. Importantly, by coexpressing mCherry-2A^pro fusion proteins, we demonstrate formally that 2A^pro activity is required to allow HRV 3CD access to the nucleus. In contrast, mCherry-3C^pro is insufficient to allow 3CD access to the nucleus. Finally, we confirm the relevance of these results to HRV infection by demonstrating that nuclear localization of 3CD correlates with 2A^pro activity and not 3C^pro activity, which is observed only later in infection. The results thus define the temporal activities of 2A^pro and 3CD/3C^pro activities in HRV serotype16 infection.