(200f) Quantitative Analysis of Human Rhinovirus 2A Protease Disruption of Nucleocytoplasmic Trafficking Pathways and Effects on Infection Spread

Infections by human rhinovirus (HRV) contribute to a diversity of human illnesses that include the common cold, bronchitis, exacerbation of asthma and other chronic lung diseases. Previous studies have shown that HRVs grouped under three different species (HRV-A, B and C) span over 150 different genotypes and vary in their virulence and the severity of their infections. In parallel with clinical studies, genome sequencing has revealed a high level of diversity in protein sequences of the HRV 2Aprotease, an enzyme involved in viral polyprotein processing and shut off of host cell functions that can inhibit viral replication. Different 2Aproteases target and cleave different nuclear pore proteins (Nups), and thereby disrupt nucleocytoplasmic signaling and transport pathways.  This suggests that the proteases might have different enzymatic activities and carry-out their anti-host functions with unique mechanistic signatures. Here we compared the ability of different HRV 2A proteases to disrupt nuclear transport pathways using a novel cellular time-lapse fluorescent transfection assay. We created stable HeLa cells expressing fluorescent proteins fused to four different nuclear localizations signals, and we transfected these host cells with RNAs encoding different HRV 2A proteases. We observed the nuclear reporter protein accumulation in the cytoplasm/nucleus in real-time, and we employed a custom-developed java-based graphical interface to extract and analyze the fluorescent intensities from individually identified cells.  This enabled us to quantify differences between HRV 2A proteases in their ability to disrupt nuclear transport pathways. We found that all proteases disrupted active import of a fluorescent reporter protein by the importin-α/β, transportin-1, and transportin-3 pathways as well as active export by the Crm1 pathway; however, different proteases inhibited these nuclear transport pathways with different efficiencies.  We extended these studies to probe the spatial and temporal dynamics of spreading HRV infections using a dual color reporter system.  Reporter HeLa cell monolayers were infected by fluorescent HRV strains where individual cells were tracked through the course of the infection; analysis of the spatial and temporal expression behaviors of individual cells was obtained for HRV strains expressing different 2A proteases, and these single-cell profiles were correlated with different spatial patterns of HRV infection spread. Overall, this work provides a foundation for linking how HRV diversity at the level of encoded 2A proteases may contribute to differential severity of HRV infections as they spread, which can guide the development of improved treatment strategies for HR-related illnesses.