Differential Disruption of Nucleocytoplasmic Trafficking Pathways by Rhinovirus 2A Proteases.
The RNA rhinoviruses (RV) encode 2A proteases (2Apro) that contribute essential polyprotein processing and host cell shutoff functions during infection, including the cleavage of Phe/Gly-containing nucleoporin proteins (Nups) within nuclear pore complexes (NPC). Within the 3 RV species, multiple divergent genotypes encode diverse 2Aprosequences that act differentially on specific Nups. Since only subsets of Phe/Gly motifs, particularly those within Nup62, Nup98, and Nup153, are recognized by transport receptors (karyopherins) when trafficking large molecular cargos through the NPC, the processing preferences of individual 2Apropredict RV genotype-specific targeting of NPC pathways and cargos. To test this idea, transformed HeLa cell lines were created with fluorescent cargos (mCherry) for the importin α/β, transportin 1, and transportin 3 import pathways and the Crm1-mediated export pathway. Live-cell imaging of single cells expressing recombinant RV 2Apro(A16, A45, B04, B14, B52, C02, and C15) showed disruption of each pathway with measurably different efficiencies and reaction rates. The B04 and B52 proteases preferentially targeted Nups in the import pathways, while B04 and C15 proteases were more effective against the export pathway. Virus-type-specific trends were also observed during infection of cells with A16, B04, B14, and B52 viruses or their chimeras, as measured by NF-κB (p65/Rel) translocation into the nucleus and the rates of virus-associated cytopathic effects. This study provides new tools for evaluating the host cell response to RV infections in real time and suggests that differential 2Aproactivities explain, in part, strain-dependent host responses and diverse RV disease phenotypes.IMPORTANCEGenetic variation among human rhinovirus types includes unexpected diversity in the genes encoding viral proteases (2Apro) that help these viruses achieve antihost responses. When the enzyme activities of 7 different 2Aprowere measured comparatively in transformed cells programed with fluorescent reporter systems and by quantitative cell imaging, the cellular substrates, particularly in the nuclear pore complex, used by these proteases were indeed attacked at different rates and with different affinities. The importance of this finding is that it provides a mechanistic explanation for how different types (strains) of rhinoviruses may elicit different cell responses that directly or indirectly lead to distinct disease phenotypes.
Institute for Molecular Virology, University of Wisconsin-Madison, Madison, Wisconsin, USA.
Kelly
K
Watters
Wisconsin Institutes for Discovery and Department of Chemical and Biological Engineering, University of Wisconsin-Madison, Madison, Wisconsin, USA.
Bahar
B
Inankur
Institute for Molecular Virology, University of Wisconsin-Madison, Madison, Wisconsin, USA.|McArdle Laboratories for Cancer Research, University of Wisconsin-Madison, Madison, Wisconsin, USA.
Jaye C
JC
Gardiner
Wisconsin Institutes for Medical Research and Department of Biomedical Engineering, University of Wisconsin-Madison, Madison, Wisconsin, USA.
Jay
J
Warrick
Institute for Molecular Virology, University of Wisconsin-Madison, Madison, Wisconsin, USA.|McArdle Laboratories for Cancer Research, University of Wisconsin-Madison, Madison, Wisconsin, USA.
Nathan M
NM
Sherer
Wisconsin Institutes for Discovery and Department of Chemical and Biological Engineering, University of Wisconsin-Madison, Madison, Wisconsin, USA.
John
J
Yin
Institute for Molecular Virology, University of Wisconsin-Madison, Madison, Wisconsin, USA acpalmen@wisc.edu.|Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin, USA.
Ann C
AC
Palmenberg
D003546:Cysteine Endopeptidases / Q000378:metabolism*; D006367:HeLa Cells; D054884:Host-Pathogen Interactions; D006801:Humans; D008856:Microscopy, Fluorescence; D028861:Nuclear Pore Complex Proteins / Q000378:metabolism*; D021381:Protein Transport; D012229:Rhinovirus / Q000201:enzymology* / Q000472:pathogenicity*; D014764:Viral Proteins / Q000378:metabolism*
D028861:Nuclear Pore Complex Proteins; D014764:Viral Proteins; D003546:Cysteine Endopeptidases; C057400:picornain 2A, Picornavirus
2A; live-cell imaging; nuclear export; nuclear localization; nuclear trafficking; protease; rhinovirus