BTV serotype 10 and BHK 21 cells were procured from the repository (CADRAD, ICAR-IVRI). The BHK 21 cells were cultured in Glasgow minimum essential medium (GMEM, Sigma, USA) supplemented with 2% foetal bovine serum (Hyclone, Thermo Scientific, USA). Fresh BTV 10 cultured on BHK 21 cells was utilized to amplify the NS3 coding region. HEK 293 T cells were employed for transfection in GST pull-down and M2H assays. A fresh sheep lung was obtained from an abattoir at IVRI, Izatnagar for library construction. The tissue was rapidly frozen in liquid nitrogen and stored at −80 °C for further use. The tissue sample was tested for the presence of BTV RNA using real-time reverse transcriptase PCR (q-RT-PCR) (Maan et al., 2016).

The frozen lung tissue was homogenized to extract total RNA using the RNeasy^® Mini kit (Qiagen, Hilden, Germany). The poly (A) mRNA was extracted using MN-NucleoTrap® mRNA kit (Machery-Nagel, Germany) according to the manufacturer’s instructions followed by single-stranded (ss) cDNA synthesis using SMART III oligo (5′- AAG​CAG​TGG​TAT​CAA​CGC​AGA​GTG​GCC​ATT​ATG​GCC​GGG-3′), CDS III primer (5′-ATTCTAGAGGCCGAGGCGGCCGACATG-d(T)30VN-3′) and SMART MMLV Reverse Transcriptase provided in Make Your Own “Mate & Plate” Library System (Clontech, USA). The double-stranded (ds) cDNA was amplified by long distance PCR (LD-PCR) using Advantage 2 PCR kit (Clontech, USA) and size-fractioned using CHROMA-SPIN TE-400 columns (Clontech, Takara, USA). Subsequently, the SmaI-linearized pGADT7-rec vector and purified ds cDNA were co-transformed into Saccharomyces cerevisiae (S. cerevisiae) Y187 cells using YeastMaker™ Yeast Transformation System 2 and cultured on SD/-Leu agar plates at 30 °C for 4 days. The resulting transformants were harvested in freezing medium and stored at −80 °C. Following this, the transformed cells (1 mL) were diluted in YPDA medium, and 100 μL of 10⁻¹, 10⁻², 10⁻³, 10⁻⁴, and 10⁻⁵ dilutions were grown on SD/-Leu plates to estimate library quality parameters. The library’s average insert size and recombination rate were calculated from randomly selected 50 colonies using Matchmaker Insert Check PCR Mix 2 (Clontech, Takara, USA) as described earlier (Mahajan et al., 2015). Plasmids rescued from the selected colonies were re-transformed in competent Escherichia coli (E. coli) DH5α cells and positive transformants were subjected to nucleotide sequencing. The assembled cDNA sequences were submitted to GenBank and annotated for gene ontology (GO) terms using the WEB-based GEneSeTAnaLysiS Toolkit (Liao et al., 2019, WebGestalt).

BTV 10 virus was introduced to BHK 21 cells, and a truncated form of NS3 (NS3_t, consisting of amino acids 1-115) was amplified and inserted into the pGBKT7-BD vector (Clontech, USA) (Chaple et al., 2021). The positive recombinants (pGBKT7-NS3_t) were validated and were re-introduced into competent Y2H Gold cells (Gietz et al., 1995). The transformed mixture was cultured on SD/-Trp, SD/-Trp/X-α-gal (SDO/X), and SD/-Trp/X-α-gal/Aureobasidin A (SDO/X/A) agar plates and then placed in incubator at 30 °C for 3-5 days. Additionally, the empty vector control, along with the positive diploid, and negative diploid controls were similarly transformed to assess the bait toxicity and auto-activation. To analyze expression, a single, large colony of pGBKT7-NS3_t bait, empty pGBKT7-53 (positive control) and Y2H gold (negative control) was cultured overnight in 5 mL SD/-Trp medium at 30 °C and 230 rpm. The overnight culture was centrifuged, re-suspended in 50 mL YPD medium and incubated again at 30 °C and 230 rpm until an OD₆₀₀ of 0.4–0.6 was achieved. The protein lysates were prepared by the TCA method according to the Yeast Protocols Handbook (Clontech), and expression was detected using anti-c-myc monoclonal (Clontech, USA) and anti-NS3 polyclonal antibodies (kindly provided by Damian Vituor, ANSES, Paris, France).

The library scale mating of viral bait protein and characterized library was carried out using Matchmaker two hybrid system (Clontech, USA) as per the manufacturer’s protocol. Briefly, a concentrated bait culture containing >1 × 10⁸ cells/mL was combined with the 1 mL aliquot of the characterized library and suspended in 45 mL of 2X YPDA liquid medium. The mixture was incubated at 30 °C with gentle shaking (30-40 rpm) for 20 h. After incubation, the culture was screened for presence of three lobed zygotes and plated on SD/-Trp, SD/-Leu, and SD/-Leu/-Trp (DDO) plates in dilutions of 10⁻¹, 10⁻², 10⁻³, and 10⁻⁴ to estimate the various mating parameters. The remaining culture was plated on SD/-Leu/-Trp/X-α-gal/AureobasidinA (DDO/X/A) agar plates and incubated at 30 °C for 3-5 days. The interacting blue colonies that developed on DDO/X/A were selected and transferred to SD/-Leu/-Trp/-Ade/-His/X-α-gal/AureobasidinA (QDO/X/A) agar plates for more rigorous screening. QDO/X/A positive colonies were subjected to colony PCR using Matchmaker Insert Check PCR Mix 2 (Clontech, USA) and amplicons were sequenced with a single pass reading from the 5′end on an ABI 3130 Genetic Analyzer using Big dye terminator v3.1 (Applied Biosystems). The colonies with repetitive sequences and the inserts shorter than 500 bp were excluded. The remaining confirmed colonies were streaked again on DDO/X/A agar plates, and plasmids were extracted from the isolated blue colonies. These prey plasmids were re-transformed into competent E. coli DH5α cells, and the plasmids were rescued for sequence analysis.

To evaluate the interactions, small-scale mating was conducted between the NS3_t bait and each selected prey and diploids were plated on DDO, DDO/X/A and QDO/X/A agar plates, alongside the positive and negative mating controls. The positive interactions were identified by development of blue colonies on QDO/X/A plates. Additionally, the interactions were qualitatively confirmed by β-Gal expression level by colony lift filter assay and ONPG liquid culture assay (Mahajan et al., 2021). To measure the β-Gal expression, a 125-mm sterile grade 40 Whatman filter paper (HiMedia, India) was gently pressed on the colonies patched out on DDO and QDO agar plates, immersed in liquid nitrogen for 10 s, thawed it, and then placed on another filter paper pre-soaked with Z-buffer containing X-gal. The filter papers were incubated at 30 °C until a blue colour appeared (within 8 h). The quantitative determination of the interaction’s strength was carried out with the ONPG assay. The β-Gal expression was determined using the formula: 1,000 × OD₄₂₀/(t × V × OD₆₀₀), where OD₄₂₀ represents optical density at 420 nm due to the yellow colour developed, OD₆₀₀ is the cell density of the culture, t is the reaction time in minutes, and V is the volume in ml. Each interaction was assessed in triplicate, and β-Gal% was calculated in comparison to the controls. The interactions exhibiting the manifold higher β-Gal% than their respective prey-pGBKT7-BD combination were marked as positive while those with no or minimal variation in respect to their corresponding control were marked as negative.

The DNA fragment NS3_t was amplified and sub-cloned in pGEX-4T-1 vector (GE healthcare, USA). Similarly, NAP1L1 was amplified from pGADT7-NAP1L1 plasmid (Supplementary Figure S7) and sub-cloned into pCMV-Myc-N vector (Clontech, USA). The resulting recombinant clones were validated by restriction enzyme (RE) analysis and sequencing. The GST-NS3_t protein was expressed in E. coli BL21 (DE3)pLysS and confirmed using an anti-GST antibody (Invitrogen, USA). In parallel, HEK 293 T cells were transfected with Myc-NAP1L1, and expression was assessed using an anti-c-Myc antibody (Clontech, USA). The protein interaction pull down was carried out using the Pierce™ GST Protein Interaction Pull-Down Kit (Thermo Scientific, USA) following the manufacturer’s instructions. Subsequently, the eluted sample was analyzed using anti-GST, anti-c-Myc, and anti-NS3 antibodies.

The DNA fragment NS3_t was amplified and sub-cloned in pCMV-HA-N vector (Clontech, USA). HA-NS3_t plasmid was co-transfected with pCMV-Myc-NAP1L1 in HeLa cells and the cell lysate was harvested 48 h post transfection. The co-IP was performed using Pierce c-Myc-TagIP/Co-IP Kit (Thermo Scientific, USA) as per manufacturer’s instructions. The eluates were then subjected to SDS-PAGE followed immunoblotting using anti-c-Myc, anti-HA and anti-β-actin antibodies.

The M2H assay utilized the Matchmaker™ Mammalian Assay Kit 2 (Clontech, USA) to quantify the secreted alkaline phosphatase (SEAP) level in the supernatant of the mammalian cell culture, providing an indication of PPI intensity. The amplified NS3_t and NAP1L1 were incorporated into the pM and pVP16 vectors, respectively. These recombinant constructs, along with the pG5SEAP reporter plasmid, were co-transfected in HEK 293 T cells. SEAP activity was measured using the Great EscAPe™ SEAP Chemiluminescence Detection Kit (Clontech, USA) after 48 h of incubation.

The quality of the total RNA extracted from sheep lung tissue was evaluated via agarose gel electrophoresis, which revealed distinct 28S and 18S rRNA bands, with the 28S intensity being more than that of 18S rRNA (Supplementary Figure S1). The total RNA concentration was found to be 738ng/ul with an A₂₆₀/A₂₈₀ ratio of 2.1. Following purification, mRNA concentration was determined to be 375 ng/ul with an A₂₆₀/A₂₈₀ ratio of 2.2 and mRNA showed a smear of across the lane with a higher intensity from 500 bp to 6 kb, indicating its high quality for library preparation (Supplementary Figure S1). The ds cDNA synthesized from ss cDNA displayed a widespread smear before purification, however the smear ranged from 500 bp to 5,000 bp post-purification (Supplementary Figure S2). The library’s transformation efficiency was calculated to be 5.6 × 10⁶/µg of pGADT7-rec vector, representing 1.68 × 10⁶ independent clones. Additional parameters for evaluating library competency were also examined (Supplementary Table S1) to determine the library index. The insert size of the library was calculated from the randomly selected 50 colonies and it ranged from 350 bp to 2,500 bp with an average fragment size of 896 bp (Supplementary Figure S3). Since 49 out of 50 colonies were positive, the recombination rate was 98%, indicating the library’s complexity (Supplementary Figure S3). The PCR positive clones from the cDNA library were sequenced by Sanger Sequencing by a commercial vendor (Eurofins India), analyzed using nucleotide BLAST against Nucleotide collection (nr/nt) database and deposited in the GenBank (Supplementary Table S2). The identified protein coding sequences were annotated in GO terms, and their biological processes, molecular function and cellular compartment were determined (Supplementary Figure S4).

The PCR amplified NS3_t amplicon of ∼345 bp was cloned into the pGBKT7-BD vector (Supplementary Figure S5). Upon transformation into Y2H Gold cells, the recombinant pGBKT7-NS3_t plasmid exhibited growth only on SD/-Trp and SDO/X but not on SDO/X/A plates (Figure 1). This confirms that the bait cannot activate the reporter genes in Y2H Gold cells, in the absence of a prey protein. Additionally, on SD/-Trp plates, the transformed pGBKT7-NS3_t plasmid exhibited robust growth with colony size similar to those of the pGBKT7-53 colonies, indicating that the constructed bait is not toxic to the yeast cells (Figure 1). The bait protein was successfully expressed in yeast, as evidenced by the appearance of a protein band at the expected size of ∼34 kDa (Figure 2).

The mating of prey library and bait resulted in a mating efficiency of 3.14%. Approximately 4.9 × 10⁶ clones underwent screening, and all the recorded mating quality parameters were found to be well above the recommended threshold values (Supplementary Table S3). During the preliminary screening, 65 colonies appeared blue on DDO/X/A agar plates. When subjected to a higher-stringency QDO/X/A screening, the number was reduced to 37. The identified interactors were PCR amplified, sequenced, and analyzed by BLAST search. Eight colonies were selected after excluding the repetitive clones and insert fragment <500 bp, and plasmids were rescued for further confirmation (Table 1).

The interaction between the rescued prey plasmids and bait was once again confirmed through small-scale mating of individual prey and bait, along with their respective prey controls. All the prey-bait diploids formed blue colonies on QDO/X/A agar plates, whereas the prey controls did not grow in same manner (Figure 3a). Additionally, the strength of these interactions was verified through β-Gal assays. Seven out of eight prey proteins produced blue colour on X-gal-soaked filter paper, while one prey (DCTN2) did not (Supplementary Figure S6). Similarly, in the ONPG assay, seven prey-bait interactions exhibited higher β-Gal activity, while DCTN2 demonstrated very low β-Gal activity, close to its prey control, and therefore identified as negative interactor (Figure 3b). The seven prey proteins that tested positive in all yeast-based assays were recognized as true interactors and their interpretation with alternative yeast-based assays is mentioned in Table 2.

The coding region of NAP1L1 was amplified, resulting in an expected size band of ∼1,179 bp (Supplementary Figure S7) observed during gel electrophoresis. NAP1L1 was then cloned in the pCMV-Myc-N vector and confirmed through RE digestion (Supplementary Figure S7) and sequencing. Expression analysis of the c-Myc-fused NAP1L1 in transfected HEK 293 T cell lysate revealed an anticipated band size of ∼45 kDa (Figure 4a). Further, NS3_t was sub-cloned in the pGEX-4T-1 vector and its expression as a GST-fused protein in E. coli BL21 (DE3)pLysS resulted in a band with an expected size of∼39 kDa (Figure 4a). A pull-down assay was performed to evaluate the binding ability between the GST-NS3_t and Myc-NAP1L1 fusion proteins. The pull-down elutes determined the formation of GST-NS3_t-Myc-NAP1L1 protein complex, validated by the observation of the targeted bands with anti-GST, anti-Myc, and anti-NS3 antibodies (Figure 4b), confirming the interaction between NS3_t and NAP1L1 in vitro.

Similarly, the interaction of NAP1L1 with NS3_t was examined by Co-IP. As seen in Figure 5, HA-tagged NS3_t co-precipitated with the Myc-tagged NAP1L1, whereas no HA-tagged protein was detected in absence of NAP1L1-Myc.

To validate the NS3_t/NAP1L1 interaction, M2H was performed. NS3_t and NAP1L1 were subcloned into the pM and pVP16 vectors, respectively, and then co-transfected in HEK 293 T cells along with reporter plasmids. The SEAP activity was measured, revealing high SEAP activity for NS3_t/NAP1L1 and pM3-VP16 (Figure 6) compared to other controls, confirming the interaction between NS3_t and NAP1L1.