Materials and methods
Phylogenetic analysis. Phylogenetic analysis based on adenovirus pVII was performed to determine the evolutionary affiliation of the genus Siadenovirus with the protein of interest. For that amino acid sequences of pVII available in GenBank for genusSiadenovirus members were extracted and aligned with MAFTT (version 7.450), using Geneious (version 22.1.1, Biomatters, Ltd., Auckland, New Zealand) (scoring matrix BLOSUM62; gap open penalty 1.53; offset value 0.123). Following the amino acids sequence alignments, maximum likelihood (ML)-based phylogenetic analysis was performed with 500 non-parametric bootstrap replicates implemented in Geneious (version 22.1.1, Biomatters, Ltd., Auckland, New Zealand).
Identification of putative NLSs on pVII. The primary sequence from FrAdV1 Pre-pVII (GenBank accession no; NC_0025011, was analysed with the cNLS mapper software21, and by visual inspection to identify putative NLSs.
Tertiary structure prediction . AlphaFold2 22was utilized to predict a model for pVII in response to the absence of structural information. A comprehensive model for the full-length pVII of FrAdV1 1 was created, emphasising the identified NLS regions. Additionally, for the purpose of structural comparison, we employed AlphaFold2 and generated a structural model for HAdV5 Pre-pVII10.
Plasmids . Bacterial expression plasmids mediating the expression of truncated version of mouse (m)IMPα2 lacking the autoinhibitory IMPβ1 binding (IBB) domain, and mIMPβ1 from a pET30a backbone were described previously 23,24. Mammalian expression plasmids pcDNA3.1-NT-GFP-TOPO and pcDNA3.1-NT-GFP-TOPO-SV40-NLS, mediating the expression of GFP cycle3, or of a fusion protein between GFP cycle3 and Simian vacuolating virus (SV) 40 large tumor antigen NLS (PKKKRKV-132), respectively, were described previously 25. Plasmid pEPI-GFP-UL44, encoding a fusion protein between GFP and human cytomegalovirus (HCMV) DNA polymerase UL44, localizing to the nucleus via the IMPα/β dependent pathway, was also previously described 26. Plasmid GST-GFP-FUS, encoding a fusion protein translocated via IMPβ227 was kindly provided from Dorothee Dormann (Mainz, Germany), plasmid pDsRed-C1-Fibrillarin 28, was kindly provided by Denis Archambault (University of Québec, Canada). Plasmid RFP-M9M, encoding from a potent and selective IMPβ2 inhibitor29 was provided by Yoshihiro Kino (Tokyo, Japan), plasmid mcherry-Bimax2, encoding for a competitive inhibitor of the IMPα/β nuclear import pathway 30 was a generous gift from Yoshihiro Yoneda and Masahiro Oka (Osaka, Japan). Plasmid DsRed-RanQ69L, encoding for a transdominant negative, GTPase deficient mutant of the Ran GTPase, which impairs Ran-dependent nuclear transport31, was a generous gift from Michael Green (University of Massachusetts, USA). Mammalian expression plasmids encoding for GFP-FrAdV1 Pre-pVII (1-149) and substitution derivatives thereof, as well as for GFP fused to FrAdV1 Pre-pVII individual NLS sequences, were synthesized by BioFab research (Rome, Italy) and GeneScript (New Jersey, USA). A list of all plasmids used in this study is available in Supplementary Table S1.
Peptides. Synthetic peptides representing the predicted amino acid residues, with an N-terminal FITC tag, were synthesized and obtained from GenScript (Supplementary Table S2).
Expression and purification of recombinant proteins. The overexpression of IMPs was carried out in E. coli pLysS BL21(DE3) as described previously 32 using the auto-induction method 33. After induction, the cultures were centrifuged at 6000 rpm for 20 minutes at 4°C, and the resulting bacterial pellets were resuspended in HIS buffer A (pH 8), which consisted of 50 mM phosphate buffer, 300 mM NaCl, and 20 mM imidazole. To lyse the cells, two freeze-thaw cycles were performed, followed by the addition of lysozyme (1 mL of 20 mg/mL) (Sigma-Aldrich, St. Louis, MI, USA) and DNase (10 μL of 50 mg/mL) (Sigma-Aldrich, St. Louis, MI, USA), and incubation at room temperature for 1 hour. The supernatants containing soluble proteins were collected by centrifugation at 12,000 rpm for 30 minutes at 4°C. The extracts were then filtered through 0.45 μm low protein affinity filters and injected into a 5 mL HisTrap HP column (GE Healthcare, Chicago, IL, USA) that had been pre-equilibrated with His buffer A, in an AKTA purifier FPLC system (GE Healthcare, USA). Followed by washes of 20 column volumes with His buffer A, the proteins of interest were eluted using a gradually increasing gradient of imidazole (ranging from 20 mM to 500 mM) (ChemSupply, Gillman, SA, Australia). The eluted protein fractions were combined and loaded onto a pre-equilibrated HiLoad 26/60 Superdex 200 column (GE Healthcare, USA) in GST buffer A (50 mM Tris and 125 mM NaCl) for further purification using size-exclusion chromatography. The fractions corresponding to the eluted volumes at the respective protein sizes were collected, and the samples were concentrated using an Amicon MWCO 10 kDa filter (Merck Millipore, Burlington, MA, USA). Prior to experimental use, the purity of the samples was assessed by performing SDS-PAGE at 165 V for 30 minutes on a 4–12% Bis-Tris Plus gel (Thermo Fisher Scientific, Waltham, MA, USA).
Fluorescence polarization (FP) assays. FrAdV1 Pre-pVII NLSs were incubated with IMPs in a two-fold serial dilution scheme, essentially as described previously 34,35. The experiment involved testing IMPαΔIBB isoform 2 and IMPβ1, separately. Starting with a low concentration of 5 µM, the dilution series was performed across 23 wells in a 96-well, black Fluotrac microplate (Greiner Bio-One, Austria) each plate containing an appropriate negative control lacking the IMP binding partner. GST buffer A (50 mM Tris and 125 mM NaCl) was added to bring the total volume of 200 µL per well and fluorescence polarization measurements were recorded immediately using a CLARIOstar Plus plate reader (BMG Labtech, Germany). The assays were repeated in triplicate to ensure consistency. The data from the three independent experiments were analysed using GraphPad Prism software (version 9.3.1) to determine the dissociation constant (Kd). A summary of FP assays results is shown in Supplementary Table S3.
Crystallization, data collection, and structure determination.The hanging drop vapor diffusion method was employed to crystallize the IMPα2ΔIBB:FrAdV1 pVII NLS protein complex. The complex was attained in a 1:1 molar ratio, with each hanging drop having a total volume of 3 μL over a well of 300 μL precipitant solution containing 650 mM sodium citrate (pH 6.5) and 10 mM DTT at a temperature of 23°C. Crystals were formed after 3 days of incubation. Formed crystals were collected and cryoprotected in a precipitant solution containing 20% glycerol, before being rapidly frozen in liquid nitrogen. X-ray diffraction data were obtained at the Australian Synchrotron using the MX2 macromolecular beam lines, utilizing an Eiger 16M detector 36. The data obtained were subjected to indexing and integration using MOSFLM37. Subsequent steps including merging, space group assignment, scaling, and Rfree calculations were carried out using AIMLESS within CCP4, a suite of programs for crystallographic data processing 38. The final model building and refinement were performed using software tools that are commonly used for model building and refinement in structural biology, COOT39, and Phenix 40. Phasing was performed using molecular replacement in Phaser 41 and PDB code 3UKX was used as the search model for IMPα2. The finalized model was subjected to validation and subsequently deposited to the Protein Data Bank (PDB) with an assigned accession number 8U36; refinement statics are detailed in Supplementary Table S3.
Cells . HEK293A cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (v/v) foetal bovine serum (FBS), 50 U/ml penicillin, 50 U/ml streptomycin, and 2 mM L-glutamine in a humidified incubator at 37°C in the presence of 5% CO2 and passaged when reached confluence as described in42.
Transfections. HEK293A cells were seeded in a 24-well plate onto glass coverslips (5×104 cells/well). The next day, cells were transfected with appropriate amounts of expression constructs (range 5-250 ng), using Lipofectamine 2000 (ThermoFisher Scientific, Monza, Italy), following the manufacturer’s recommendations and further incubated at 37 °C and 5% CO2 in complete medium as described 43, until being processed for CLSM.
CLSM and image analysis. Cells were transfected and incubated for 24 hours to allow expression of spontaneously fluorescent proteins. Afterward, the cells were treated with DRAQ5 (#62251, ThermoFisher Scientific, Monza, Italy) at a dilution of 1:5000 in DMEM without phenol red for 30 minutes 44. Following incubation, the cells were washed twice with PHEM 1x solution (60 mM PIPES, 25 mM HEPES, 10 mM EGTA, and 4 mM MgSO4) and fixed with 4% paraformaldehyde (v/v) for 10 minutes at room temperature (RT). After three washes with PBS 1x, coverslips were mounted on glass slides using Fluoromount G (Southern Biotech, Birmingham, AL, USA). The subcellular localization of fusion proteins was examined using a Nikon A1 confocal laser scanning microscope (Nikon, Tokyo, Japan) equipped with a 60x oil immersion objective, following the established protocol outlined in45,46. To determine the levels of nuclear accumulation of the proteins of interest, the FiJi public domain software (https://doi.org/10.1038/nmeth.2019) was utilized, and single-cell measurements were taken for nuclear (Fn), nucleolar (Fno), and cytoplasmic (Fc) fluorescence. DRAQ5 and fibrillarin were used to define nuclear and nucleolar masks, respectively, while a small area close to DRAQ5 was used to define a cytosolic mask as previously47. The fluorescence attributed to autofluorescence/background (Fb) was subtracted from the measurements, to calculate the Fn/c and Fno/Fn ratios according to the formulas Fn/c = (Fn-Fb)/(Fc-Fb) and Fno/Fn = (Fno-Fb)/(Fn-Fb). Cells with oversaturated signals were excluded from analysis. To allow easier detection of nucleoli, cells were co-transfected with DsRed-fibrillarin expression plasmid, and rgb profile plots calculated with FiJi. Statistical analysis was performed using GraphPad Prism 9 software (GraphPad, San Diego, CA, USA) applying either Student’s t-test, one-way ANOVA, or two-way ANOVA, as appropriate.