Three cardiovirus Leader proteins equivalently inhibit four different nucleocytoplasmic trafficking pathways

Ciomperlik, Jessica J.; Basta, Holly A.; Palmenberg, Ann C.

doi:10.1016/j.virol.2015.06.004

Cited by 22 publications

(28 citation statements)

References 47 publications

(97 reference statements)

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“…For the Crm1 export pathway, the C15 and B04 proteases were the most efficient, and again, the relative rates were dependent on the selection of 2A pro , with the highest (C15) exceeding the lowest (B52) by nearly 4-fold. Interestingly, in reported nonquantitative immunofluorescence (IF) assays with poliovirus 2A pro , it was suggested that this particular enzyme targeted importin ␣/␤ and transportin 1 import pathways, but not Crm1 export (9,39). Our data found all RV enzymes readily impacted the export pathway, albeit some did it more slowly and with a lower apparent priority.…”

Section: Discussionmentioning

confidence: 59%

Differential Disruption of Nucleocytoplasmic Trafficking Pathways by Rhinovirus 2A Proteases

Watters

İnankur

Gardiner

et al. 2017

J Virol

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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

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Section: Discussionmentioning

confidence: 59%

Differential Disruption of Nucleocytoplasmic Trafficking Pathways by Rhinovirus 2A Proteases

Watters

İnankur

Gardiner

et al. 2017

J Virol

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“…The possible interaction between L protein (or its interacting cellular partners) and nucleoporins is supported by a recently published report that L protein of SAFV induces Phe/Gly-containing nucleoporins to undergo phosphorylation. 10 The phosphorylation of nucleoporins by L protein requires the phosphorylation of L protein in advance. 25 Furthermore, our study demonstrates that the phosphorylation of Thr 58 in the Ser/Thr-rich domain of L protein has a critical role in the cellular distribution of SAFV L in HEp-2 cells, which also suggests the possible role of L protein in the phosphorylation of nucleoporins.…”

Section: Discussionmentioning

confidence: 99%

“…8 The L protein of SAFV and Theiler's murine encephalitis virus (TMEV)—the phylogenetically closest cardiovirus relative of SAFV—share 62% similarity in sequence. Currently, the associated functions of L protein of SAFV include interfering with the transcription of immediate-early alpha/beta interferon, 9 inducing the phosphorylation of Phe/Gly-containing nucleoporins 10 and inhibiting stress granule assembly. 11 …”

Section: Introductionmentioning

confidence: 99%

Intracellular localization of Saffold virus Leader (L) protein differs in Vero and HEp-2 cells

Victorio

et al. 2016

Emerging Microbes & Infections

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The Saffold virus (SAFV) genome is translated as a single long polyprotein precursor and co-translationally cleaved to yield 12 separate viral proteins. Little is known about the activities of SAFV proteins although their homologs in other picornaviruses have already been described. To further support research on functions and activities of respective viral proteins, we investigated the spatio-temporal distribution of SAFV proteins in Vero and HEp-2 cells that had been either transfected with plasmids that express individual viral proteins or infected with live SAFV. Our results revealed that, with the exception of the Leader (L) protein, all viral proteins were localized in the cytoplasm at all the time points assayed. The L protein was found in the cytoplasm at an early time point but was subsequently translocated to the nucleus of HEp-2, but not Vero, cells. This was observed in both transfected and infected cells. Further mutational analysis of L protein revealed that Threonine 58 of the Ser/Thr-rich domain of L protein is crucial for protein trafficking between the cytoplasm and nucleus in HEp-2 cells. These findings contribute to a deeper understanding and stimulate investigation of the differetial cellular responses of HEp-2 cells in comparison to other mammalian cell lines during SAFV infection.

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“…Plasmids encoding GST- L E , GST-L E ΔA, GST-L E W 40 A, and related derivatives are described (7, 8, 12), as are analogous L X -GST proteins from Saffold 2 and Theiler’s virus (BeAn), L S -GST, L T -GST, L E -GST and L E -GST Thr 41 Ala/Tyr 47 Ala (5, 20). L E -GST 3D3A was made similarly by introducing point mutations to alter the 3 codons for Asp 48 , Asp 51 and Asp 52 to Ala. For recombinant proteins, these cDNAs were transformed into BL21 E. coli.…”

Section: Methodsmentioning

confidence: 99%

Cardiovirus Leader proteins bind exportins: Implications for virus replication and nucleocytoplasmic trafficking inhibition

2016

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Cardiovirus Leader proteins (LX) inhibit cellular nucleocytoplasmic trafficking by directing host kinases to phosphorylate Phe/Gly-containing nuclear pore proteins (Nups). Resolution of the Mengovirus LM structure bound to Ran GTPase, suggested this complex would further recruit specific exportins (karyopherins), which in turn mediate kinase selection. Pull-down experiments and recombinant complex reconstitution now confirm that Crm1 and CAS exportins form stable dimeric complexes with encephalomyocarditis virus LE, and also larger complexes with LE:Ran. shRNA knockdown studies support this idea. Similar activities could be demonstrated for recombinant LS and LT from Theiloviruses. When mutations were introduced to alter the LE zinc finger domain, acidic domain, or dual phosphorylation sites, there was reduced exportin selection. These regions are not involved in Ran interactions, so the Ran and Crm1 binding sites on LE must be non-overlapping. The involvement of exportins in this mechanism is important to viral replication and the observation of trafficking inhibition by LE.

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Three cardiovirus Leader proteins equivalently inhibit four different nucleocytoplasmic trafficking pathways

Cited by 22 publications

References 47 publications

Differential Disruption of Nucleocytoplasmic Trafficking Pathways by Rhinovirus 2A Proteases

Differential Disruption of Nucleocytoplasmic Trafficking Pathways by Rhinovirus 2A Proteases

Intracellular localization of Saffold virus Leader (L) protein differs in Vero and HEp-2 cells

Cardiovirus Leader proteins bind exportins: Implications for virus replication and nucleocytoplasmic trafficking inhibition

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