LiteFold/DisProt huggingface dataset:
Protein: DP03000 (UniProt Q997F2)
Region: residues 1	to 456
Annotation: pre-molten globule (Structural state)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
The spectra are typical of proteins predominantly unfolded, as judged from their large negative ellipticity at 200 nm, low amplitude in the 210–230 nm region, and low ellipticity at 190 nm (Fig. 3A).
As shown in the inset of Fig. 3A, HeV and NiV V fall closer to the PMG-like region than to the RC-like region.
Protein: DP03000 (UniProt Q997F2)
Region: residues 1	to 456
Annotation: disorder (Structural state)
Evidence: small-angle X-ray scattering evidence used in manual assertion
Supporting statements:
The strong discrepancy between the experimentally observed Rg values and those expected for a globular/spherical form, together with the fact that they are even larger than the values expected for IDPs indicate that the V proteins are highly disordered.
Protein: DP03000 (UniProt Q997F2)
Region: residues 1	to 456
Annotation: protein binding (Molecular function)
Evidence: microscale thermophoresis evidence used in manual assertion
Supporting statements:
In an attempt at quantifying the V-DDB1 binding affinity, we used microscale thermophoresis (MST). Labeled DDB1 was then titrated with unlabeled V. Results allowed us to infer an equilibrium dissociation constant (KD) of 32 ± 6 nM for HeV V and of 35 ± 3 nM for NiV V (Fig. 6C and D).
Protein: DP03000 (UniProt Q997F2)
Region: residues 1	to 406
Annotation: disorder (Structural state)
Evidence: small-angle X-ray scattering evidence used in manual assertion
Supporting statements:
The flexible nature of NiV PNT was qualitatively assessed by using the normalized Kratky plot, where the absence of a well-defined bell shape indicates a protein with intrinsically disordered regions (Fig. 4C).
The resulting final Rg distribution, broader than the one generated from the NMR-based pool, indicates that NiV PNT exists in solution as a randomly distributed ensemble of non-compact and highly flexible conformations.
Cross-references: SASBDB:SASDJB5
Protein: DP03000 (UniProt Q997F2)
Region: residues 1	to 406
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
NiV PNT is a very large intrinsically disordered protein (412 residues including the C-terminal His tag and the initial methionine), as can be inferred by inspection of the two 2D spectra, the 1H–15N HSQC (Fig. 2A) and the 13C′–15N CON (Fig. 2B).
The relaxation rates show an overall trend consistent with the considerably disordered conformation of PNT. 1H–15N NOE values are all well below 0.5, indicating high flexibility of the backbone (Supplementary Figure S1A).
Cross-references: BMRB:50370
Protein: DP03000 (UniProt Q997F2)
Region: residues 1	to 456
Annotation: disorder (Structural state)
Evidence: near-UV circular dichroism evidence used in manual assertion
Supporting statements:
The near-UV CD spectra of the V proteins are rather smooth suggesting a predominantly disordered nature (Fig. 3B).
Protein: DP03000 (UniProt Q997F2)
Region: residues 409	to 456
Annotation: protein binding (Molecular function)
Evidence: co-immunoprecipitation evidence used in manual assertion
Supporting statements:
To confirm the result of the mass-spectrometric analysis, the same immunoprecipitation assay was followed by western blotting with an anti-myc antibody and an anti-UBXN1 antibody. UBXN1 was detected as the protein binding to wild-type NiV V, but not to ΔCT (Fig. 1B). The interaction was also verified by the coimmunoprecipitation of NiV V with the endogenous UBXN1 (Fig. 1C). In HeLa cells, UBXN1 was also coimmunoprecipitated with NiV V, indicating that UBXN1 is a binding partner of NiV V in various cell types (Fig. 1D). 
The wild-type NiV V, but not ΔCT, was coimmunoprecipitated with UBXN1 (Fig. 1E).
Two protein bands coimmunoprecipitated with wild-type NiV V, but not with ΔCT were detected (Supplementary Fig. 1A). One of the proteins with a mass of approximately 42.56 kDa was identified as UBX domain-containing protein 1 (UBXN1) by a mass-spectrometric analysis (Fig. 1A).
Protein: DP03000 (UniProt Q997F2)
Region: residues 1	to 456
Annotation: protein binding (Molecular function)
Evidence: immunofluorescence confocal microscopy evidence used in manual assertion
Supporting statements:
To confirm their intracellular interaction, an immunofluorescence assay was also performed after NiV V or UBXN1 was expressed in HEK293T cells. As previously reported, NiV V was mainly localized in cytoplasm (Fig. 1F, upper lane)24. UBXN1 was also localized in cytoplasm, and formed spot-like structures (Fig. 1F, middle lane), which is reportedly attributable to its association with the ubiquitin–proteasome system27,28. The coexpression of both proteins caused the accumulation of UBXN1 in the cytoplasm, and its signal localized strongly with that of the V protein (Fig. 1F, lower lane). Furthermore, NiV V was co-localized with endogenous UBXN1 in the cytoplasm (Fig. 1G). These results suggest that V protein interacts intracellularly with UBXN1.
Protein: DP03000 (UniProt Q997F2)
Region: residues 409	to 426
Annotation: protein stabilization (Biological process)
Evidence: far-Western blotting evidence used in manual assertion
Supporting statements:
When NiV V was expressed together with UBXN1, the degradation of UBXN1 was inhibited (Fig. 3E,F). This result suggests that NiV V increases the stability of UBXN1, causing its intracellular accumulation.
The mutants containing Domain1 (Del1, -2 and -5) clearly increased the expression level of UBXN1, whereas the others did not (Fig. 4A).
These results indicate that the stabilization of UBXN1 requires Domain1 of NiV V and the UBX domain of UBXN1, which are identical to the domains required for the interaction of the two proteins (Fig. 2C,E). Therefore, we infer that the stabilization of UBXN1 is caused by its interaction with NiV V.
Protein: DP03000 (UniProt Q997F2)
Region: residues 412	to 426
Annotation: protein stabilization (Biological process)
Evidence: loss-of-function mutant phenotype evidence used in manual assertion
Supporting statements:
To confirm the stabilization of UBXN1 by the myc-tagged alanine-substitution mutants of NiV V, these mutants were coexpressed with HA-tagged UBXN1. RRE stabilized UBXN1 as effectively as did wild-type NiV V, but GKR and EEW stabilized it less effectively, and ISI, CWD, and AWV did not stabilize it at all (Fig. 5C). Therefore, residues ISI, CWD, and AWV are important for UBXN1 stabilization.
Protein: DP03000 (UniProt Q997F2)
Region: residues 409	to 423
Annotation: protein binding (Molecular function)
Evidence: loss-of-function mutant phenotype evidence used in manual assertion
Supporting statements:
The alanine-substitution mutants CWD, GKR, and EEW reduced the activity of MDA5 as strongly as did wild-type NiV V, whereas the interference activities of RRE, ISI, and AWV were weaker than that of wild-type NiV V (Fig. 5D). These results suggest that MDA5 and UBXN1 share the amino acid residues in ISI and AWV to interact with NiV V.
Protein: DP03000 (UniProt Q997F2)
Region: residues 1	to 456
Annotation: host cell cytoplasm (Cellular component)
Evidence: immunofluorescence confocal microscopy evidence used in manual assertion
Supporting statements:
As previously reported, NiV V was mainly localized in cytoplasm (Fig. 1F, upper lane)24.
Protein: DP03000 (UniProt Q997F2)
Region: residues 412	to 423
Annotation: protein binding (Molecular function)
Evidence: loss-of-function mutant phenotype evidence used in manual assertion
Supporting statements:
The mutants were expressed in HEK293T cells and an immunoprecipitation assay was performed. Mutants RRE and EEW maintained the interaction with UBXN1, although it was weaker than that of intact NiV V. However, the interactions of mutants ISI, CWD, GKR, and AWV were eliminated (Fig. 5B).
Protein: DP03000 (UniProt Q997F2)
Region: residues 1	to 456
Annotation: suppression by virus of host type I interferon-mediated signaling pathway (Biological process)
Evidence: luciferase reporter gene assay evidence used in manual assertion
Supporting statements:
MDA5 induced the activation of the IFNB promoter, and wild-type NiV V interrupted MDA5 activity (Fig. 5D).
HEK293T cells were transfected with an IFNβ reporter vector together with vectors expressing FLAG-tagged MDA5 and wild-type NiV V or its alanine-substitution mutants.
Protein: DP03000 (UniProt Q997F2)
Region: residues 1	to 456
Annotation: suppression by symbiont of host innate immune response (Biological process)
Evidence: luciferase reporter gene assay evidence used in manual assertion
Supporting statements:
MDA5 induced the activation of the IFNB promoter, and wild-type NiV V interrupted MDA5 activity (Fig. 5D).
HEK293T cells were transfected with an IFNβ reporter vector together with vectors expressing FLAG-tagged MDA5 and wild-type NiV V or its alanine-substitution mutants.
Protein: DP03000 (UniProt Q997F2)
Region: residues 1	to 456
Annotation: protein binding (Molecular function)
Evidence: competitive binding evidence used in manual assertion
Supporting statements:
Although the coimmunoprecipitation of UBXN1 and MDA5 was slightly decreased in the competitive binding, both proteins were coimmunoprecipitated with NiV V together (Fig. 5E). These results indicated that the binding sites of NiV V to MDA5 and UBXN1 were close but not identical, and NiV V could interact with both proteins together.
Protein: DP03000 (UniProt Q997F2)
Region: residues 1	to 456
Annotation: suppression by symbiont of host receptor-mediated signal transduction (Biological process)
Evidence: luciferase reporter gene assay evidence used in manual assertion
Supporting statements:
The coexpression of UBXN1 increased MDA5-suppression activity of NiV V, depending on the expression level (Fig. 6A). To examine whether RIG-I signaling was suppressed by the stabilized UBXN1, the constitutively activated RIG-I (RIG-IΔ) was expressed with NiV V and UBXN1, and IFN reporter assay was performed. As previously reported19, NiV V did not suppress RIG-I signaling directly (Fig. 6B). However, when coexpressed with UBXN1, NiV V suppressed RIG-I signaling depending on the expression level (Fig. 6B). This suppression effect of NiV V was also observed in 293UBXN1- cells, indicating that it is not due to the artifacts derived from a high amount UBXN1 (Fig. 6C).
Protein: DP03000 (UniProt Q997F2)
Region: residues 1	to 456
Annotation: suppression by symbiont of host innate immune response (Biological process)
Evidence: combinatorial experimental and author inference evidence contained in single publication used in manual assertion
Supporting statements:
Furthermore, NiV V suppressed RIG-I and MDA5-dependent interferon signaling by stabilizing UBXN1 and increasing the interaction between MAVS and UBXN1 in addition to directly interrupting the activation of MDA5. Our results suggest a novel molecular mechanism by which the induction of interferon is potentially suppressed by NiV V protein via UBXN1.
Protein: DP03001 (UniProt O55777)
Region: residues 1	to 457
Annotation: disorder (Structural state)
Evidence: sodium dodecyl sulfate polyacrylamide gel electrophoresis evidence used in manual assertion
Supporting statements:
This aberrant migration during electrophoresis is a hallmark of intrinsically disordered proteins (IDPs) and is often due to their typically high content of acidic residues and/or low compaction.
Protein: DP03001 (UniProt O55777)
Region: residues 1	to 457
Annotation: pre-molten globule (Structural state)
Evidence: chromatography evidence used in manual assertion
Supporting statements:
The experimentally observed values of the Rs are very close to the values expected for a PMG form (i.e. an extended conformation possessing nevertheless some residual structure
Protein: DP03001 (UniProt O55777)
Region: residues 1	to 457
Annotation: pre-molten globule (Structural state)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
The spectra are typical of proteins predominantly unfolded
HeV and NiV V fall closer to the PMG-like region than to the RC-like region
Protein: DP03001 (UniProt O55777)
Region: residues 1	to 457
Annotation: disorder (Structural state)
Evidence: near-UV circular dichroism evidence used in manual assertion
Supporting statements:
The near-UV CD spectra of the V proteins are rather smooth suggesting a predominantly disordered nature.
Protein: DP03001 (UniProt O55777)
Region: residues 1	to 457
Annotation: disorder (Structural state)
Evidence: small-angle X-ray scattering evidence used in manual assertion
Supporting statements:
observed Rg values and those expected for a globular/spherical form, together with the fact that they are even larger than the values expected for IDPs indicate that the V proteins are highly disordered
Protein: DP03001 (UniProt O55777)
Region: residues 1	to 457
Annotation: protein binding (Molecular function)
Evidence: microscale thermophoresis evidence used in manual assertion
Supporting statements:
Labeled DDB1 was then titrated with unlabeled V. Results allowed us to infer an equilibrium dissociation constant (KD) of 32 +/- 6 nM for HeV V
Protein: DP03001 (UniProt O55777)
Region: residues 173	to 178
Annotation: intracellular transport of virus (Biological process)
Evidence: loss-of-function mutant phenotype evidence used in manual assertion
Supporting statements:
In untreated cells the HeV V double leucine mutant (VL174A/L177A) showed a significant increase (p < 0.0001) in nuclear localization compared to wild-type V (Fig. 1c,d), suggesting loss of exportin-1-mediated nuclear export of V. Consistent with this, HeV VL174A/L177A nuclear localization was not strongly affected by LMB treatment (Fig. 1c,d). Together, these data indicate that exportin-1-mediated nuclear export of HeV V is dependent on residues L174 and L177, and that the NES from NiV V is conserved in HeV V.
Protein: DP03001 (UniProt O55777)
Region: residues 173	to 178
Annotation: protein binding (Molecular function)
Evidence: loss-of-function mutant phenotype evidence used in manual assertion
Supporting statements:
As expected, the absence of a larger sedimenting species when VL174A/L177A was incubated with exportin-1/Ran-GTP (Fig. 3c) confirmed that L174 and L177 within the HeV V NES are critical to exportin-1/Ran-GTP binding.
Protein: DP03001 (UniProt O55777)
Region: residues 173	to 178
Annotation: small GTPase binding (Molecular function)
Evidence: loss-of-function mutant phenotype evidence used in manual assertion
Supporting statements:
As expected, the absence of a larger sedimenting species when VL174A/L177A was incubated with exportin-1/Ran-GTP (Fig. 3c) confirmed that L174 and L177 within the HeV V NES are critical to exportin-1/Ran-GTP binding.
Protein: DP03001 (UniProt O55777)
Region: residues 1	to 457
Annotation: protein binding (Molecular function)
Evidence: analytical ultracentrifugation evidence used in manual assertion
Supporting statements:
The formation of a larger sedimenting species when HeV V was incubated with equimolar concentrations of importin α2/β1 (8.1 S) or exportin-1/Ran-GTP (6.7 S) confirmed HeV V bound either importin α2/β1 and exportin-1/Ran-GTP directly (Fig. 3a,b and Supplementary Table 1).
Protein: DP03001 (UniProt O55777)
Region: residues 1	to 457
Annotation: small GTPase binding (Molecular function)
Evidence: analytical ultracentrifugation evidence used in manual assertion
Supporting statements:
The formation of a larger sedimenting species when HeV V was incubated with equimolar concentrations of importin α2/β1 (8.1 S) or exportin-1/Ran-GTP (6.7 S) confirmed HeV V bound either importin α2/β1 and exportin-1/Ran-GTP directly (Fig. 3a,b and Supplementary Table 1).
Protein: DP03001 (UniProt O55777)
Region: residues 1	to 457
Annotation: importin-alpha family protein binding (Molecular function)
Evidence: analytical ultracentrifugation evidence used in manual assertion
Supporting statements:
The formation of a larger sedimenting species when HeV V was incubated with equimolar concentrations of importin α2/β1 (8.1 S) or exportin-1/Ran-GTP (6.7 S) confirmed HeV V bound either importin α2/β1 and exportin-1/Ran-GTP directly (Fig. 3a,b and Supplementary Table 1).
Protein: DP03001 (UniProt O55777)
Region: residues 51	to 405
Annotation: protein binding (Molecular function)
Evidence: analytical ultracentrifugation evidence used in manual assertion
Supporting statements:
In contrast, binding was observed when both V1-405 (Fig. 3e) or V51-457 (Fig. 3g) were mixed with exportin-1/Ran-GTP, indicating that neither the C-terminal region (residues 406–457) nor N-terminal 50 residues of V are critical for exportin-1/Ran-GTP binding.
Protein: DP03001 (UniProt O55777)
Region: residues 51	to 405
Annotation: small GTPase binding (Molecular function)
Evidence: analytical ultracentrifugation evidence used in manual assertion
Supporting statements:
In contrast, binding was observed when both V1-405 (Fig. 3e) or V51-457 (Fig. 3g) were mixed with exportin-1/Ran-GTP, indicating that neither the C-terminal region (residues 406–457) nor N-terminal 50 residues of V are critical for exportin-1/Ran-GTP binding.
Protein: DP03001 (UniProt O55777)
Region: residues 1	to 50
Annotation: importin-alpha family protein binding (Molecular function)
Evidence: analytical ultracentrifugation evidence used in manual assertion
Supporting statements:
Similarly, we observed binding when V1–405 was incubated with importin α2/β1 (Fig. 3f), but not upon incubation of V51-457 with importin α2/β1 (Fig. 3h). This suggests that the first 50 residues of V are required for interaction with importin α2/β1.
Protein: DP03001 (UniProt O55777)
Region: residues 1	to 457
Annotation: intracellular transport of virus (Biological process)
Evidence: confocal laser scanning microscopy evidence used in manual assertion
Supporting statements:
In untreated cells, HeV P and V were excluded from the nucleus, whereas W showed strong nuclear localization, but was excluded from structures consistent with nucleoli (Fig. 1a,b). Following LMB treatment HeV V showed a marked increase in nuclear localization (p < 0.0001), but remained excluded from nucleoli (Fig. 1a,b), providing the first empirical evidence that HeV V undergoes exportin-1-dependent nuclear export.
Protein: DP03001 (UniProt O55777)
Region: residues 1	to 457
Annotation: intracellular transport of virus (Biological process)
Evidence: small interfering RNA knockdown evidence used in manual assertion
Supporting statements:
A significant (p < 0.0001) increase in the extent of nuclear accumulation of wild-type HeV V was observed in cells transfected with XPO1 siRNA, compared to mock-treated cells and those transfected with Scr siRNA (Fig. 1f,g).
Protein: DP03001 (UniProt O55777)
Region: residues 1	to 405
Annotation: disorder (Structural state)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
Using CD we observed full-length HeV V displayed a single minimum at around 200 nm, which is indicative of a largely unstructured protein. Also apparent was broad but weak negative ellipticity between 215 and 235 nm, indicative of a small proportion of residual secondary structure that is absent in the CD spectra of V1-405. Based on this data we concur HeV V1-405 is predominantly intrinsically disordered (Supplementary Fig. 7a).
Protein: DP03001 (UniProt O55777)
Region: residues 1	to 50
Annotation: disorder to order (Structural transition)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
In contrast, V structural gain was negligible upon mixing VL174/L177A or V51-457 with equimolar exportin-1/Ran-GTP (Fig. 4c,g magenta vs gray). This was expected for VL174/L177A since it does not bind to exportin-1/Ran-GTP (Fig. 3c), but not for V51-457, since the first 50 residues are dispensable for exportin-1/Ran-GTP binding (Fig. 3g). These studies suggest that HeV V residues 1–50, which are the only residues with predicted secondary structure in the shared P/V/W-N-terminal region, are critical to the disorder-to-order transition of V upon binding to exportin-1/Ran-GTP.
Protein: DP03001 (UniProt O55777)
Region: residues 1	to 457
Annotation: disorder (Structural state)
Evidence: small-angle X-ray scattering evidence used in manual assertion
Supporting statements:
In contrast, the Kratky plot of V (Fig. 5e,f, black) lacked a well-defined peak and plateaued at high s values, as is typical of an unfolded protein.
Protein: DP03001 (UniProt O55777)
Region: residues 1	to 457
Annotation: response to leptomycin B (Biological process)
Evidence: confocal laser scanning microscopy evidence used in manual assertion
Supporting statements:
In untreated cells, HeV P and V were excluded from the nucleus, whereas W showed strong nuclear localization, but was excluded from structures consistent with nucleoli (Fig. 1a,b). Following LMB treatment HeV V showed a marked increase in nuclear localization (p < 0.0001), but remained excluded from nucleoli (Fig. 1a,b), providing the first empirical evidence that HeV V undergoes exportin-1-dependent nuclear export.
Protein: DP03001 (UniProt O55777)
Region: residues 1	to 457
Annotation: protein binding (Molecular function)
Evidence: co-immunoprecipitation evidence used in manual assertion
Supporting statements:
Consistent with the above results, coimmunoprecipitation revealed endogenous exportin-1 could readily interact with HA-fused wild-type HeV V, but the interaction with HA-tagged VL174A/L177A appeared reduced in comparison (Fig. 1h, Supplementary Fig. 4).
Protein: DP03001 (UniProt O55777)
Region: residues 1	to 457
Annotation: importin-alpha family protein binding (Molecular function)
Evidence: co-immunoprecipitation evidence used in manual assertion
Supporting statements:
Consistent with this result, GFP-fused V coimmunoprecipitated endogenous importin α1 to levels comparable to those of a GFP-fusion protein containing the prototypic importin α/β1-recognized NLS from simian virus (SV40) large tumour antigen GFP-T-Ag NLS (Fig. 2e, Supplementary Fig. 6).
Protein: DP03001 (UniProt O55777)
Region: residues 1	to 457
Annotation: intracellular transport of virus (Biological process)
Evidence: combinatorial experimental and author inference evidence contained in single publication used in manual assertion
Supporting statements:
Taken together, results suggest HeV V shuttles dynamically between the host cell nucleus and cytosol, accessing the nucleus by subverting importin α1 of the host nuclear transport system.
Protein: DP03001 (UniProt O55777)
Region: residues 1	to 457
Annotation: host cell cytoplasm (Cellular component)
Evidence: confocal microscopy evidence used in manual assertion
Supporting statements:
In untreated cells, HeV P and V were excluded from the nucleus, whereas W showed strong nuclear localization, but was excluded from structures consistent with nucleoli (Fig. 1a,b).
Despite the fact that P, V and W share the exportin-1 recognized NES shown to be active for V here, there are inherent differences in the steady-state localization of the P-gene encoded products, whereby V is predominantly cytoplasmic and W is predominantly nuclear, implying the unique C-terminal regions of these proteins contribute strongly to nucleocytoplasmic trafficking (see16,33).
Protein: DP03002 (UniProt P59636)
Region: residues 26	to 37
Annotation: disorder (Structural state)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
in the electron density map, two regions near the N terminus (residues 1–8 and 26–37) are not resolved. Both of these disordered segments are located on the periphery of the structure, whereas the inner core of the protein is reasonably well ordered.
Cross-references: PDB:2CME