Importin Alpha Is Implicated in the Nuclear Import of Novel Duck Reovirus Protein p18

TL;DR

This study finds that a duck virus protein, p18, enters the cell nucleus with the help of importin alpha proteins, and its nuclear entry does not require active viral infection.

Contribution

The study identifies importin alpha as a key transporter for p18 nuclear import and shows that this process is independent of viral replication.

Findings

p18 localizes to the nucleus in transfected cells.

Importin α1, α3, α4, α5, and α7 interact with p18.

The C-terminal region of p18 is essential for its nuclear localization.

Abstract

Novel duck reovirus encodes a new nucleus-localized protein, p18. We aimed to investigate whether the nuclear entry of p18 is dependent on viral replication, identify the cellular proteins that interact with p18, and determine the transporters involved in the nuclear import. The subcellular localization of p18 was observed by confocal microscopy. Cellular proteins interacting with p18 were identified through data-independent acquisition qualitative proteomics. The interaction between p18 and importin α was determined by confocal microscopy, co-immunoprecipitation (Co-IP) and molecular docking. We observed that p18 was localized to the nucleus in transfected cells. Importin α1, α3, α4, α5, and α7 were colocalized and co-immunoprecipitated with p18. The importin α/β1 pathway inhibitor reduced the nuclear distribution of p18. The truncated form of p18, lacking the C-terminal region, was predominantly distributed in the cytoplasm. Collectively, our research suggests that the nuclear entry of p18 is independent of viral infection, importin α is implicated in the nuclear import of p18, and the C-terminal region of p18 is crucial for nuclear localization.

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1. Introduction

Novel duck reovirus (NDRV) is a member of Orthoreovirus avis in the genus Orthoreoviridae of the family Spinareoviridae. NDRV causes “hemorrhagic–necrotic hepatitis” in Muscovy ducklings and “spleen necrosis disease” in Pekin ducklings [1,2]. The mortality of NDRV-associated disease can reach 50%, while the mortality caused by the classical Muscovy duck reovirus (MdRVC) is usually under 20% [2,3]. Different duck-origin NDRVs possess similar genomic sequences and genomic electrophoretic patterns. NDRV contains 10 double-stranded RNA genome segments, which are divided into large (L1–L3), medium (M1–M3), and small (S1–S4) groups. With the exception of S1, the nine genomic segments express the primary translation products of λ (λA, λB, and λC), μ (μA, μB, and μNS), and σ (σA, σB, and σNS) [2,4]. The tricistronic S1 genomic segment contains three overlapping ORFs, encoding for proteins p10.2, p18, and σC [5,6,7]. P18 is a functionally unknown protein with no counterpart in MdRVC, as the bicistronic S4 genomic segment of MdRVC encodes only p10.8 and σC [8,9]. While the tricistronic S1 genomic segment of chicken-origin avian reovirus (ARV) encodes proteins p10, p17, and σC, no sequence homology exists between p17 and p18 [2].

Virus-encoded nucleus-targeting proteins typically hijack the nuclear–cytoplasmic transport system of the host to perform functions such as regulating cell proliferation, evading host immune responses, and facilitating viral replication [10,11]. The MdRVC p10.8 protein localizes to the nucleus and triggers apoptosis [12,13]. The nucleocytoplasmic shuttling protein p17 of ARV is involved in transcriptional regulation, cell cycle arrest, and autophagy induction, thereby promoting viral replication [14,15,16,17,18,19,20]. The nuclear targeting protein σ1s of mammalian orthoreovirus serotype 3 (MRV 3) is associated with cell cycle arrest (in G2/M phase) and the induction of apoptosis [10,21]. NDRV p18 has been reported to be detectable in NDRV-infected duck tissues and cells, and it accumulates in both the nucleus and cytoplasm [22]. The shared capacity for nuclear translocation implies that p18 may be functionally analogous to the aforementioned proteins.

The nuclear localization signal (NLS) mediates the transport of proteins from the cytoplasm into the nucleus [23]. Classical NLS (cNLS) includes the monopartite and bipartite types. The former is usually composed of 4–6 basic amino acids (e.g., arginine and lysine), and its core sequence can be denoted as (K/R)4–6 [23,24,25,26]. The bipartite type comprises two fundamental basic amino acid regions, which are separated by 10 to 12 non-conserved amino acids and can be denoted as (K/R)2-X_(10–12)_-(K/R)3 [23,24,27]. The cNLSs on cargo are recognized by the adaptor protein importin α, which bridges the cargo to importin β, and the trimeric Imp α-Imp β-cNLS-cargo complex enters the nucleus [23]. The nonclassical NLSs (ncNLSs) lack strictly conserved consensus sequences and mediate the nuclear import of cargo via direct binding to specific members of the karyopherin β family [28]. It has been confirmed that MdRVC p10.8 and MRV 3 σ1s harbor functional ncNLS and cNLS, respectively, which are essential for their nuclear import [13,29]. ARV p17 possesses a putative monopartite NLS and an interaction site for heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1). HnRNP A1 interacts directly with p17 and Transportin 1 to form a carrier–cargo complex, thereby modulating the nuclear import of p17 [15,30]. The C-terminal of NDRV p18 contains an acidic amino acid-rich region (^114^PLPVKRRRSFDDTDQIPPKRRRLTQRI^140^) with NLS-like characteristics, and its function requires experimental verification.

Early studies have suggested that p18 could be detected in the nucleus during NDRV infection; however, definitive evidence regarding p18’s nuclear localization, the associated transport receptor, and the potential NLS region in p18 has been lacking. In the present study, we aim to confirm that the nuclear entry of p18 is independent of viral replication, importin α is involved in nuclear import of p18 as a transport receptor, and the C-terminal region of p18 is crucial for its nuclear localization.

2. Materials and Methods

2.1. Cells, Plasmid Construction, and Transfection

BHK-21 (ATCC, CCL-10) cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS) (Thermo Fisher Scientific, Waltham, MA, USA). The cells were maintained at 37 °C in an incubator with 5% CO_2_. To generate the recombinant plasmid pEGG, expressing EGFP-GST fusions, the GST sequence from the pGEX-4T-1 vector was amplified and inserted into the pEGFP-C1 vector. For the construction of plasmids pEGG-p18 and pFlag-p18, the p18 gene was amplified using the DRV 091 strain RNA as the template and cloned into the vectors pEGG and p3×Flag-CMV. pEGG-p18m (118–121, 132–135) was created by mutating amino acids ^118^KRRR^121^ and ^132^KRRR^135^ of p18 to ^118^AAAA^121^ and ^132^AAAA^135^. pEGG-p18N (1–97), expressing the N-terminal 1–97 amino acids of p18, was constructed via the insertion of the synthesized sequence into the plasmid pEGG. To produce a series of plasmids, namely, pCDNA3.1-importin α1, α3, α4, α5, and α7-RFP and pCAGGS-HA-importin α1, α3, α4, α5, and α7, full-length importin α1, α3, α4, α5, and α7 (accession: NM_002266.4, NM_002268.5, NM_002267.4, BC002374.2, and AF060543.1) were synthesized and inserted into vectors pcDNA3.1-RFP and pCAGGS-HA by Beijing Rui Biotech Co., Ltd., Beijing, China. Transfection was performed on preconfluent monolayer cells using Lipofectamine^TM^ 2000 (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions.

2.2. Immunofluorescence and Confocal Microscopy

For the immunofluorescence assays, BHK-21 cells were seeded onto glass coverslips (Thermo Fisher Scientific, Waltham, MA, USA) in 24-well plates and cultured overnight. The plasmids pEGG and pEGG-p18 were individually transfected into the cells, while the plasmids pEGG-p18 and pCDNA3.1-importin α1, α3, α4, α5, and α7-RFP were co-transfected into the cells. After 24 h, the cells were first fixed with 4% paraformaldehyde (Solarbio, Beijing, China) for 20 min, then permeabilized with 0.2% TritonX-100 for 5 min (Beyotime Biotechnology, Shanghai, China), and finally blocked with skim milk for 30 min at 37 °C. The nucleoli were stained with rabbit pAbs to fibrillarin and Cy3-conjugated goat anti-rabbit IgG (H+L) (XMJ Scientific, Beijing, China). The nuclei were stained with DAPI before being observed under a Nikon A1 confocal microscope (Nikon, Tokyo, Japan). The images were exported to and analyzed using NIS-Elements Viewer 5.22.

2.3. Immunoprecipitation (IP)

The plasmid pEGFP-18 and vector pEGFP-C1 were separately transfected into the preconfluent monolayer of BHK-21 cells. After 24 h of transfection, the cells were rinsed with cold PBS (Solarbio, Beijing, China) and lysed using a cell lysate with RIPA (X-BLOT LifeScience, Suzhou, China) at 4 °C for 15 min. Then, the cell debris was removed via centrifugation at 12,000 g for 10 min. The cleared lysate was added to equilibrated GFP-Trap agarose beads (Proteintech, Wuhan, China) and rotated end-over-end at 4 °C overnight. The beads were sedimented using centrifugation at 2500 g for 5 min and washed four times. They were then resuspended in loading buffer and heated at 95 °C for 5 min to dissociate the immunocomplexes from the beads. The IP proteins were separated by 12% SDS-PAGE and stained with Coomassie blue. The distinct bands in the pEGFP-18 group were collected for subsequent protein analysis.

2.4. Data-Independent Acquisition (DIA) Qualitative Proteomics

The gel was destained using a destaining solution (50 mM triethylammonium bicarbonate (TEAB) and 50% acetonitrile (ACN)) and dehydrated with 100% ACN. It was then treated with 10 mM DTT for 40 min at 56 °C, alkylated with 50 mM iodoacetamide for 30 min in the dark, and finally washed with the destaining solution and treated with ACN as described above.

In-gel proteins were digested with 100 mM TEAB and trypsin overnight at 37 °C. Proteins were extracted from the gel with ACN and 0.1% formic acid (FA) and then lyophilized. The powder was dissolved in 0.1% FA and desalinated using a C18 desalting column. The eluents were collected and lyophilized.

Ultra-high-performance liquid chromatography-MS/MS (UHPLC-MS/MS) analysis was conducted on an Orbitrap Astral mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). The mobile phases were as follows: A (99.9% H_2_O and 0.1% FA) and B (80% ACN and 0.1% FA). The powder was dissolved with phase A and then centrifuged at 14,000 g for 20 min at 4 °C. The supernatant sample was injected into the sample for liquid quality detection. A Vanquish Neo UHPLC system was used with a C18 pre-column of 174500 (5 mm × 300 μm, 5 μm; Thermo Fisher Scientific, Waltham, MA, USA) heated at 50 °C and a C18 analytical column of ES906 (150 µm × 15 cm, 2 μm; Thermo Fisher Scientific, Waltham, MA, USA). An easy-spray ion source was used with the ion spray voltage set at 2.0 kV and the ion transfer tube temperature at 290 °C. The MS operated in data-independent acquisition mode with a full first-stage MS scanning range of 380 to 980 m/z. The primary MS resolution was set to 240,000 (200 m/z), AGC to 500%, the parent ion window size to 2 Th, the number of DIA windows to 300, and NCE to 25%. The acquisition range of the secondary MS was from 150 to 2000 m/z, and the sub-ion resolution was 80,000.

The raw data collected using the UHPLC-MS/MS were imported into the Pulsar module of Spectronaut 19.5 for database search and the identification of peptides and proteins. The proteins were qualitatively identified by means of alignment with the UniProt database. The subcellular localization of identified proteins was predicted by Cell-PLoc 2.0 (http://www.csbio.sjtu.edu.cn/bioinf/Cell-PLoc-2/), accessed on 26 March 2025. The functional analysis of proteins in terms of Gene Ontology (GO) and InterPro (IPR) was conducted using InterProScan 5.22-61.0 against databases such as Pfam, ProDom, and SMART [31]. The protein family and pathway analysis was based on the Clusters of Orthologous Groups (COG) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases (http://www.genome.jp/kegg/), accessed on 26 March 2025 [32]. All procedures were carried out in the laboratory of Novogene Co., Ltd., Beijing, China.

2.5. Co-Immunoprecipitation (Co-IP) and Western Blotting

BHK-21 cells were seeded onto a 10 cm dish, and the pFlag-p18 and pCAGGS-HA-importin α1, α3, α4, α5, and α7 plasmids were sequentially co-transfected into the cells. After 24 h, the cells were lysed and centrifuged at 12,000 g for 10 min to obtain the supernatant. The cleared lysate was incubated with HA antibody-conjugated magnetic beads (MedChemExpress, Monmouth Junction, NJ, USA) at 4 °C overnight. The beads were washed and then resuspended in loading buffer and kept at 95 °C for 5 min. The Co-IP proteins were subjected to 12% SDS-PAGE and analyzed through Western blotting with mouse mAbs against Flag (Merck, Darmstadt, Germany) and mouse mAbs to HA (Proteintech, Wuhan, China). HRP-conjugated goat anti-mouse IgG (H+L) (Proteintech, Wuhan, China) was used as a secondary antibody.

2.6. Cell Treatment, Cell Counting Kit-8 Assays and Subcellular Fractionation

To determine the effect of the importin α/β1 pathway inhibitor ivermectin (IVM, MedChemExpress, Monmouth Junction, NJ, USA) on the subcellular distribution of p18, the pFlag-p18 plasmid was transfected into BHK-21 cells for 4 h. Subsequently, the cells were treated with different concentrations of IVM (1 μM and 5 μM) for an additional 24 h. IVM dissolved in DMSO was diluted to the working concentration with culture medium. An equivalent volume of DMSO was added to the cells as a vehicle control.

The Cell Counting Kit-8 (CCK-8) assays were used to assess the cell vitality. BHK-21 cells were seeded in 96-well plates and incubated for 12 h. Subsequently, cells were treated with 1, 5, 10, 15, and 20 μM IVM for 24 h. Then, 10 μL of CCK-8 solution (Beyotime, Shanghai, China) was added to each well, followed by an additional hour of incubation at 37 °C. The absorbance at 450 nm was measured using a microplate reader (BioTek, Winooski, VT, USA).

The nuclear and cytoplasmic fractions of cells were obtained in accordance with the instructions accompanying NE-PER^TM^ Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific, Waltham, MA, USA) and analyzed with a Western blotting assay using rabbit pAbs against Lamin B1, rabbit mAbs against GAPDH (Proteintech, Wuhan, China), and mouse mAbs against Flag (Proteintech, Wuhan, China).

2.7. Molecular Docking

The three-dimensional structures of p18 and importin α1, α3, α4, α5, and α7 proteins were predicted using the AlphaFold3 web server (https://alphafoldserver.com/), which enables high-accuracy protein structure prediction based on deep learning algorithms. For each protein, ten structural models were generated, and the model with the highest confidence score was then selected for subsequent analyses. The processed p18 and importin α1, α3, α4, α5, and α7 structures were imported into the receptor and ligand modules of HDOCK for molecular docking [33]. During the docking process, the entire surface of the protein was defined as the potential binding interface, and all proteins were treated as rigid bodies. The resulting protein–protein interaction models were analyzed using PDBePISA (http://www.ebi.ac.uk/msd-srv/prot_int/), accessed on 12 November 2025, to characterize hydrogen bond interactions and identify key binding residues. The visualization of the docking complexes was carried out using PyMOL 3.1.

3. Results

3.1. P18 Accumulates in the Nucleus of the Transfected Cells

To verify whether the nuclear localization of p18 was dependent on viral infection, the recombinant plasmids pEGFP-GST-p18 and pEGFP-GST were transfected into BHK-21 cells. After 24 h, the subcellular localization of p18 was detected using confocal microscopy. Fluorescence visualization showed that the EGFP-GST-tagged p18 was localized in the nucleus, whereas the EGFP-GST control was predominantly localized in the cytoplasm (Figure 1). Fusion of EGFP-GST to the N-terminus of p18 increased its molecular weight to above 70 kDa, thereby preventing nuclear entry via passive diffusion. This result demonstrated that nuclear targeting of p18 was independent of viral infection, and that p18 was capable of guiding fused proteins into the nucleus.

3.2. Category and Function Prediction of Cellular Proteins Interacting with p18

IP-UHPLC-MS/MS was used to identify cellular proteins interacting with p18. In electrophoretic gels, distinct protein bands were detected in GFP-p18 immunoprecipitates compared with the GFP control (Figure 2a). A total of 1839 proteins were identified from the distinct protein bands and matched against the UniProt database. The subcellular localization of the identified proteins was annotated through the Cell-PLOC 2.0 website. Of 1322 annotated proteins, 513 were nuclear proteins (38.80%), 246 were cytoplasmic proteins (18.61%), and 147 were mitochondrial proteins (11.12%) (Figure 2b; Table S1a).

The Eukaryotic Orthologous Groups (KOGs) annotation analysis showed that a total of 1758 proteins were categorized into 25 subcategories. Among these, “General function prediction only”, “Translation, ribosomal structure and biogenesis”, “Signal transduction mechanisms”, “RNA processing and modification”, “Posttranslational modification, protein turnover, and chaperones”, “Cytoskeleton”, and “Intracellular trafficking, secretion, and vesicular transport” were the top functional classes (Figure 2c; Table S1b).

According to the GO enrichment analyses, 127, 112, and 1001 proteins were assigned to the categories of biological process, cellular component and molecular function. The top three enriched terms for proteins in biological processes were “translation”, “phosphorylation”, and “oxidation–reduction”. In the cellular component, “nucleus”, “intracellular”, and “ribosome” were the top three terms. For molecular function, “protein binding”, “ATP binding”, and “nucleic acid binding” were the top three enriched terms (Table S1c). The top 10 GO terms of each category are shown in Figure S1a.

With regard to IPR enrichment analysis, 873 proteins were aligned to 1475 domain terms, including 90 proteins with “RNA recognition motif domain”, 44 proteins with “WD40 repeat”, and 41 proteins with “protein kinase domain” (Table S1d). The top 20 protein domains are shown in Figure S1b.

3.3. Importin α Is Involved in the Nuclear Transport of p18

We identified a series of importin α subunits by DIA-based qualitative proteomics. To veirify this, the plasmids expressing EGFP-GST-tagged p18 (EGFP-GST-p18) and RFP-tagged importin α (Imp α1-RFP, Imp α3-RFP, Imp α4-RFP, Imp α5-RFP, and Imp α7-RFP) were sequentially co-transfected into BHK-21 cells, and the subcellular localization of the proteins was observed by confocal microscopy. As shown in Figure 3a, EGFP-GST-p18 and Imp α1, α3, α4, α5, and α7–RFP were co-localized in the nucleus. Co-IP assays were used to verify the interaction between importin α and p18. Flag-p18 was detected in proteins co-immunoprecipitated with HA–importin α through Western blotting (Figure 3b). To investigate the effects of the importin α/β1 pathway inhibitor IVM on the subcellular distribution of p18, BHK-21 cells transfected with Flag-p18 were then treated with 1 μM or 5 μM IVM for 24 h. The CCK-8 assays showed that treatment with IVM at concentrations lower than 10 μM did not affect cell vitality (Figure S2). Compared with the untreated and DMSO-treated groups, nuclear accumulation of p18 was decreased in the IVM-treated groups (Figure 3c), indicating that the inhibitor impaired the nuclear transport of p18.

3.4. Prediction of the Interaction Between p18 and Importin α by Molecular Docking

We predicted the protein structures of p18 and importin α based on their amino acid sequences. The docking score was used to evaluate the binding affinity between p18 and importin α (Table S2a). The model with the highest confidence score was selected for subsequent molecular docking analysis. In comparison to importin α3 (ΔiG = −4.9 kcal/mol), α4 (ΔiG = −3.7 kcal/mol), and α7 (ΔiG = −5.3 kcal/mol), importin α1 (ΔiG = −10.8 kcal/mol) and α5 (ΔiG = −9.9 kcal/mol) exhibited stronger binding affinity to p18, as reflected by the calculated binding free energy. The complexes primarily interacted via hydrogen bonds. The key binding residues of p18 interacting with importin α1 were predicted to be Gln23, Tyr54, Tyr55, Glu61, Arg119, and Arg120, while those interacting with importin α5 were Met1, Tyr54, Tyr55, Thr107, Asp109, Thr110, Leu115, and Arg119 (Figure 4; Table S2b).

3.5. The C-Terminal Region Is Crucial for the Nuclear Localization of p18

Sequence analysis of p18 suggested the presence of a putative NLS-containing region (^114^PLPVKRRRSFDDTDQIPPKRRRLTQRI^140^) near the C-terminus of the protein. To determine whether this R/K-rich region was functional, a C-terminal deletion mutant of p18 (EGFP-GST-p18N (1–97)) was constructed. Furthermore, site-directed mutagenesis was performed to convert residues ^118^KRRR^121^ and ^132^KRRR^135^ to ^118^AAAA^121^ and ^132^AAAA^135^ in the EGFP-GST-p18 construct, generating the mutant EGFP-GST-p18m (118–121, 132–135) (Figure 5a). BHK-21 cells were transfected with pEGG-p18m (118–121, 132–135) or pEGG-p18N (1–97) and analyzed at 24 h post-transfection by confocal microscopy. Mutations at the two “KRRR” sites remarkably altered the nucleocytoplasmic distribution of p18 and severely impaired its ability to transport proteins into the nucleus (Figure 5b). Removal of the C-terminus disrupted p18-mediated nuclear localization, as the truncated mutant p18N(1–97) was predominantly localized in the cytoplasm (Figure 5b). These results indicate that the C-terminus of p18 is essential for nuclear localization.

4. Discussion

Previous studies have detected p18 in NDRV-infected cells and duck spleen tissues using specific antibodies and observed the accumulation of p18 in both the nucleus and cytoplasm [22]. We have confirmed that p18 is capable of translocating into the nucleus independently of viral replication or the presence of other viral proteins. For mammalian cells, the permeability of nuclear pore complexes (NPCs) is considered to be 30–60 kDa; small molecules below this molecular weight range (or with a diameter of less than 9 nm) can enter the nucleus via passive diffusion [34]. NPCs allow large molecules with a diameter greater than 26 nm to pass through the channel via active transport [35,36]. The theoretical molecular weight of p18 is approximately 18 kDa. To exclude the possibility that p18 freely diffuses into the nucleus, one tracer protein (EGFP) and one tag protein (GST) were fused to the N-terminus of p18, resulting in the fusion protein exceeding 70 kDa. We observed that the fusion protein EGFP-GST was distributed in the cytoplasm, while EGFP-GST-p18 was predominantly localized in the nucleus. As p18 is highly likely to pass through NPCs via active transport, this suggests that a specific transport receptor is required.

A major functional category of p18-interacting cellular proteins is associated with intracellular trafficking, among which we identified the transport receptor importin α. Given that we predicted a putative NLS region at the C-terminus of p18, this suggests that p18 may employ importin α for its nuclear translocation. We confirmed that the importin α subunits (α1, α3, α4, α5, and α7) interact sequentially with p18 and colocalize with this protein in the nucleus. The importin α/β1 pathway mediates the translocation of proteins harboring cNLSs into the nucleus [37]. Inhibition of the importin α/β1 pathway using IVM reduced nuclear accumulation of p18, indirectly indicating that importin α mediates p18 nuclear translocation. Molecular docking analyses revealed that multiple amino acid residues of p18 are capable of binding to importin α, which provides a framework for the precise validation of the p18-importin α interaction sites. The C-terminus of p18 contains a basic amino acid enrichment domain, with a core sequence ^118^KRRR^121^-X_(10–12)_-^132^KRRR^135^, which conforms to the cNLS. The substitution of residues K/R with A significantly impaired p18’s nuclear localization capacity, suggesting this region constitutes a functional domain involved in nuclear import. However, the crucial functional sites require further validation. Based on the count of stripped sequence identifications derived from UHPLC-MS/MS analysis, hnRNP H1 and hnRNP K accounted for relatively high proportions. Given that hnRNP H1 is a transportin-1 cargo and hnRNP K has been implicated in the replication of viruses [28,38], we further performed Co-IP and IFA to examine their interaction with p18 for reference (Figure S3).

Viral nuclear localization proteins exert multiple biological functions through their interactions with cellular proteins. Thus, functional characterization of the cellular proteins interacting with p18 will facilitate the elucidation of p18’s biological functions. In this study, the first important functional category of p18-interacting proteins is “Translation, ribosomal structure and biogenesis”. Nuclear-localized viral proteins typically hijack host ribosome biogenesis and translation to facilitate viral replication. For instance, Hantavirus nucleocapsid protein recruits activated P58^IPK^ to inhibit the activity of PKR and prevent the phosphorylation of eIF2α, thereby preventing host translation shutoff during viral infection [39]. Henipavirus matrix protein interacts with treacle protein in the nucleolus and mimics the nucleolar DNA damage response to suppress ribosomal RNA synthesis [40]. The second functional category is “Signal transduction mechanisms”. For example, nuclear-localized Zika virus NS5 interacts with IRF3 to repress IRF3-mediated transcriptional activation and downstream signaling [41]. Porcine epidemic diarrhea virus N protein binds to p53 to induce S-phase cell cycle arrest, thereby establishing a permissive microenvironment for viral replication [42]. The third functional category is “RNA processes and modifications”. Human herpesvirus ICP27 protein interacts with spliceosome proteins and functions as an mRNA splicing regulator to inhibit pre-mRNA splicing [43,44]. The fourth functional category is “Posttranslational modification, protein turnover, and chaperones”. The immediate-early ICP0 protein of herpes simplex virus type 1 is essential for the formation of virus-induced nuclear foci and mediates the recruitment and redistribution of Hsc70 and the 26S proteasome to these discrete nuclear foci [45]. In addition, “Cytoskeleton” is another important category. For example, the adenovirus E1B protein specifically interacts with intermediate filaments and the nuclear lamina to disrupt their organization; such perturbation of the nuclear lamina may affect the structure of the nucleus and chromatin, thereby inhibiting DNA degradation and enhancing viral gene expression [46]. We therefore postulate that p18 exerts multiple biological functions through host protein interactions, and the underlying molecular mechanisms warrant further investigation. In this study, mammalian cells were employed to establish comprehensive protein interaction profiles. However, NDRV p18 interacts with avian host proteins during natural infection, which may lead to differential binding affinities for some of the identified interactors.

5. Conclusions

Collectively, our work supports the view that the nuclear entry of NDRV p18 is independent of viral infection. Importin α, as the transport receptor, is implicated in the nuclear import of p18. The C-terminal region of p18 is crucial for nuclear localization.