Differential Disruption of Nucleocytoplasmic Trafficking Pathways by Rhinovirus 2A Proteases

Abstract

The RNA rhinoviruses (RV) encode 2A proteases (2A pro ) 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 2A pro sequences 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 2A pro predict 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 ␣/␤, transportin 1, and transportin 3 import pathways and the Crm1-mediated export pathway. Live-cell imaging of single cells expressing recombinant RV 2A pro (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-B (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 2A pro activities explain, in part, strain-dependent host responses and diverse RV disease phenotypes.IMPORTANCE Genetic variation among human rhinovirus types includes unexpected diversity in the genes encoding viral proteases (2A pro ) that help these viruses achieve antihost responses. When the enzyme activities of 7 different 2A pro were 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.KEYWORDS 2A, live-cell imaging, nuclear export, nuclear localization, nuclear trafficking, protease, rhinovirus T he controlled trafficking of protein and RNA across the nuclear membrane is essential for many eukaryotic cellular processes. Nuclear pore complexes (NPC) are large proteinaceous channels embedded in the nuclear envelope that restrict and

Three cardiovirus Leader proteins equivalently inhibit four different nucleocytoplasmic trafficking pathways

“…Averaged pixel scans centered over the width of individual nuclei showed that the mCherry signals, compared to steady-state DAPI, diminished measurably after infection of cells, if the reporter was linked to the SV40 NLS, the M9 NLS (26), or the RS domain NLS from an SR protein, splicing factor 2 (27). Previous characterization of these cells with rhinovirus reagents confirmed the cell-wide stability of the total mCherry signal (22). The reporter signal redistributes out of nuclei during infection, but is not degraded.…”

“…The impact of L X on other characterized transport pathways was assessed with HeLa cell lines transduced with mCherry reporter genes (~30 kD) linked to additional NLS/NES segments (~15-45 aa; (22)). After infection with vEC 9 (3 hr), the cells and controls were fixed, stained with DAPI and imaged (Fig 2).…”

“…SV40) with non-specific nuclear import localization signals (NLS), or tried to follow common cellular mRNAs as the metric for nuclear egress (8). These previous experiments are less sensitive than the newer, path-specific assays recently described for the RV (22). Application of those new systems now provides clarification on the scope of L X disruption of trafficking pathways for the L E , L S and L T proteins, and as described here, show all 3 of these viruses act ubiquitously against 4 tested pathways, including a path dependent upon a nuclear export signal (NES) for protein egress.…”

“…The sequence differences in specific 2A pro which are characteristic of the multiple RV genotypes allow individual viruses to preferentially cleave selected cohorts of Nup substrate panels with different avidities and rates (21). Consequently, not all import/export pathways are equivalently disabled by every RV, allowing each 2A pro sequence to manifest as a strain-specific Nup degradation pattern (22). …”

The roles of the nuclear pore complex in cellular dysfunction, aging and disease

“…Different strains of the rhinovirus produce variants of the 2A proteases, which cleave Nup62 in distinct ways. Nup62 cleavage changes nucleocytoplasmic transport receptors depending on how the nucleoporin is cleaved, which partially explains some of the differences in disease phenotypes and host responses to the different rhinovirus strains [109]. The nuclear basket nucleoporin Nup153 is also degraded during picornavirus infections such as poliovirus and rhinovirus [105,106].…”