Structural basis for importin alpha 3 specificity of W proteins in Hendra and Nipah viruses

Smith, Kate; Tsimbalyuk, S.; Edwards, Megan R.; Cross, E.M.; Batra, Jyoti; Costa, Tatiana P. Soares da; Aragão, D.; Basler, Christopher F.; Forwood, Jade K.

doi:10.1038/s41467-018-05928-5

Cited by 50 publications

(87 citation statements)

References 62 publications

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“…Expression and purification of codon-optimized, His-tagged ∆Impα3 (residues 64-521) were carried out using BL21 (DE3) cells [ 25 , 40 ]. The DNA of the codon-optimized, intact Impα3 was synthesized by NZYtech (Lisbon, Portugal) and cloned into the pHTP1 vector (kanamycin resistance), and with a His-tag at the protein N terminus.…”

Section: Methodsmentioning

confidence: 99%

The Paralogue of the Intrinsically Disordered Nuclear Protein 1 Has a Nuclear Localization Sequence that Binds to Human Importin α3

Neira

Rizzuti

Jiménez-Alesanco

et al. 2020

IJMS

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Numerous carrier proteins intervene in protein transport from the cytoplasm to the nucleus in eukaryotic cells. One of those is importin α, with several human isoforms; among them, importin α3 (Impα3) features a particularly high flexibility. The protein NUPR1L is an intrinsically disordered protein (IDP), evolved as a paralogue of nuclear protein 1 (NUPR1), which is involved in chromatin remodeling and DNA repair. It is predicted that NUPR1L has a nuclear localization sequence (NLS) from residues Arg51 to Gln74, in order to allow for nuclear translocation. We studied in this work the ability of intact NUPR1L to bind Impα3 and its depleted species, ∆Impα3, without the importin binding domain (IBB), using fluorescence, isothermal titration calorimetry (ITC), circular dichroism (CD), nuclear magnetic resonance (NMR), and molecular docking techniques. Furthermore, the binding of the peptide matching the isolated NLS region of NUPR1L (NLS-NUPR1L) was also studied using the same methods. Our results show that NUPR1L was bound to Imp α3 with a low micromolar affinity (~5 μM). Furthermore, a similar affinity value was observed for the binding of NLS-NUPR1L. These findings indicate that the NLS region, which was unfolded in isolation in solution, was essentially responsible for the binding of NUPR1L to both importin species. This result was also confirmed by our in silico modeling. The binding reaction of NLS-NUPR1L to ∆Impα3 showed a larger affinity (i.e., lower dissociation constant) compared with that of Impα3, confirming that the IBB could act as an auto-inhibition region of Impα3. Taken together, our findings pinpoint the theoretical predictions of the NLS region in NUPR1L and, more importantly, suggest that this IDP relies on an importin for its nuclear translocation.

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Section: Methodsmentioning

confidence: 99%

The Paralogue of the Intrinsically Disordered Nuclear Protein 1 Has a Nuclear Localization Sequence that Binds to Human Importin α3

Neira

Rizzuti

Jiménez-Alesanco

et al. 2020

IJMS

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“…Much remains unknown about the contributions of viral factors to the respiratory and encephalitic components of henipavirus infection. The ability of henipaviruses to evade the immune system and establish systemic infection was suggested to be due to inhibition of IFN-α/β activation and signalling, with W playing a significant role [ 28 , 29 ]. However, in vivo NiV studies using a ferret model suggests that V plays a major role in determination of viral pathogenesis and lethality [ 28 ].…”

Section: Innate Immune Antagonism and Virus Pathogenesismentioning

confidence: 99%

The Intrinsically Disordered W Protein Is Multifunctional during Henipavirus Infection, Disrupting Host Signalling Pathways and Nuclear Import

Tsimbalyuk

Cross

Hoad

et al. 2020

Cells

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Nipah and Hendra viruses are highly pathogenic, zoonotic henipaviruses that encode proteins that inhibit the host’s innate immune response. The W protein is one of four products encoded from the P gene and binds a number of host proteins to regulate signalling pathways. The W protein is intrinsically disordered, a structural attribute that contributes to its diverse host protein interactions. Here, we review the role of W in innate immune suppression through inhibition of both pattern recognition receptor (PRR) pathways and interferon (IFN)-responsive signalling. PRR stimulation leading to activation of IRF-3 and IFN release is blocked by henipavirus W, and unphosphorylated STAT proteins are sequestered within the nucleus of host cells by W, thereby inhibiting the induction of IFN stimulated genes. We examine the critical role of nuclear transport in multiple functions of W and how specific binding of importin-alpha (Impα) isoforms, and the 14-3-3 group of regulatory proteins suggests further modulation of these processes. Overall, the disordered nature and multiple functions of W warrant further investigation to understand henipavirus pathogenesis and may reveal insights aiding the development of novel therapeutics.

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“…In contrast, C/NP TAIL presents a higher affinity for importin-α3. Importin-α3 has a low auto-inhibition and a unique intrinsic flexibility, acting as an adapter for unusual and complex NLS sequences or NLS close to globular domains [53,54]. Such NLSs could not associate with a less flexible importin-α due to steric hindrance.…”

Section: Discussionmentioning

confidence: 99%

Differential Behaviours and Preferential Bindings of Influenza Nucleoproteins on Importins-α

Donchet

Vassal-Stermann

Gérard

et al. 2020

Viruses

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Influenza viruses are negative single-stranded RNA viruses with nuclear transcription and replication. They enter the nucleus by using the cellular importin-α/-β nuclear import machinery. Influenza nucleoproteins from influenza A, B, C and D viruses possess a nuclear localization signal (NLS) localized on an intrinsically disordered extremity (NPTAIL). In this paper, using size exclusion chromatography (SEC), SEC-multi-angle laser light scattering (SEC-MALLS) analysis, surface plasmon resonance (SPR) and fluorescence anisotropy, we provide the first comparative study designed to dissect the interaction between the four NPTAILs and four importins-α identified as partners. All interactions between NPTAILs and importins-α have high association and dissociation rates and present a distinct and specific behaviour. D/NPTAIL interacts strongly with all importins-α while B/NPTAIL shows weak affinity for importins-α. A/NPTAIL and C/NPTAIL present preferential importin-α partners. Mutations in B/NPTAIL and D/NPTAIL show a loss of importin-α binding, confirming key NLS residues. Taken together, our results provide essential highlights of this complex translocation mechanism.

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Structural basis for importin alpha 3 specificity of W proteins in Hendra and Nipah viruses

Cited by 50 publications

References 62 publications

The Paralogue of the Intrinsically Disordered Nuclear Protein 1 Has a Nuclear Localization Sequence that Binds to Human Importin α3

The Paralogue of the Intrinsically Disordered Nuclear Protein 1 Has a Nuclear Localization Sequence that Binds to Human Importin α3

The Intrinsically Disordered W Protein Is Multifunctional during Henipavirus Infection, Disrupting Host Signalling Pathways and Nuclear Import

Differential Behaviours and Preferential Bindings of Influenza Nucleoproteins on Importins-α

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