Comparative Analysis of Rhinovirus 2A Protease Cleavage of the Nuclear Pore Complex

Several active nucleocytoplasmic transport pathways are disrupted in picornavirus infected cells. Recent studies have shown that rhinoviruses (RV) use encoded 2A protease, 2Apro, to cleave nucleoporins (Nups) in nuclear pore complexes (NPC) to inhibit nucleocytoplasmic transport pathways. Although they share similar structures and carry out the same functions during infection, the 2Apro sequences are highly diverse among different RV clades, which suggests that the proteases might have different enzymatic activities and carryout their anti-host functions with unique mechanistic signatures. The avidity and specificity with which different RV 2As cleave Nups and their ability to disrupt nuclear transport pathways could alter antiviral signaling and affect virus replication levels, ultimately triggering different disease phenotypes and host immune responses. In this thesis, we sought to compare the proteolytic activities of different RV 2Apros in NPCs.;In vitro assays with recombinant proteases showed that different RV 2A pros have different Nup substrate specificities and target and cleave Nup62, Nup98, and Nup153 at different relative rates. Relative Nup degradation kinetics in infected cells between different RVs (A16 and B14) were similar to the relative Nup degradation kinetics we observed in the in vitro assays between corresponding 2Apros. Development of a cellular time-lapse fluorescent assay of nuclear import and export allowed for the comparison of nucleocytoplasmic transport disruption efficiencies of different RV 2A pros. RV 2Apros expressed alone in cells inhibited importin alpha/beta, transportin-1, transportin-3, and Crm1 nuclear transport pathways. However, the proteases inhibited these nuclear transport pathways at different rates. The RV-B proteases, especially B52 2Apro, disrupted nuclear import more efficiently than RV-A or RV-C proteases. Comparison of results from 2Apro nucleocytoplasmic transport inhibition experiments and results of clinical studies on RV illness severities revealed an inverse correlation between RV 2Apro nuclear import disruption efficiency and RV illness severity, which suggested that RVs with 2A pros that more efficiently disrupt nuclear import pathways are less virulent.;Collectively, the results presented in this thesis show that different RV 2Apros differentially disrupt nucleocytoplasmic trafficking pathways by differentially targeting and cleaving nucleoporins. Additionally, correlations between results of 2Apro nucleocytoplasmic disruption experiments and results of clinical studies suggest that 2A proteolytic activity in NPCs affects RV virulence.