Mechanistic Consequences of hnRNP C Binding to Both RNA Termini of Poliovirus Negative-Strand RNA Intermediates

Ertel, Kenneth J.; Brunner, Jo Ellen; Semler, Bert L.

doi:10.1128/jvi.02198-09

Cited by 61 publications

(88 citation statements)

References 57 publications

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“…Among them, several members of the hnRNPs family have been previously shown to play a role in regulating viral replication. 7,8,39,[44][45][46][47] For examples, hnRNP C and K were documented to interact with the non-coding regions of enteroviral RNA to facilitate translation initiation and RNA synthesis. 44,45,47 Similar to our findings in the current study, enteroviral infection causes cytoplasmic redistribution and cleavage of hnRNP D and deletion of this protein results in enhanced viral replication, suggesting that hnRNA D acts as a host restriction factor against enterovirus infection.…”

Section: Discussionmentioning

confidence: 99%

“…7,8,39,[44][45][46][47] For examples, hnRNP C and K were documented to interact with the non-coding regions of enteroviral RNA to facilitate translation initiation and RNA synthesis. 44,45,47 Similar to our findings in the current study, enteroviral infection causes cytoplasmic redistribution and cleavage of hnRNP D and deletion of this protein results in enhanced viral replication, suggesting that hnRNA D acts as a host restriction factor against enterovirus infection. 7,8,39 The antiviral mechanisms of hnRNP D appear to involve negative regulation of internal ribosome entry site (IRES)-mediated translation of viral RNA via direct interaction with the IRES of enteroviruses, 7,46 and viral RNA degradation by targeting the 3′-untranslated region (UTR) of enteroviral genome.…”

Section: Discussionmentioning

confidence: 99%

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Cytoplasmic translocation, aggregation, and cleavage of TDP-43 by enteroviral proteases modulate viral pathogenesis

et al. 2015

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We have previously demonstrated that infection by coxsackievirus B3 (CVB3), a positive-stranded RNA enterovirus, results in the accumulation of insoluble ubiquitin-protein aggregates, which resembles the common feature of neurodegenerative diseases. The importance of protein aggregation in viral pathogenesis has been recognized; however, the underlying regulatory mechanisms remain ill-defined. Transactive response DNA-binding protein-43 (TDP-43) is an RNA-binding protein that has an essential role in regulating RNA metabolism at multiple levels. Cleavage and cytoplasmic aggregation of TDP-43 serves as a major molecular marker for amyotrophic lateral sclerosis and frontotemporal lobar degeneration and contributes significantly to disease progression. In this study, we reported that TDP-43 is translocated from the nucleus to the cytoplasm during CVB3 infection through the activity of viral protease 2A, followed by the cleavage mediated by viral protease 3C. Cytoplasmic translocation of TDP-43 is accompanied by reduced solubility and increased formation of protein aggregates. The cleavage takes place at aminoacid 327 between glutamine and alanine, resulting in the generation of an N-and C-terminal cleavage fragment of~35 and~8 kDa, respectively. The C-terminal product of TDP-43 is unstable and quickly degraded through the proteasome degradation pathway, whereas the N-terminal truncation of TDP-43 acts as a dominant-negative mutant that inhibits the function of native TDP-43 in alternative RNA splicing. Lastly, we demonstrated that knockdown of TDP-43 results in an increase in viral titers, suggesting a protective role for TDP-43 in CVB3 infection. Taken together, our findings suggest a novel model by which cytoplasmic redistribution and cleavage of TDP-43 as a consequence of CVB3 infection disrupts the solubility and transcriptional activity of TDP-43. Our results also reveal a mechanism evolved by enteroviruses to support efficient viral infection. Coxsackievirus B3 (CVB3) is a small, positive-stranded RNA enterovirus. 1 The single open reading frame of CVB3 is translated into a viral polypeptide that is subsequently cleaved by two virus-encoded proteases 2A and 3C to generate structural and non-structural proteins. 2 In addition to processing viral polyprotein, 2A and 3C target host proteins important for maintenance of protein translation and transcription, antiviral activity, and cellular architecture and signaling, contributing to virus-induced pathogenesis. [3][4][5] Although enteroviral replication takes place exclusively in the cytoplasm, viral infection has been demonstrated to lead to cytoplasmic translocation of nuclear proteins. 6 For example, heterogeneous ribonucleoprotein D (hnRNP D) has been shown to translocate from the nucleus to the cytoplasm during enteroviral infection. 5,7,8 Moreover, hnRNP D is cleaved by 3C and has an antiviral function against enteroviral infection. 5,7,8 Cytoplasmic translocation after enteroviral infection has also been demonstrated for several other hnRNPs (A1, C, and K)...

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Cytoplasmic translocation, aggregation, and cleavage of TDP-43 by enteroviral proteases modulate viral pathogenesis

et al. 2015

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“…It is known that the host protein hnRNP C can bind both the 3= and 5= termini of poliovirus negative-strand RNA intermediates, an interaction proposed to promote the synthesis of positive-strand enterovirus RNA molecules (12,70,71). Thus, the 5=-terminal deletions of positive-strand RNAs would lead to deletions in the 3= ends of negative-strand RNAs, possibly reducing the binding of hnRNP C to these replication intermediates.…”

Section: Discussionmentioning

confidence: 99%

Functional Consequences of RNA 5′-Terminal Deletions on Coxsackievirus B3 RNA Replication and Ribonucleoprotein Complex Formation

Lévêque

Garcia

Bouin

et al. 2017

J Virol

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Group B coxsackieviruses are responsible for chronic cardiac infections. However, the molecular mechanisms by which the virus can persist in the human heart long after the signs of acute myocarditis have abated are still not completely understood. Recently, coxsackievirus B3 strains with 5=-terminal deletions in genomic RNAs were isolated from a patient suffering from idiopathic dilated cardiomyopathy, suggesting that such mutant viruses may be the forms responsible for persistent infection. These deletions lacked portions of 5= stem-loop I, which is an RNA secondary structure required for viral RNA replication. In this study, we assessed the consequences of the genomic deletions observed in vivo for coxsackievirus B3 biology. Using cell extracts from HeLa cells, as well as transfection of luciferase replicons in two types of cardiomyocytes, we demonstrated that coxsackievirus RNAs harboring 5= deletions ranging from 7 to 49 nucleotides in length can be translated nearly as efficiently as those of wild-type virus. However, these 5= deletions greatly reduced the synthesis of viral RNA in vitro, which was detected only for the 7-and 21-nucleotide deletions. Since 5= stem-loop I RNA forms a ribonucleoprotein complex with cellular and viral proteins involved in viral RNA replication, we investigated the binding of the host cell protein PCBP2, as well as viral protein 3CD pro , to deleted positive-strand RNAs corresponding to the 5= end. We found that binding of these proteins was conserved but that ribonucleoprotein complex formation required higher PCBP2 and 3CD pro concentrations, depending on the size of the deletion. Overall, this study confirmed the characteristics of persistent CVB3 infection observed in heart tissues and provided a possible explanation for the low level of RNA replication observed for the 5=-deleted viral genomes-a less stable ribonucleoprotein complex formed with proteins involved in viral RNA replication.IMPORTANCE Dilated cardiomyopathy is the most common indication for heart transplantation worldwide, and coxsackie B viruses are detected in about one-third of idiopathic dilated cardiomyopathies. Terminal deletions at the 5= end of the viral genome involving an RNA secondary structure required for RNA replication have been recently reported as a possible mechanism of virus persistence in the human heart. These mutations are likely to disrupt the correct folding of an RNA secondary structure required for viral RNA replication. In this report, we demonstrate that transfected RNAs harboring 5=-terminal sequence deletions are able to direct the synthesis of viral proteins, but not genomic RNAs, in human and murine cardiomyocytes. Moreover, we show that the binding of cellular and viral replication factors to viral RNA is conserved despite genomic deletions but that the impaired RNA synthesis as-

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“…Despite this spatial compartmentalization, evidence suggests that picornaviruses rely on activities provided by host nuclear RNA binding proteins for efficient translation and RNA replication. The strongest support for this comes from experiments using either in vitro or in vivo systems that have demonstrated interaction of host nuclear factors with viral proteins or RNA elements, along with modulation of translation and/or replication of the viral genome (2)(3)(4)(5)(6)(7)(8). Consistent with a role for host nuclear factors in viral replication, many have been shown to redistribute to the cytoplasm following infection (2,4,(7)(8)(9)(10)(11)(12)(13).…”

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confidence: 99%

Selective Removal of FG Repeat Domains from the Nuclear Pore Complex by Enterovirus 2A ^pro

Park

Schweers

Gustin

2015

J Virol

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Enteroviruses proteolyze nuclear pore complex (NPC) proteins (Nups) during infection, leading to disruption of host nuclear transport pathways and alterations in nuclear permeability. To better understand how enteroviruses exert these effects on nuclear transport, the mechanisms and consequences of Nup98 proteolysis were examined. The results indicate that Nup98 is rapidly targeted for degradation following enterovirus infection and that this is mediated by the enterovirus 2A protease (2A pro ). Incubation of bacterially expressed or in vitro-translated Nup98 with 2A pro results in proteolytic cleavage at multiple sites in vitro, indicating that 2A pro cleaves Nup98 directly. Site-directed mutagenesis of putative cleavage sites identified Gly374 and Gly552 as the sites of 2A pro proteolysis in Nup98 in vitro and in infected cells. Indirect immunofluorescence assays using an antibody that recognizes the N terminus of Nup98 revealed that proteolysis releases the N-terminal FG-rich region from the NPC. In contrast, similar analyses using an antibody to the C terminus indicated that this region is retained at the nuclear rim. Nup88, a core NPC component that serves as a docking site for Nup98, also remains at the NPC in infected cells. These findings support a model whereby the selective removal of Nup FG repeat domains leads to increased NPC permeability and inhibition of certain transport pathways, while retention of structural domains maintains the overall NPC structure and leaves other transport pathways unaffected. IMPORTANCEEnteroviruses are dependent upon host nuclear RNA binding proteins for efficient replication. This study examines the mechanisms responsible for alterations in nuclear transport in enterovirus-infected cells that lead to the cytoplasmic accumulation of these proteins. The results demonstrate that the enterovirus 2A protease directly cleaves the nuclear pore complex (NPC) protein, Nup98, at amino acid positions G374 and G552 both in vitro and in infected cells. Cleavage at these positions results in the selective removal of the FG-containing N terminus of Nup98 from the NPC, while the C terminus remains associated. Nup88, a core component of the NPC that serves as a docking site for the C terminus of Nup98, remains associated with the NPC in infected cells. These findings help to explain the alterations in permeability and nuclear transport in enterovirus-infected cells and how NPCs remain functional for certain trafficking pathways despite significant alterations to their compositions. Members of the Picornaviridae are small nonenveloped viruses with positive-strand RNA genomes that are responsible for a variety of diseases in humans and animals (reviewed in reference 1). The family includes enteroviruses, such as poliovirus and rhinovirus, along with cardioviruses, such as encephalomyocarditis virus and Theiler's murine encephalomyelitis virus. Following entry of these viruses into the host cell, translation and replication of the RNA genome occur exclusively in the cytoplasm. Desp...

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Mechanistic Consequences of hnRNP C Binding to Both RNA Termini of Poliovirus Negative-Strand RNA Intermediates

Cited by 61 publications

References 57 publications

Cytoplasmic translocation, aggregation, and cleavage of TDP-43 by enteroviral proteases modulate viral pathogenesis

Cytoplasmic translocation, aggregation, and cleavage of TDP-43 by enteroviral proteases modulate viral pathogenesis

Functional Consequences of RNA 5′-Terminal Deletions on Coxsackievirus B3 RNA Replication and Ribonucleoprotein Complex Formation

Selective Removal of FG Repeat Domains from the Nuclear Pore Complex by Enterovirus 2A ^pro

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Mechanistic Consequences of hnRNP C Binding to Both RNA Termini of Poliovirus Negative-Strand RNA Intermediates

Cited by 61 publications

References 57 publications

Cytoplasmic translocation, aggregation, and cleavage of TDP-43 by enteroviral proteases modulate viral pathogenesis

Cytoplasmic translocation, aggregation, and cleavage of TDP-43 by enteroviral proteases modulate viral pathogenesis

Functional Consequences of RNA 5′-Terminal Deletions on Coxsackievirus B3 RNA Replication and Ribonucleoprotein Complex Formation

Selective Removal of FG Repeat Domains from the Nuclear Pore Complex by Enterovirus 2A pro

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Selective Removal of FG Repeat Domains from the Nuclear Pore Complex by Enterovirus 2A ^pro