Nuclear Import of the Respiratory Syncytial Virus Matrix Protein Is Mediated By Importin β1 Independent of Importin α

Ghildyal, Reena; Ho, Adeline; Wagstaff, Kylie M.; Dias, Manisha M; Barton, Chenoa; Jans, Patricia; Bardin, Phillip; Jans, David A.

doi:10.1021/bi050701e

Cited by 104 publications

(129 citation statements)

References 42 publications

(88 reference statements)

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“…these range from essential replication activities such as Dna replication, integration, or transcription to more broad-spectrum effects such as dampening of the host cell immune responses, which are nonetheless critical to virus replication/survival. [1][2][3][4] as such, the transport of viral proteins from the cytoplasm, through the nuclear envelope-embedded nuclear pore complexes (npcs) and into the nucleus, remains a highly attractive, although largely overlooked, target for the development of antiviral therapeutics.…”

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confidence: 99%

An AlphaScreen®-Based Assay for High-Throughput Screening for Specific Inhibitors of Nuclear Import

et al. 2011

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Specific viral proteins enter the nucleus of infected cells to perform essential functions, as part of the viral life cycle. The integrase (IN) molecule of human immunodeficiency virus (HIV)–1 is of particular interest in this context due to its integral role in integrating the HIV genome into that of the infected host cell. Most IN-based antiviral compounds target the IN/DNA interaction, but since IN must first enter the nucleus before it can perform these critical functions, nuclear transport of IN is also an attractive target for therapeutic intervention. Here the authors describe a novel high-throughput screening assay for identifying inhibitors of nuclear import, particularly IN, based on amplified luminescent proximity homogeneous assay (AlphaScreen®) technology, which is high throughput, requires low amounts of material, and is efficient and cost-effective. The authors use the assay to screen for specific inhibitors of the interaction between IN and its nuclear transport receptor importin α/β, successfully identifying several inhibitors of the IN/importin α/β interaction. Importantly, they demonstrate that one of the identified compounds, mifepristone, is effective in preventing active nuclear transport of IN in transfected cells and hence may represent a useful anti-HIV therapeutic. The screen also identified broad-spectrum importin α/β inhibitors such as ivermectin, which may represent useful tools for nuclear transport research in the future. The authors validate the activity and specificity of mifepristone and ivermectin in inhibiting nuclear protein import in living cells, underlining the utility of the screening approach.

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An AlphaScreen®-Based Assay for High-Throughput Screening for Specific Inhibitors of Nuclear Import

et al. 2011

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“…To test 3C's nuclear targeting ability, the coding sequences for HRV16 3CD and 3C were both cloned into the pEPI-DESTC Gateway vector (9) and expressed as C-terminal green fluorescent protein (GFP) fusion derivatives in transfected Vero cells with GFP alone (pEPI-GFP) as a control. Localization of GFP fluorescence was monitored by live-cell confocal laser scanning microscopy (CLSM) at 18 h posttransfection, and the ImageJ1.…”

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confidence: 99%

“…This system, also applicable to other adherent cell types, is completely comparable to digitonin-permeabilized cells in terms of dependence for transport on importins, Ran, etc., and has been used successfully to study the nuclear transport of various cargos (4,5,7,9,12,13,18). Nuclear localization signal (NLS)-dependent nuclear protein import can be reconstituted in this system through the exogenous addition of cytosolic extract, an ATPregenerating system (0.125 mg/ml creatine kinase, 30 mM creatine phosphate, 2 mM ATP), and GTP (2 mM).…”

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Rhinovirus 3C Protease Can Localize in the Nucleus and Alter Active and Passive Nucleocytoplasmic Transport

Ghildyal

Jordan

et al. 2009

J Virol

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The degradation of nuclear pore components and disruption of nucleocytoplasmic trafficking during rhinovirus infection have been attributed to viral 2A protease. Here we show for the first time that rhinovirus 3C protease may also have a role. Specifically, we show that 3C and its precursor, 3CD, can target green fluorescent protein to the nucleus of living cells, leading to degradation of nuclear pore components, and that incubation with recombinant 3C disrupts active and passive nucleocytoplasmic transport in a semi-intact cell nuclear transport system dependent on 3C protease activity. 3C may thus contribute to host cell shutoff in infected cells by localizing in the nucleus and facilitating nuclear pore breakdown.Human rhinoviruses (HRVs) are positive-strand RNA viruses belonging to the Picornaviridae family, which includes poliovirus. Although picornavirus replication is completed within the cytoplasm, many nuclear factors have been implicated in the life cycle of both HRVs and polioviruses. Gustin and Sarnow (11) previously showed mislocalization of cellular proteins late in infection in HRV-infected cells that was attributed to inhibited nuclear import due to degradation of several nucleoporins (Nup153 and Nup62) that are critical components of the nuclear pore, the only avenue for transport into and out of the nucleus. Degradation of Nup153 and Nup62 has also been observed in poliovirus infection (10). Recent work (17) indicates that another nucleoporin, Nup98, is also degraded in poliovirus-infected cells and that this precedes cleavage of Nup153 and Nup62. Importantly, results from in vitro cleavage experiments using HRV 2A protease were used to implicate picornavirus 2A protease as the mediator of cleavage of Nup98 (17), but 2A protease cleavage of Nup98 alone was not found to be sufficient to induce alterations in nuclear pore permeability. Importantly, a role in this context was not considered for the other major protease of HRV, 3C. Both 3C protease and its precursor form, 3CD, have been observed in the nucleus of cells infected with HRV or transfected to express 3CD (1), meaning that 3C is an ideal candidate to mediate effects on host cell nuclear transport.Here we present evidence for the first time that HRV 3C protease has intrinsic nuclear targeting potential, and, dependent on its protease activity, is able to disrupt both active and passive nucleocytoplasmic transport. The results suggest that 3C protease, like 2A (17), is likely to contribute to the disruption of host cell nuclear transport, a key factor in host cell shutdown.To test 3C's nuclear targeting ability, the coding sequences for HRV16 3CD and 3C were both cloned into the pEPI-DESTC Gateway vector (9) and expressed as C-terminal green fluorescent protein (GFP) fusion derivatives in transfected Vero cells with GFP alone (pEPI-GFP) as a control. Localization of GFP fluorescence was monitored by live-cell confocal laser scanning microscopy (CLSM) at 18 h posttransfection, and the ImageJ1.62 shareware was used to analyze the digital im...

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“…In particular, a nuclear targeting signal within M is able to be recognised by a specific member of the importin superfamily of cellular nuclear transporters -importin β1 [50] -which is believed to transport M into the nucleus early in infection [2,50]. The role of nuclear M appears to be dual; to inhibit host cell transcription, and at the same time, to sequester M from RSV transcription occurring in the cytoplasm [14]; inactivation by mutation of the nuclear import signal in the context of RSV itself reduces virus production markedly [51], underlying the importance of RSV nuclear import to viral infection.…”

Section: B Additional Roles Of M Proteinmentioning

confidence: 99%

Protein-Protein Interactions in RSV Assembly: Potential Targets for Attenuating RSV Strains

Ghildyal

Jans²,

Bardin

et al. 2012

IDDT

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Respiratory syncytial virus (RSV) is the major respiratory pathogen of infants and children worldwide, with no effective treatment or vaccine available. Steady progress has been made in understanding the respiratory syncytial virus lifecycle and the consequences of infection, but some areas of RSV still remain poorly understood. Although many of the interactions between virus proteins that are required for efficient RSV assembly have been elucidated, many questions still remain regarding viral assembly, as well as the mechanisms of RSV budding. This review will summarise the current understanding of RSV assembly, including the various interactions between virus proteins and the involvement of cellular factors with a view to identifying possible attenuation and/or drug targets within the assembly pathway.

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Nuclear Import of the Respiratory Syncytial Virus Matrix Protein Is Mediated By Importin β1 Independent of Importin α

Cited by 104 publications

References 42 publications

An AlphaScreen®-Based Assay for High-Throughput Screening for Specific Inhibitors of Nuclear Import

An AlphaScreen®-Based Assay for High-Throughput Screening for Specific Inhibitors of Nuclear Import

Rhinovirus 3C Protease Can Localize in the Nucleus and Alter Active and Passive Nucleocytoplasmic Transport

Protein-Protein Interactions in RSV Assembly: Potential Targets for Attenuating RSV Strains

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