Archives of Virology (2020) 165:2561–2587 https://doi.org/10.1007/s00705-020-04789-y

ORIGINAL ARTICLE

Inhibition of orf virus replication in goat skin fbroblast cells by the HSPA1B protein, as demonstrated by iTRAQ‑based quantitative proteome analysis

Jun‑hong Hao1 · Han‑jin Kong1 · Ming‑hao Yan1 · Chao‑chao Shen1 · Guo‑wei Xu1 · Da‑jun Zhang1 · Ke‑shan Zhang1 · Hai‑xue Zheng1 · Xiang‑tao Liu1

Received: 8 March 2020 / Accepted: 26 July 2020 / Published online: 2 September 2020 © Springer-Verlag GmbH Austria, part of Springer Nature 2020

Abstract Orf virus (ORFV) infects sheep and goat tissues, resulting in severe proliferative lesions. To analyze cellular protein expres- sion in ORFV-infected goat skin fbroblast (GSF) cells, we used two-dimensional liquid chromatography-tandem mass spectrometry coupled with isobaric tags for relative and absolute quantifcation (iTRAQ). The proteomics approach was used along with quantitative reverse transcription polymerase chain reaction (RT-qPCR) to detect diferentially expressed proteins in ORFV-infected GSF cells and mock-infected GSF cells. A total of 282 diferentially expressed proteins were identifed. It was found that 222 host proteins were upregulated and 60 were downregulated following viral infection. We confrmed that these proteins were diferentially expressed and found that heat shock 70-kDa protein 1B (HSPA1B) was diferentially expressed and localized in the cytoplasm. It was also noted that HSPA1B caused inhibition of viral proliferation, in the mid- dle and late stages of viral infection. The diferentially expressed proteins were associated with the biological processes of viral binding, cell structure, signal transduction, cell adhesion, and cell proliferation.

Introduction factor kappa B (NF‐κB) inhibitory protein (ORFV125), and deoxyuridine 5′-triphosphate pyrophosphatase (dUTPase, Orf, which is caused by orf virus (ORFV), is one of the ORFV007) [38]. most widespread viral diseases worldwide. Although ORFV Like other poxviruses, ORFV encodes a range of mol- mainly infects sheep and goats, it can also infect other rumi- ecules that play vital roles in immune evasion by the pro- nants and other mammals [18]. Genes involved in virulence duction of anti-infammatory proteins [13]. These proteins, and pathogenesis are distributed in the ITR regions of the which are mainly involved in the interaction with host ORFV genome. The virus encodes involved in immunomod- defense mechanisms, include OVIFNR, GIF, vIL-10, and ulation, including viral-interleukin 10 (vIL‐10, ORFV127), VEGF. A number of these proteins are orthologues of known vascular endothelial growth factor (VEGF, ORFV132), orf mammalian proteins, whereas others do not appear to have virus interferon resistance protein (OVIFNR, ORFV020), mammalian counterparts [7, 11–13]. ORFV encodes GIF, chemokine binding protein (CBP, ORFV112), granulo- which is a novel secreted inhibitor of the cytokines GM- cyte–macrophage colony-stimulating factor/interleukin-2 CSF and IL-2 [7]. GIF co-localizes with ORFV in infected (GM-CSF/IL-2) inhibitory protein (GIF, ORFV117), nuclear epidermal cells, as detected by immunohistochemistry (IHC) [13]. Both ORFV and ovine IL-10 (vIL-10 and ovIL- 10) inhibit production of tumor necrosis factor-α (TNF-α) Handling Editor: William G Dundon. and IL-8 from macrophages and keratinocytes as well as production of interferon gamma (IFN-γ) from activated * Ke‑shan Zhang [email protected] lymphocytes [10]. The viral VEGF is important for viru- lence [25, 34, 40]. It is mitogenic to endothelial cells and 1 State Key Laboratory of Veterinary Etiological Biology, promotes angiogenesis in the underlying dermis as well as National Foot-and-Mouth Disease Reference Laboratory, proliferation of epidermal cells and activation of VEGF Lanzhou Veterinary Research Institute of Chinese Academy of Agriculture Science, No. 1, Xujiaping, Lanzhou 730046, receptors. Aside from an array of immunomodulatory fac- Gansu, People’s Republic of China tors produced by ORFV, poxviruses also produced a class

Vol.:(0123456789)1 3 2562 J. Hao et al. of proteins, namely ankyrin (ANK) repeat proteins [28], corresponding to the ORFV B2L gene was sequenced, and which can also promote viral survival. A proteomic analy- found to be identical to an ORFV B2L gene sequence pub- sis of host cellular responses to viral infection may provide lished in the Gene Bank database (GU320351). new insights into the cellular mechanisms involved in viral pathogenesis. Growth curve of ORFV in GSF cells The isobaric tag for relative and absolute quantitation (iTRAQ) technique allows comprehensive, comparative, To analyze the growth of ORFV in GSF cells, the cells were and quantitative determination of protein expression [33]. infected at an MOI of 0.1 in a 24-well plate, and uninfected This technique has been extensively applied to proteome cells served as a control. Samples were collected at 2, 12, 18, analysis [3, 14, 15, 33, 39]. iTRAQ simultaneously identi- 24, 36, 48, and 60 h postinfection. The ORFV copy number fes and quantifes peptides by measuring peak intensities was estimated using RT-qPCR. of reporter ions using tandem mass spectroscopy (MS/MS) and has been developed to identify biomarkers for various Sample preparation viral diseases [2, 41]. This method has been widely utilized to study the mechanisms of viral infection through the com- The cultured GSF cells were divided into an experimental parative analysis of cellular protein profles. In the present group and a control group. When the cells reached about study, a comparative iTRAQ-based proteomic analysis was 80% confuency, 500 μL of ORFV suspension (MOI, 0.1) conducted to analyze the changes in cellular proteins of goat was added to the cells in the experimental group, and skin fbroblast (GSF) cells exposed to ORFV in vitro at spe- DMEM was added to the cells in the control group. The cifc time points. The experiments were performed to inves- cells were incubated for 1 h and washed three times with tigate functional changes occurring in GSF cells infected by phosphate-bufered saline (PBS), and DMEM medium con- ORFV. This is the frst study on the interactions of ORFV taining 2% FBS was added. The cells were collected and pro- with its host using a proteomics approach. The purpose of tein samples were prepared after 35 h. The culture medium the present study was to investigate the adaptability and was discarded, and the cells were washed three times with proliferation of ORFV in goat skin fbroblasts. Hence, the cold PBS, scraped with a cell scraper, and collected in a iTRAQ-2D-LC–MS/MS technology was used to analyze centrifuge tube. The cells were pelleted by centrifugation proteomic changes in ORFV-infected GSF cells. Quantita- at 2000 rpm for 3 min, resuspended and washed two times, tive reverse transcription polymerase chain reaction (RT- and the fnal cell pellet was stored at -80 °C for further qPCR) and other methods were applied to identify diferen- experiments. tially expressed proteins and to further analyze and confrm the function of the diferentially expressed proteins and heat Protein extraction shock 70-kDa protein 1B (HSPA1B) in ORFV-infected host cells. These data provide a foundation for mapping of gene Five hundred μL of protein lysate was added to the sample regulatory networks that are afected by ORFV infection. and lysed on ice. Phenylmethylsulfonyl fuoride (PMSF) and dithiothreitol (DTT) with fnal concentrations of 1 and 10 mM, respectively, were added. After sonication in an ice Materials and methods bath for 15 min, the sample was centrifuged, and the super- natant was treated at 56 °C for 1 h to reduce disulfde bonds. Cell culture and virus infection Next, iodoacetamide (IAM) was added to a fnal concen- tration of 55 mM, and the sample was kept in the dark for GSF cells were purchased from the Kunming Institute of 45 min to block cysteine alkylation. An appropriate amount Zoology, Chinese Academy of Sciences (Kunming, China) of cold acetone was added, and the sample was stored at and cultured in complete Dulbecco’s modified Eagle’s -20 °C for 2 h. The sample centrifuged at 14,000 rpm for medium (DMEM; HyClone Laboratories Inc., Logan, UT, 20 min, the supernatant was discarded, and 200 μl of 0.5 mM USA), containing 10% fetal bovine serum (FBS; Gibco, New tetraethylammonium borohydride (TEAB) was added to the York, NY, USA) in an incubator with 5% ­CO2. The GSF pellet, which was sonicated for 15 min, and centrifuged at cell line was tested for mycoplasma contamination, which 14,000 rpm for 20 min. showed an appropriate microscopic morphology. ORFV was isolated and stored in our laboratory for further experi- Protein concentration measurement ments. ORFV was passaged continuously for 15 generations on GSFs, virus samples were selected after the 1st, 5th, 10th, Protein quantifcation was done by the Bradford method, and 15th generations, and the viral genome was extracted using prepare a standard curve made from series of dilutions for B2L gene detection. A 1137-bp fragment of the genome

1 3 Inhibition of orf virus replication by HSPA1B 2563 of bovine serum albumin (BSA) and measuring absorbance energy of 27 (± 12). Secondary fragments were detected in at 595 nm. Orbi with a resolution of 17,500 FWHM. In addition, 15 secondary spectra were plotted for primary precursor ions Protein digestion and iTRAQ labeling with peak intensities in excess of 20,000, and primary and secondary scanning was performed. Scanned mass-to-charge One hundred μg of protein was trypsinized for 12 h. After ratios ranged from 350 to 2000 Da. enzymatic digestion, the peptide was dried using a vacuum centrifugal pump and reconstituted with 0.5 M TEAB. MS data analysis iTRAQ labeling was done according to the manufacturer’s instructions. Labeling reagent 116 was used to label the The original mass spectrum fle was converted to MGF for- experimental group, and labeling reagent 119 was used mat. The quantitative data were analyzed by iTRAQ Result to label the control group. After incubation at room tem- Multiple File Distiller, and the MGF fle was used as an perature for 2 h, the labeled peptides of each group were original fle with Mascot2.3.02 protein identifcation soft- mixed and separated by liquid-phase chromatography using ware. A selected sheep genome annotation database (22134 a strong cation-exchange (SCX) column. sequences) and ORFV virus from the NCBI database (150 sequences) were used for searching and combined with the SCX chromatography quantitative results.

An LC-20AB high-performance liquid chromatography Bioinformatics analysis (HPLC) pump system (Shimadzu, Tokyo, Japan) and a 4.6 × 250-mm separation column were used for liquid-phase Functional annotation, subcellular localization, and meta- separation of the samples. The mixed peptides were labeled bolic pathway analysis of the identified differentially were reconstituted and 4 ml of bufer A. Gradient elution expressed proteins were carried out. The hypergeometric was performed at a rate of 1 ml/min. The frst elution was test was used to fnd Gene Ontology (GO) entries that were performed with bufer A for 10 min, which was gradually enriched compared with other proteins. These diferentially mixed with 5–35% bufer B for 11 min, and fnally mixed expressed proteins were compared with the Clusters of with 35–80% bufer B and eluted for 1 min. The entire elu- Orthologous Groups of proteins (COG) database to predict tion process was monitored by measuring the absorbance the possible functions of these proteins and perform statisti- at 214 nm, and 20 components were eventually selected. cal analysis on their functional classifcation. Each component was desalted separately using a Strata-X C18 column (Phenomenex, Torrance, CA, USA) and then RT‑qPCR freeze-dried. Specifc primers (Table 1) were designed to amplify various Capillary HPLC target genes simultaneously according to the correspond- ing gene sequences of MS/MS-identifed proteins, and the The concentration of each component that was adjusted to available genetic information was deposited in the Gen- about 0.5 µg/µl with bufer C and centrifuged at 20,000 rpm Bank database. The analysis was conducted using Laser- for 10 min to remove insoluble matter. Eight microliters of gene sequence analysis software (DNASTAR, Inc., Madison, each component (about 4 µg of protein) was separated using WI, USA). GSF monolayers were inoculated with ORFV for a Shimadzu LC-20AD HPLC system (Shimadzu, Tokyo, 35 h and washed three times with ice-cold PBS. They were Japan). The column included a trap column and an analytical then harvested by centrifugation at 1,000 rpm for 10 min. column. The separation parameters were as follows: injec- Total cellular RNA was extracted using an RNeasy Mini tion for 4 min at a fow rate of 8 nL/min, washing at a fow Kit (QIAGEN GmbH, Hilden, Germany) according to the rate of 300 nL/min for 40 min, washing with gradient bufer manufacturer’s protocol. The RNA concentration was meas- D from 2–35% and then from 35–80%, and washing with ured using a spectrophotometer (260/280 nm). RT-qPCR 80% bufer D for 4 min and bufer C for 1 min. was performed using an Mx3005P Real-Time PCR System (Agilent Technologies, Inc., Santa Clara, CA, USA). A Electrospray ionization mass spectrometry (ESI‑MS) reverse transcription step was frst performed at 42 °C for 5 min to make cDNA from total RNA. The PCR procedure The peptides were separated using a Q-Exactive mass spec- consisted of a denaturation step at 95 °C for 10 s, followed trometer. The primary MS resolution was set to 70,000 by 40 cycles of amplifcation, each consisting of an extra full width at half maximum (FWHM). The peptides were denaturation step at 95 °C for 5 s and a primer annealing screened using a high-energy collision mode with a collision step at 60 °C for 34 s. Melting curves were plotted, and

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Table 1 Primers used for the goat target genes based on the corre- in 100 μL of serum-free DMEM, was mixed for 20 min at sponding gene sequences of the identifed proteins. Specifc primers room temperature and subsequently added to each well with were designed to simultaneously amplify various target genes accord- ing to the corresponding gene sequences of identifed proteins, and complete medium for 24 h. The cells were evaluated for pro- the available gene information was deposited in the GenBank data- tein expression by fuorescence microscopy at 24–48 h post- base transfection and then were fxed by conventional methods ′ Gene name Primer sequence (5′ -3′) and stained with 4 ,6-diamidino-2-phenylindole (DAPI). The cellular distribution of HSPA1B was analyzed by confocal BCLF1 F: AAG​ATA​CAT​TTG​AAC​ACG​ACCCG​ laser scanning microscopy (CLSM). R: ATC​CAT​TTC​CAA​CAG​AAC​CAGAC​ PARP1 F: TCG​GGC​TCG​TGG​ACA​TCG​T Measurement of the efect of HSPA1B on ORFV R: GGC​ATC​TGC​TCC​AGT​TTG​T replication COR1B F: TTG​CCC​TTC​TAC​GAC​CCT​GAT​ R: GGC​TCC​TTG​CTG​GTG​AAT​GTA​ In order to examine the infuence of HSPA1B on the prolif- MAP4 F: GGC​TCC​CAA​TGC​TTC​TGC​ eration of ORFV, GSF cells were transiently transfected with R: CCC​GTA​GGC​GGT​TTC​TGT​ pEFGP-HSPA1B plasmids as described previously. Subse- 6PGD F: ATG​GCT​TTG​TGG​TCT​GTG​C quently, the cells were infected with ORFV at an MOI of 0.1. R: CCG​TCT​CAT​GGT​ATC​CCT​GTAT​ After incubation for 1 h, the cells were washed three times G3P F: AAG​TTC​CAC​GGC​ACA​GTC​A with PBS and DMEM containing 2% FBS was added. The R: TGG​TTC​ACG​CCC​ATC​ACA​A cultured cells were collected at 15, 24, and 36 h postinfec- SC24D F: GCT​ATT​ATG​CGG​GTTCG​ tion and the viral DNA was quantitated using RT-qPCR. R: ATC​AAG​GCT​CCA​GTGTC​ ZN207 F: TAC​CTG​GGA​GAA​CAG​ACA​T RNA interference R: GCT​GCG​GTT​GAA​ATG​AAG​T FIS1 F: ACA​GAG​CCG​CAG​AAC​AAC​CA Small interfering RNA (siRNA) was chemically synthe- R: TCC​GAT​GAG​TCC​AGC​CAG​TC sized by Gene Pharma (Shanghai, China). The knockdown CDV3 F: CCT​CCT​GCT​CCA​GTA​GTT​GTT​ of endogenous HSPA1B was carried out by transfection of R: TTG​TGG​TGC​TTT​CCT​TGT​TGT​ GSF cells with HSPA1B siRNA (517) (5′- UUU​GUA​GCU​ VMA5A F: CCA​ACT​GCT​CCT​TGA​GTC​CTA​ CAC​CUG​CAC​CTT-3′), HSPA1B siRNA (1258) (5′- GUU​ R: AGC​TTC​TCC​CAT​CTC​CAC​GA GAA​GAA​GUC​CUG​CAG​CTT-3′), and HSPA1B siRNA PSME2 F: CCC​ACC​CAA​GGA​TGA​TGA​GAT​ (1486) (5′- GUA​GGU​GGU​GAA​GAU​CUG​CTT-3′) using R: GAA​AGC​CGC​ACT​TAG​GGA​CTG​ Lipofectamine 2000. Nontargeting siRNA (NC siRNA) was ACTB F: ACA​CGG​TGC​CCA​TCT​ACG​A used as a negative control. R: TTG​ATG​TCA​CGG​ACG​ATT​TC GAPDH F: AAG​TTC​CAC​GGC​ACA​GTC​A Western blot R: TGG​TTC​ACG​CCC​ATC​ACA​A B2L F: GGG​GCG​GCG​TAT​TCT​TCT​ For Western blotting, target proteins were resolved by R: GCT​GTT​CTT​GGC​GTT​CTC​G SDS-PAGE and transferred to an Immobilon-P membrane HSPA1B F: GGG​AGG​ACT​TCG​ACA​ACA​GG (Millipore). The membrane was blocked and incubated R: GAC​AAG​GTT​CTC​TTG​GCC​CG with appropriate primary antibodies and secondary anti- bodies. Antibody-antigen complexes were visualized using enhanced chemiluminescence detection reagents (Thermo) quantitative analysis of the data was conducted using the [42]. Mouse anti-HSPA1B anti-β-actin antibodies were 7500 Fast System SDS 1.3.1 software (Applied Biosystems, purchased from Abbkine. Mouse anti-B2L antibody was Foster City, CA, USA). Mock-infected GSF cells were used obtained from the Lanzhou Veterinary Research Institute. as a control. The RT-PCR products were analyzed by elec- trophoresis in a 2% agarose gel. Results Subcellular localization of HSPA1B Growth of ORFV in GSF cells Cells were seeded into 6-well plates and cultured overnight in DMEM in the presence of 10% FBS. The transfection In order to assess the growth of ORFV in GSF cells, a mixture, which contained 2.0 μg of plasmid DNA and 6 one-step growth curve was carried out using RT-qPCR. μL of transfection reagent (Invitrogen, Carlsbad, CA, USA) ORFV was adapted to GSF infection by passaging for 15

1 3 Inhibition of orf virus replication by HSPA1B 2565 generations. At diferent time points, the supernatants and Kinetics of ORFV‑induced cytopathology in cultured the cells were collected, and the copy number of viral DNA GSF cells was determined by RT-qPCR to prepare a one-step growth curve. The results indicated that the intracellular viral DNA One of the key parameters for determining virus-induced content increased from 2 to 36 h postinfection and reached alterations is the length of time until a cytopathic efect the highest level at 36 h. The extracellular viral DNA con- (CPE) is observed in the model system. GSF cells were tent gradually increased (Fig. 1A), while the intracellular infected with ORFV at an MOI of approximately 0.1, and viral DNA content decreased during this time period, and monitored for cell viability and CPE. As shown in Fig. 2, the extracellular viral DNA content increased to form an the cells infected with ORFV that were cultured for less than S-shaped curve. The eclipse phase was from 0 to 12 h postin- 24 h demonstrated no detectable CPE (Fig. 2A-B). At 24 h fection, during which the viral content remained at a low postinfection, a small number of cells began to swell and had level. The logarithmic phase was from 12 to 48 h, and the a round shape (Fig. 2C-D). At 60 h, the cells detached from viral DNA content increased, reaching a peak at 48 h, slow- the plate (Fig. 2E) and were completely destroyed after 72 h ing from 48 to 60 h postinfection, and gradually reaching a postinfection (Fig. 2F-G). plateau (Fig. 1B).

Fig. 1 One-step growth curve of ORFV infection in GSF cells. GSF postinfection. RT-qPCR was used to make a one-step growth curve cells were infected with ORFV at an MOI of 0.1 and cell supernatants to measure the copy number of the intracellular virus (Fig. 1A) and and infected cells were collected at 2, 12, 18, 24, 36, 48, and 60 h extracellular virus (Fig. 1B)

Fig. 2 Kinetics of the ORFV-induced cytopathic efect (CPE) in GSF the control group, and after 1 h, the cells were washed three times cells. Cells at about 80% confuency were infected with ORFV at an with PBS, and DMEM 2% FBS was added. Morphological changes MOI of 0.1 or mock infected, and DMEM was added to the cells in were then examined at diferent time points

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Diferentially expressed proteins analyzed ATP hydrolase activity. Molecular-function-based enrich- by iTRAQ‑coupled 2D LC/MS‑MS ment analysis demonstrated that binding, catalytic activ- ity, structural molecule activity, and enzymatic regulation The cellular proteins in ORFV-infected and mock-infected were infuenced by viral infection (Fig. 3B). Biological GSF cells were extracted for iTRAQ analysis. In total, processes ontology refers to the ordered combination of 10,630 peptides and 2,776 proteins were detected. Of these, molecular functions to achieve a wider range of biological 282 proteins were found to be diferentially expressed in functions, such as mitosis or purine metabolism. Enrich- ORFV-infected GSF cells compared with the mock-infected ment analysis using biological processes indicated that viral GSF cells, including 222 signifcantly upregulated proteins infection mainly afected cellular and metabolic processes and 60 signifcantly downregulated proteins. The upregu- (Fig. 3C). A protein may have multiple GO annotations. lated proteins are listed in Table 2, and the downregulated To determine which biological functions were associated proteins are listed in Table 3. Three proteins that are involved with diferentially expressed proteins, a signifcant enrich- in cell killing showed altered expression levels. The levels ment analysis of GO functions was carried out on these of CXCL6, IFNG (interferon gamma [IFNγ]), and ALBU diferentially expressed proteins. The results revealed that increased after infection. In addition, 28 proteins involved these diferentially expressed proteins were mainly local- in cell proliferation exhibited diferential expression. NFIP1, ized in the cytoplasm, ribosomes and the nucleus (Fig. 3D). IFNG, TKT, CD9, BAK, HS71B, CSK2B, NPM, HDGF, Molecular enrichment analysis showed that these diferen- PA2G4, CDV3, RL23A, THIO, MCM7, CXCL6, YBOX1, tially expressed proteins included proteins with nucleic acid RS4X, CYR61, HMOX1, APOD, TANA, RS6, and PAI1 binding activity, threonine peptidase activity, and growth were upregulated, while CAV1, GBG2, ZPR1, MK01, and factor binding activity (Fig. 3E). Gene set enrichment analy- LAMB2 were downregulated. CXCL6 and IFNG are associ- sis revealed that these diferentially expressed proteins were ated with pathways involved in apoptosis [5, 22] and prolif- mainly involved in the initiation and termination of tran- eration [23, 29]. Changes in the levels of biological adhesion scription, which are related to virus replication and signal proteins were also detected, including PAI1, APOD, CTGF, transduction processes (Fig. 3F). The identifed diferentially ADAM9, EZRI, CYR61, CD9, and TKT, which were upreg- expressed proteins were compared with the COG database, ulated, and TBCD, VINC, CO3A1, 2AAA, LAMB2, and and possible functions of these proteins were predicted. PARVA were downregulated. The abundance of the 70-kDa COG annotation classifcation indicated that the diferen- heat shock proteins (HSPs) HSPA6 and HSPA1B increased tially expressed proteins were mainly involved in functions after infection. Moreover, several ribosomal proteins, includ- such as post-translational modifcation, protein folding, ing RPS6, RPS17, RPS3, RPS21, RPS18, RPS3A, Rps16, molecular chaperones, translation, ribosome structure and Rps8, RPS4X, Rps23, RPS2, RPS19, RPS12, Rps13, and biosynthesis, energy generation and conversion, and signal RPS20 were upregulated (Table 2). However, no ribosomal transduction (Fig. 3G). proteins were downregulated after infection (Table 3). Confrmation of proteomic data by RT‑qPCR Functional classifcation of diferentially expressed proteins Changes in transcription of 12 genes selected from the dif- ferentially expressed proteins were analyzed by quantify- To understand the implications of the cellular responses to ing their mRNA transcripts. The “β-actin” (ACTB) gene ORFV infection, these proteins were categorized into three was used as a control. The pattern of diferences in mRNA main types using the UniProt Knowledgebase (Swiss-Prot/ abundance for these genes between infected and control TrEMBL) and the GO databases: cellular components, meta- GSF cells was similar to the pattern observed for the cor- bolic functions, and biological processes. Cell component responding proteins based on LC–MS/MS data. As shown ontology refers to subcellular structures, locations, and in Fig. 4, the abundance of BCLF1, MAP4, 6PGD, G3P, macromolecular complexes, such as nucleoli, telomeres, ZN207, and CDV3 mRNA increased. The SC24D, FIS1, and complexes that recognize initiation. Cellular-compo- COR1B, and PSME2 genes were downregulated, whereas nent-based enrichment analysis identified differentially the PARP1 gene was upregulated. An inconsistency between expressed proteins that are well distributed in cell compo- the RT-qPCR data and the LC–MS/MS data was observed nents (Fig. 3A) and are mainly involved in morphogene- for VMA5A, which was downregulated in the RT-qPCR sis, protein synthesis, metabolism, the stress response, the analysis and upregulated in the LC–MS/MS analysis. This ubiquitin–proteasome pathway, cellular processes, metabolic inconsistency might have been due to post-translational processes, biological regulation, and response to stimuli. modifcations, such as methylation, phosphorylation, or Molecular function ontology refers to the function of an acetylation, or to diferences in protein degradation rates individual gene product, such as carbohydrate binding or for unknown reasons. These data provide transcriptional

1 3 Inhibition of orf virus replication by HSPA1B 2567 lase EF-1beta domain) domain) domain) thioredoxins domain) diketogulonate reductase diketogulonate protein COG function descriptionCOG Ribosomal protein S6E (S10) Ribosomal protein - - hydro S-adenosylhomocysteine - Ribosomal protein S17E Ribosomal protein - Glucan phosphorylase Translation elongation factor factor elongation Translation Ribosomal protein S3 Ribosomal protein Transketolase RNA-binding proteins (RRM proteins RNA-binding RNA-binding proteins (RRM proteins RNA-binding - - RNA-binding proteins (RRM proteins RNA-binding - Thiol-disulfde isomerase and Thiol-disulfde isomerase RNA-binding proteins (RRM proteins RNA-binding Histone H2A Histone - - Cold shock proteins Cold shock - Aldo/keto reductases, to related Aldo/keto Leucine-rich (LRR) repeat form protein 70 kDa protein nucleoprotein M nucleoprotein protein 2 protein 1 binding protein 1 binding protein domain-containing protein domain-containing protein 5/6 +)] [NADP( protein 59 protein 1.2 relative to the to group at the control same time postinfection 1.2 relative Protein descriptionProtein 40S ribosomal S6 protein Interferon gamma Interferon Adenosylhomocysteinase Protein FAM98B Protein 40S ribosomal S17 protein CD9 antigen Glycogen phosphorylase, liver phosphorylase, liver Glycogen Elongation factor 1-delta factor Elongation 40S ribosomal S3 protein Transketolase U1 small nuclear ribonucleo - Heterogeneous nuclear ribo - Heterogeneous Selenium-binding protein 1 Selenium-binding protein Far upstream element-binding upstream Far Polyadenylate-binding protein protein Polyadenylate-binding LDLR chaperone MESD LDLR chaperone Thioredoxin ELAV-like protein 1 protein ELAV-like Histone H2A.V Histone Tropomyosin beta chain Tropomyosin 40S ribosomal S21 protein Nuclease-sensitive element- Nuclease-sensitive RANBP2-like and GRIP RANBP2-like Alcohol dehydrogenase Alcohol dehydrogenase Leucine-rich repeat-containing > Protein Protein abbreviation RPS6 IFNG AHCY FAM98B RPS17 CD9 PYGL EEF1D RPS3 TKT SNRNP70 HNRNPM SELENBP1 Khsrp PABPC1 MESDC2 TXN ELAVL1 h2afv TPM2 RPS21 YBX1 RGPD5 AKR1A1 LRRC59 vorfM_116: CK_119 1.263 1.472 1.234 1.319 1.219 1.392 1.247 1.347 1.337 1.267 1.231 1.343 1.226 1.261 1.398 1.317 1.246 1.291 1.352 1.335 2.11 1.974 1.418 1.32 1.332 3 1 5 1 2 4 7 4 8 5 3 9 8 4 5 3 5 1 2 1 2 1 4 5 13 Unique Unique peptide 4 1 5 1 2 4 7 4 8 5 3 9 9 7 5 3 5 3 8 1 3 1 4 5 13 Peptide (95%) 4.2 2.8 9.3 0.3 21.1 14.2 25.2 25.1 12.3 39.9 14.7 16.1 12.7 41.7 16.3 12.8 18.1 34 18.4 %Cov (95%) 20.5 19.1 12.3 20.6 16.6 19 82 45 58 61 48 71 94 50 19 63 185 130 190 306 244 131 361 379 166 266 241 208 167 188 1243 Score Accession no Accession 1::GOAT_ENSP00000369757 1::GOAT_ENSBTAP00000016634 1::GOAT_ENSBTAP00000024092 1::GOAT_ENSBTAP00000029157 1::GOAT_ENSBTAP00000000814 1::GOAT_ENSBTAP00000019643 1::GOAT_ENSBTAP00000015277 1::GOAT_ENSP00000410059 1::GOAT_ENSP00000278572 1::GOAT_ENSP00000391481 1::GOAT_ENSBTAP00000002587 1::GOAT_ENSP00000325376 1::GOAT_ENSP00000408263 1::GOAT_ENSBTAP00000027991 1::GOAT_ENSP00000313007 1::GOAT_ENSBTAP00000002045 1::GOAT_ENSBTAP00000041860 1::GOAT_ENSP00000264073 1::GOAT_ENSBTAP00000022576 1::GOAT_ENSP00000367550 1::GOAT_ENSBTAP00000027713 1::GOAT_ENSBTAP00000023094 1::GOAT_ENSBTAP00000021345 1::GOAT_ENSBTAP00000000630 1::GOAT_ENSP00000225972 ratios had expression GSF cells. These proteins of ORFV-infected analysis iTRAQ identifed by proteins Upregulated 2 Table Group ID Group 1 3 2 4 5 6 8 7 9 12 11 10 13 15 14 18 17 16 19 20 21 22 23 25 24

1 3 2568 J. Hao et al. protein, contains CCCH-type protein, protein Zn-fnger ferases (snRNP) homolog domain) cases, SNF2 family subunits helicases COG function descriptionCOG - - Ribosomal protein S13 Ribosomal protein Uncharacterized conserved conserved Uncharacterized - methyltrans SAM-dependent Small nuclear ribonucleoprotein RNA-binding proteins (RRM proteins RNA-binding Superfamily II DNA/RNA heli - II DNA/RNA Superfamily - - FOG: TPR repeat FOG: - - - Glucose-6-phosphate isomerase - - Ribosomal protein S3AE Ribosomal protein 20S proteasome, alpha and beta 20S proteasome, Superfamily II DNA and RNA and RNA II DNA Superfamily 1 - = phosphate phosphodiesterase phosphate phosphodiesterase beta-3 15 SV containing protein transferase 1 transferase tein F factor 7 factor associated actin-dependent of chromatin regulator A member 5 subfamily factor 1-beta factor nucleoprotein H nucleoprotein copeptide repeat-containingcopeptide alpha protein hydrolase isozyme L5 hydrolase protein 1B protein ase type-1 Protein descriptionProtein 1-phosphatidylinositol 4,5-bis - 1-phosphatidylinositol Target of EGR1 protein 1 of EGR1 protein Target 40S ribosomal S18 protein Zinc fnger CCCH domain- Zinc fnger - N-methyl arginine Protein Small nuclear ribonucleopro - Serine/arginine-rich splicing SWI/SNF-related matrix- SWI/SNF-related Transcription intermediaryTranscription Heterogeneous nuclear ribo - Heterogeneous Small glutamine-rich tetratri - Ubiquitin carboxyl-terminal carboxyl-terminal Ubiquitin A-kinase anchor protein 12 protein anchor A-kinase Microtubule-associated Microtubule-associated Glucose-6-phosphate isomer Protein CYR61 Protein RNA-binding protein 45 protein RNA-binding 40S ribosomal S3a protein Proteasome subunit beta Proteasome Nucleolar RNA helicase 2 RNA Nucleolar Protein Protein abbreviation PLCB3 TOE1 RPS18 ZC3H15 PRMT1 Snrpf Srsf7 SMARCA5 TRIM28 HNRNPH1 ​ SGTA UCHL5 AKAP12 MAP1B GPI CYR61 RBM45 RPS3A PSMB1 DDX21 vorfM_116: CK_119 1.578 1.441 1.474 1.288 1.221 1.376 1.527 1.24 1.274 1.44 1.488 1.266 1.236 1.206 1.771 1.266 1.424 1.425 1.381 1.294 1 2 3 3 3 2 1 4 3 2 1 3 6 8 5 2 6 5 5 6 Unique Unique peptide 1 2 3 3 3 2 2 4 3 8 1 3 6 9 5 2 6 5 5 6 Peptide (95%) 0.7 5.8 4.9 6.6 5.3 7.8 6.3 5.5 7.6 13.8 11.2 10 10.9 10.9 25.2 12.2 %Cov (95%) 14.7 22.7 27.4 11 75 80 87 38 26 64 111 262 102 113 169 485 194 297 137 141 103 234 101 179 Score Accession no Accession 1::GOAT_ENSP00000279230 1::GOAT_ENSBTAP00000040666 1::GOAT_ENSP00000416110 1::GOAT_ENSBTAP00000029007 1::GOAT_ENSP00000246789 1::GOAT_ENSBTAP00000021643 1::GOAT_ENSP00000325905 1::GOAT_ENSBTAP00000004408 1::GOAT_ENSP00000327539 1::GOAT_ENSBTAP00000020081 1::GOAT_ENSP00000356420 1::GOAT_ENSP00000253332 1::GOAT_ENSP00000296755 1::GOAT_ENSBTAP00000008386 1::GOAT_ENSBTAP00000012977 1::GOAT_ENSBTAP00000008111 1::GOAT_ENSBTAP00000013079 1::GOAT_ENSP00000262193 1::GOAT_ENSP00000346120 (continued) 1::GOAT_ENSP00000253024 2 Table ID Group 26 27 33 32 31 30 29 28 34 35 36 37 38 39 40 41 42 45 44 43

1 3 Inhibition of orf virus replication by HSPA1B 2569 plex, subunit RPN10/PSMD4 plex, replication control, Cdc46/Mcm control, replication family aldolase subunits domain) subunits COG function descriptionCOG - 26S proteasome regulatory26S proteasome com - Predicted ATPase involved in involved ATPase Predicted Fructose-1,6-bisphosphate Ribosomal protein L2 Ribosomal protein - 20S proteasome, alpha and beta 20S proteasome, Ferritin-like protein - - - - - RNA-binding proteins (RRM proteins RNA-binding PPE-repeat proteins PPE-repeat - Bacterial lipocalin - Ribosomal protein L14 Ribosomal protein Casein kinase II, beta subunit - 20S proteasome, alpha and beta 20S proteasome, - 3B regulatory subunit 4 factor MCM7 factor lase A homolog type-7 homolog G 1 protein 4 protein homolog beta nucleoprotein U nucleoprotein mitochondrial type-1 Protein descriptionProtein Bone morphogenetic protein Bone morphogenetic protein 26S proteasome non-ATPase non-ATPase 26S proteasome DNA replication licensing replication DNA Fructose-bisphosphate aldo - 60S ribosomal L8 protein UPF0539 protein C7orf59UPF0539 protein Proteasome subunit alpha Proteasome Ferritin (Fragment) light chain Olfactory receptor 10AG1 receptor Olfactory Suppressor of SWI4 1 of SWI4 Suppressor Caldesmon V-type proton ATPase subunit ATPase proton V-type Microtubule-associated Microtubule-associated Transformer-2 protein protein Transformer-2 Ribosome-binding protein 1 Ribosome-binding protein Protein HEXIM1 Protein Apolipoprotein D Apolipoprotein Heterogeneous nuclear ribo - Heterogeneous 39S ribosomal L14, protein Casein kinase II subunit beta Serum albumin Proteasome subunit alpha Proteasome Tight junction protein ZO-1 junction protein Tight Protein Protein abbreviation GDF10 PSMD4 MCM7 ALDOA RPL8 CG059 PSMA7 FTL OR10AG1 Ppan CALD1 ATP6V1G1 MAP4 TRA2B RRBP1 HEXIM1 APOD HNRNPU MRPL14 Csnk2b ALB PSMA1 TJP1 vorfM_116: CK_119 1.413 1.404 1.245 1.216 1.511 1.675 1.265 1.255 1.46 1.34 1.666 1.205 1.511 1.695 1.379 1.415 1.454 1.325 1.289 1.332 1.359 1.571 1.357 2 3 6 2 1 5 1 1 1 8 2 5 8 2 2 6 2 2 7 4 4 11 13 Unique Unique peptide 2 3 6 2 1 5 3 1 1 8 2 5 8 2 2 6 2 2 7 4 4 11 13 Peptide (95%) 6.7 4.6 2.3 1.8 8.4 9.7 6.6 8.9 8.1 2.7 10.3 11 40.9 14 31.5 25.7 17.9 16.9 24.7 %Cov (95%) 13.2 10.3 13.3 20.5 90 69 65 23 45 61 46 80 41 49 71 47 382 162 134 255 107 300 232 364 162 293 1208 Score Accession no Accession 1::GOAT_ENSBTAP00000001351 1::GOAT_ENSP00000357876 1::GOAT_ENSP00000307288 1::GOAT_ENSP00000378669 1::GOAT_ENSP00000262584 1::GOAT_ENSBTAP00000019980 1::GOAT_ENSP00000359910 1::goat_GLEAN_10016260 1::GOAT_ENSP00000324111-D9 1::GOAT_ENSP00000377385 1::GOAT_ENSBTAP00000018566 1::GOAT_ENSBTAP00000000240 1::GOAT_ENSP00000407602 1::GOAT_ENSP00000376290 1::GOAT_ENSBTAP00000053740 1::GOAT_ENSP00000328773 1::GOAT_ENSBTAP00000043789 1::goat_GLEAN_10013438 1::GOAT_ENSBTAP00000015619 1::GOAT_ENSP00000415615 1::GOAT_ENSBTAP00000022763 1::GOAT_ENSP00000315309 1::GOAT_ENSP00000281537 (continued) 2 Table Group ID Group 49 48 47 46 50 52 51 53 54 55 56 57 59 58 60 61 62 63 64 65 67 66 68

1 3 2570 J. Hao et al. chaperone implicated in de novo in de novo implicated chaperone folding protein specifcities (related to short- to specifcities (related alcohol dehydrogenases) chain specifcities (related to short- to specifcities (related alcohol dehydrogenases) chain COG function descriptionCOG Methionine aminopeptidase Methionine - Predicted prefoldin, molecular prefoldin, Predicted Dehydrogenases with diferent Dehydrogenases - - Ribosomal protein S9 Ribosomal protein - - - - Ribosomal protein S8E Ribosomal protein DnaJ-class molecular chaperone Dehydrogenases with diferent Dehydrogenases - - - - - FOG: TPR repeat FOG: - tein 2G4 SDR family member 1 SDR family tion factor 3 subunit D tion factor ribonucleoprotein U-like ribonucleoprotein U-like 2 protein protein 1 protein protein member 4 nase-like protein 2 protein nase-like beta-2 factor PE = 2 = 1 SV protein Protein descriptionProtein - pro Proliferation-associated Nucleophosmin Prefoldin subunit 5 Prefoldin Dehydrogenase/reductase Dehydrogenase/reductase Matrin-3 Eukaryotic translation initia - translation Eukaryotic 40S ribosomal S16 protein Tanabin Heterogeneous nuclear Heterogeneous CUB domain-containing Protein kinase C delta-binding Protein 40S ribosomal S8 protein DnaJ homolog subfamily B DnaJ homolog subfamily - dehydroge Hydroxysteroid Sodium channel subunit Sodium channel RNA-binding protein 12B protein RNA-binding Hepatoma-derived growth growth Hepatoma-derived OS = Bos taurus GN = HDGF Hemopexin 182 kDa tankyrase-1-binding Nephrocystin-3 Clathrin B light chain PUL2 Protein Protein abbreviation PA2G4 NPM1 PFDN5 DHRS1 MATR3 EIF3D Rps16 TANA HNRN - CDCP1 PRKCDBP Rps8 DNAJB4 HSDL2 SCN2B RBM12B HDGF HPX TNKS1BP1 NPHP3 CLTB vorfM_116: CK_119 1.42 1.45 1.228 1.234 1.235 1.455 1.283 1.307 1.207 1.261 1.488 1.564 1.303 1.239 1.225 1.363 1.591 1.455 1.274 2.199 1.414 5 3 2 2 2 1 6 8 2 7 3 7 1 4 2 3 3 1 2 11 25 Unique Unique peptide 5 5 2 2 2 1 6 8 2 7 3 7 1 4 2 3 3 1 2 11 25 Peptide (95%) 8.6 4 7.5 9.3 4.1 5.3 6.3 2.9 0.5 14.4 24.9 20.8 13.8 27.6 12.7 12 14.9 42.5 20.1 %Cov (95%) 12.4 12 75 56 83 44 37 51 46 67 27 69 125 229 102 217 192 285 147 111 104 1401 1438 Score Accession no Accession 1::GOAT_ENSBTAP00000013796 1::goat_GLEAN_10013034 1::GOAT_ENSBTAP00000011762 1::GOAT_ENSBTAP00000013522 1::GOAT_ENSP00000422319 1::GOAT_ENSP00000380362 1::GOAT_ENSP00000367806 1::GOAT_ENSP00000357206 1::GOAT_ENSP00000301785 1::GOAT_ENSBTAP00000016560 1::GOAT_ENSBTAP00000026323 1::GOAT_ENSP00000379888 1::GOAT_ENSBTAP00000028994 1::GOAT_ENSP00000381785 1::GOAT_ENSBTAP00000021229 1::GOAT_ENSBTAP00000017837 1::GOAT_ENSBTAP00000008609 1::GOAT_ENSBTAP00000004635 1::GOAT_ENSP00000350990 1::GOAT_ENSBTAP00000007584 1::GOAT_ENSP00000309415 (continued) 2 Table ID Group 69 72 71 70 73 74 75 76 77 78 79 80 82 81 83 84 85 86 87 88 89

1 3 Inhibition of orf virus replication by HSPA1B 2571 domain) protein fmbrin/plastin (EF- protein Hand superfamily) COG function descriptionCOG - Enolase RNA-binding proteins (RRM proteins RNA-binding - - Serine inhibitor protease - Ribosomal protein S4E Ribosomal protein Ca2 + -binding actin-bundling - Ribosomal protein S12 Ribosomal protein - - - - Molecular chaperone - - Cold shock proteins Cold shock Ribosomal protein S5 Ribosomal protein - factor 1 factor killer tor 1 tor [ubiquinone] favoprotein 3, favoprotein [ubiquinone] mitochondrial isoform factor 2 factor sapiens family member 1 family binding protein 1 binding protein protein 1 protein Protein descriptionProtein Protein SET Protein Beta-enolase Serine/arginine-rich splicing Bcl-2 homologous antagonist/ C-X-C motif chemokine 6 chemokine motif C-X-C Plasminogen activator inhibi - activator Plasminogen NADH dehydrogenase dehydrogenase NADH 40S ribosomal S4, X protein Cytospin-A Interleukin enhancer-binding enhancer-binding Interleukin 40S ribosomal S23 protein Histone Histone H1x OS = Homo RNA-binding protein FUS protein RNA-binding Tropomyosin alpha-3 chain Tropomyosin 60S ribosomal L22 protein Heat shock 70 kDa protein 6 70 kDa protein Heat shock ELKS/Rab6-interacting/CAST ELKS/Rab6-interacting/CAST Histone H1.2 Histone Nuclease-sensitive element- Nuclease-sensitive 40S ribosomal S2 protein Polypyrimidine tract-binding tract-binding Polypyrimidine Protein Protein abbreviation SET ENO3 Srsf1 BAK1 CXCL6 SERPINE1 NDUFV3 RPS4X SPECC1L ILF2 Rps23 H1FX FUS Tpm3 RPL22 HSPA6 ERC1 HIST1H1C YBX1 RPS2 PTBP1 vorfM_116: CK_119 1.376 1.632 1.348 1.216 1.322 1.397 1.211 1.229 1.258 1.272 1.38 1.293 1.36 1.342 1.232 1.497 1.325 1.6 1.46 1.519 1.222 4 1 9 2 3 5 5 6 3 5 2 1 3 3 3 2 9 3 2 9 3 Unique Unique peptide 4 6 9 2 3 5 5 6 3 5 2 1 3 5 3 4 9 3 3 9 4 Peptide (95%) 3.2 6.2 6.4 9.6 9.2 16.8 22.3 30.6 11.5 50 18.2 16.6 28.6 21.1 15.5 %Cov (95%) 48.1 29.3 14.6 19.6 37.4 11 31 52 67 71 96 94 136 682 515 109 208 206 353 210 721 327 133 151 182 283 212 Score Accession no Accession 1::GOAT_ENSP00000361777 1::GOAT_ENSP00000370739 1::GOAT_ENSP00000401336 1::GOAT_ENSP00000353878 1::GOAT_ENSBTAP00000012939 1::GOAT_ENSBTAP00000019232 1::GOAT_ENSBTAP00000041265 1::GOAT_ENSP00000362744 1::GOAT_ENSBTAP00000029400 1::GOAT_ENSP00000355011 1::GOAT_ENSP00000296674 1::GOAT_ENSBTAP00000053088 1::GOAT_ENSBTAP00000007571 1::goat_GLEAN_10000207 1::GOAT_ENSBTAP00000019184 1::GOAT_ENSBTAP00000051168 1::GOAT_ENSBTAP00000053339 1::GOAT_ENSBTAP00000051256-D3 1::GOAT_ENSBTAP00000012735 1::GOAT_ENSBTAP00000012544 1::GOAT_ENSP00000349428 (continued) 2 Table Group ID Group 90 92 91 93 94 95 96 98 97 99 100 101 102 103 104 105 106 107 108 109 110

1 3 2572 J. Hao et al. pol III transcript stabilizing La-motif- and related protein in involved containing proteins translation ing a von Willebrand factor type factor Willebrand ing a von domain A (vWA) subunits amidase superfamily domain) COG function descriptionCOG - La protein, small RNA-binding small RNA-binding La protein, Ribosomal protein L18E Ribosomal protein - - - - Uncharacterized protein contain- protein Uncharacterized - Ribosomal protein L18 Ribosomal protein - 20S proteasome, alpha and beta 20S proteasome, Putative intracellular protease/ intracellular Putative - Permeases of the major facilitator Permeases facilitator of the major Ribosomal protein L30/L7E Ribosomal protein Splicing factor 3a, subunit 3 Splicing factor Ribosomal protein L36E Ribosomal protein - - RNA-binding proteins (RRM proteins RNA-binding - cyclin-dependent kinase cyclin-dependent mediate chain 2 mediate chain protein 1 protein heavy chain H3 chain heavy phosphatase 4 regulatory subunit 4 type-4 factor 1 factor tated transporter glucose member 1 3 Protein descriptionProtein Paralemmin-2 Lupus La protein homolog La protein Lupus 60S ribosomal L18 protein Succinate receptor 1 Succinate receptor substrate 1 substrate Nuclear ubiquitous casein and ubiquitous Nuclear Cytoplasmic dynein 1 inter Cytoplasmic Fragile X mentalFragile retardation Inter-alpha-trypsin inhibitor inhibitor Inter-alpha-trypsin Serine/threonine-protein 60S ribosomal L5 protein Transferrin receptor protein 1 protein receptor Transferrin Proteasome subunit alpha Proteasome Protein DJ-1 Protein Bcl-2-associated transcription - 2, facili Solute carrier family 60S ribosomal L7 protein Splicing factor 3A subunit 3 Splicing factor 60S ribosomal L36 protein Ezrin Kinectin Putative RNA-binding protein protein RNA-binding Putative Protein Protein abbreviation PALM2 SSB RPL18 SUCNR1 NUCKS1 DYNC1I2 FMR1 ITIH3 PPP4R4 RPL5 TFRC PSMA4 PARK7 BCLAF1 SLC2A1 RPL7 SF3A3 RPL36 EZR KTN1 RBM3 vorfM_116: CK_119 1.418 1.485 1.273 1.352 1.616 1.525 1.509 1.368 1.349 1.535 1.262 1.334 1.292 2.17 1.218 1.512 1.27 1.235 1.206 1.239 1.656 3 6 3 1 2 2 3 5 1 7 2 5 2 2 6 6 2 6 8 1 18 Unique Unique peptide 3 6 3 1 2 2 4 5 1 7 2 5 3 2 9 6 2 8 1 18 Peptide (95%) 10 4.3 1.9 8.4 7 8.8 6.1 0.7 6.9 4.5 3.8 7.5 8.1 19.5 19.7 25 27.8 38.6 38.8 %Cov (95%) 15.6 28.4 16.4 47 28 55 89 32 129 300 286 100 106 154 186 133 776 249 155 174 137 518 262 111 Score Accession no Accession 1::GOAT_ENSBTAP00000053296 1::GOAT_ENSBTAP00000011484 1::GOAT_ENSBTAP00000020452 1::GOAT_ENSBTAP00000049167 1::GOAT_ENSBTAP00000049804 1::goat_GLEAN_10006421 1::GOAT_ENSP00000359506 1::GOAT_ENSP00000415769 1::GOAT_ENSBTAP00000042575 1::GOAT_ENSP00000359345 1::GOAT_ENSP00000353224 1::GOAT_ENSBTAP00000019203 1::GOAT_ENSP00000340278 1::GOAT_ENSP00000376159 1::GOAT_ENSBTAP00000040563 1::GOAT_ENSP00000379733 1::GOAT_ENSP00000362110 1::GOAT_ENSBTAP00000002349 1::goat_GLEAN_10017787 1::GOAT_ENSP00000378720 1::GOAT_ENSP00000365950 (continued) 2 Table ID Group 112 111 113 114 115 116 118 117 119 120 123 122 121 125 124 126 127 128 129 130 131

1 3 Inhibition of orf virus replication by HSPA1B 2573 dependent) and related alpha- dependent) and related acid dehydrogenases hydroxy subunits domain) in cytokinesis, containsin cytokinesis, TGc (transglutaminase/protease-like) domain COG function descriptionCOG - L-lactate dehydrogenase (FMN- L-lactate dehydrogenase Triosephosphate isomerase Triosephosphate 20S proteasome, alpha and beta 20S proteasome, - - Histidyl-tRNA synthetase Histidyl-tRNA - RNA-binding proteins (RRM proteins RNA-binding - - - - FOG: AnkyrinFOG: repeat Ubiquitin-like protein (sentrin) protein Ubiquitin-like - - Ribosomal protein L22 Ribosomal protein Uncharacterized protein involved involved protein Uncharacterized type-3 nucleoprotein H3 nucleoprotein 3 ′ -phosphodiesterase plasmic tosaminyltransferase 13 tosaminyltransferase nucleoprotein A0 nucleoprotein multiple splicing associated protein 3 associated protein homolog (Fragment) fer 2 KIAA1967 Protein descriptionProtein Fibrinogen beta Fibrinogen chain Hydroxyacid oxidase 2 oxidase Hydroxyacid Triosephosphate isomerase Triosephosphate Proteasome subunit alpha Proteasome Heterogeneous nuclear ribo - Heterogeneous -cyclic-nucleotide 2 ′ ,3 -cyclic-nucleotide - cyto ligase, Histidine–tRNA - N-acetylgalac Polypeptide Heterogeneous nuclear ribo - Heterogeneous RNA-binding protein with protein RNA-binding Keratin, type I cytoskeletal 10 type I cytoskeletal Keratin, Thyroid hormone receptor- Thyroid Enhancer of rudimentary Myotrophin Small ubiquitin-related modi - Small ubiquitin-related Clathrin A light chain DBIRD complex subunit DBIRD complex 60S ribosomal L17 protein Kyphoscoliosis peptidase Kyphoscoliosis Protein Protein abbreviation FGB HAO2 TPI1 PSMA3 HNRNPH3 CNP HARS GALNT13 HNRNPA0 RBPMS KRT10 THRAP3 ERH MTPN SUMO2 CLTA KIAA1967 RPL17 Ky vorfM_116: CK_119 1.534 1.38 1.897 1.325 1.234 1.27 1.206 1.564 1.23 1.208 1.328 1.551 1.307 1.337 2.004 1.49 1.344 1.562 1.589 2 1 5 2 4 3 3 1 2 3 4 4 3 1 1 1 2 8 4 4 1 Unique Unique peptide 2 1 6 2 4 3 3 1 2 3 6 5 3 1 1 1 2 8 4 4 1 Peptide (95%) 4.5 1.7 8.5 5.7 9.4 1.2 6.2 8.4 1 47.2 17.3 14.4 17.7 17.5 32.7 14.4 %Cov (95%) 13.5 10.1 11.2 23.7 23.9 42 31 55 37 49 68 90 82 82 78 91 56 278 186 163 136 243 159 148 380 380 Score Accession no Accession 1::GOAT_ENSP00000306099 1::GOAT_ENSP00000354314 1::GOAT_ENSP00000229270 1::GOAT_ENSBTAP00000003636 1::GOAT_ENSBTAP00000019039 1::GOAT_ENSBTAP00000036278 1::GOAT_ENSBTAP00000025094 1::GOAT_ENSP00000393738-D2 1::GOAT_ENSP00000316042 1::GOAT_ENSP00000340176 1::goat_GLEAN_10004749 1::GOAT_ENSP00000346634 1::GOAT_ENSBTAP00000023664 1::GOAT_ENSP00000349140 1::GOAT_ENSP00000405965 1::GOAT_ENSP00000409666 1::GOAT_ENSBTAP00000001518 1::GOAT_ENSP00000373930 1::GOAT_ENSBTAP00000050222 1::goat_GLEAN_10016995 1::GOAT_ENSBTAP00000041518 (continued) 2 Table Group ID Group 133 132 135 134 136 137 138 140 139 141 142 143 144 145 146 146 147 148 149 149 150

1 3 2574 J. Hao et al. (snRNP) homolog domain) L45/RPP1/RPP2 2, beta subunit (eIF-2beta)/ eIF-5 N-terminal domain subunits EF-1beta nase to NACalpha-BTF3 to COG function descriptionCOG - Small nuclear ribonucleoprotein - - RNA-binding proteins (RRM proteins RNA-binding Malate/lactate dehydrogenases - - Ribosomal protein L12E/L44/ Ribosomal protein - Translation initiation factor initiation factor Translation - 20S proteasome, alpha and beta 20S proteasome, - - Translation elongation factor factor elongation Translation - dehydroge 6-phosphogluconate Transcription factor homologous factor Transcription factor 10 factor tein Sm D2 [ubiquinone] 1 alpha [ubiquinone] ment) chain protein 1 protein factor P2 (Fragment) subunit 4 tion factor 2 subunit tion factor interacting protein 2 protein interacting type-6 - II transcriptional coactiva p15 tor genase, decarboxylating genase, ated complex subunit alpha ated complex Protein descriptionProtein Serine/arginine-rich splicing Small nuclear ribonucleopro - subcomplex assembly factor 4 factor assembly subcomplex NADH dehydrogenase dehydrogenase NADH - Mem5 (Frag chain Ig heavy Nucleolin L-lactate dehydrogenase A L-lactate dehydrogenase NEDD4 family-interacting NEDD4 family-interacting Connective tissue growth tissue growth Connective 60S acidic ribosomal protein Proteasome activator complex complex activator Proteasome Eukaryotic translation initia - translation Eukaryotic Leucine-rich fightless- repeat Proteasome subunit beta Proteasome Calpastatin Activated RNA polymerase polymerase RNA Activated Elongation factor 1-beta factor Elongation - dehydro 6-phosphogluconate - polypeptide-associ Nascent ​ Protein Protein abbreviation Srsf10 SNRPD2 NDUFAF4 HVM63 NCL LDHA NDFIP1 CTGF RPLP2 PSME4 EIF2S2 Lrrfp2 PSMB6 CAST SUB1 EEF1B PGD NACA vorfM_116: CK_119 1.259 1.275 1.369 1.407 1.499 1.382 1.578 1.435 1.386 1.477 1.263 1.279 1.414 1.553 1.723 1.226 1.531 1.247 4 5 2 2 6 2 2 7 3 1 3 1 3 4 4 5 5 4 13 Unique Unique peptide 4 5 2 2 7 2 2 7 3 1 3 1 3 4 4 5 5 4 15 Peptide (95%) 9.8 7.5 7.5 0.4 1.9 7.7 16.7 47.9 12.6 22.8 20.5 85.2 13.5 13.5 12.6 30.7 38.7 10.4 26.1 %Cov (95%) 61 48 34 50 43 90 32 90 93 57 93 102 675 217 699 117 195 158 158 Score Accession no Accession 1::GOAT_ENSP00000420195 1::GOAT_ENSBTAP00000016153 1::GOAT_ENSBTAP00000004122 1::goat_GLEAN_10000538 1::GOAT_ENSP00000318195 1::GOAT_ENSBTAP00000047729-D2 1::GOAT_ENSP00000253814 1::GOAT_ENSBTAP00000008357 1::GOAT_ENSBTAP00000002326 1::goat_GLEAN_10014323 1::GOAT_ENSBTAP00000027001 1::GOAT_ENSP00000364119 1::GOAT_ENSP00000338727 1::GOAT_ENSBTAP00000017816 1::GOAT_ENSBTAP00000011029 1::GOAT_ENSBTAP00000029284 1::GOAT_ENSP00000376055 1::GOAT_ENSBTAP00000017988 1::GOAT_ENSP00000377469 (continued) 2 Table ID Group 152 151 153 155 154 156 157 159 158 160 161 160 163 162 164 167 166 165 168

1 3 Inhibition of orf virus replication by HSPA1B 2575 - (S12/L30/L7a) cinyl-diaminopimelate desuc - cinyl-diaminopimelate deacylases and related cinylase ase (rotamase)—cyclophilin ase (rotamase)—cyclophilin family subunits L27E COG function descriptionCOG Ribosomal protein S19E (S16A) Ribosomal protein Pyruvate kinase - Fibrillarin-like methylase rRNA Ribosomal protein L16/L10E Ribosomal protein Ribosomal protein HS6-type Ribosomal protein Transaldolase - deacetylase/Suc Acetylornithine Peptidyl-prolyl cis–trans isomer cis–trans Peptidyl-prolyl Malate/lactate dehydrogenases Molecular chaperone Transthyretin-like protein Transthyretin-like Peroxiredoxin - - 20S proteasome, alpha and beta 20S proteasome, - - Ribosomal protein L14E/L6E/ Ribosomal protein M1/M2 phosphoprotein 32 family 32 family phosphoprotein member B fbrillarin dase isomerase A isomerase plasmic tion factor 3 subunit C tion factor 6-phosphate receptor type-2 10 factor 3 factor Protein descriptionProtein 40S ribosomal S19 protein Pyruvate kinase isozymes Acidic leucine-rich nuclear Acidic -O-methyltransferase 2 ′ -O-methyltransferase rRNA 60S ribosomal L10 protein 40S ribosomal S12 protein Transaldolase (Fragment) Transaldolase - non-specifc dipepti Cytosolic Peptidyl-prolyl cis–trans cis–trans Peptidyl-prolyl - cyto Malate dehydrogenase, Heat shock 70 kDa protein 1B 70 kDa protein Heat shock Transthyretin Peroxiredoxin-6 (Fragments) Peroxiredoxin-6 Eukaryotic translation initia - translation Eukaryotic Cation-independent mannose- Proteasome subunit alpha Proteasome Alpha-(1,3)-fucosyltransferase Alpha-(1,3)-fucosyltransferase Interleukin enhancer-binding enhancer-binding Interleukin 60S ribosomal L14 protein Protein Protein abbreviation RPS19 PKM2 ANP32B Fbl RPL10 RPS12 TALDO1 CNDP2 PPIA MDH1 HSPA1B TTR ​ PRDX6 EIF3C IGF2R PSMA2 FUT10 ILF3 RPL14 vorfM_116: CK_119 1.596 1.606 1.755 1.221 1.21 1.252 1.866 1.215 1.541 1.247 1.231 1.518 1.414 1.307 1.345 1.291 2.041 1.298 1.273 4 3 4 2 3 2 2 4 2 2 3 1 6 3 3 3 1 8 3 21 11 Unique Unique peptide 4 5 5 2 3 2 2 4 5 2 9 1 6 3 3 3 1 8 3 21 11 Peptide (95%) 6.9 8.9 1.6 1.1 24.2 41.3 24.3 23 18.7 23.5 18.1 12.1 11 32.8 26.2 52.2 10.1 %Cov (95%) 20.1 19.7 11.1 13.7 68 86 96 96 92 73 71 21 108 137 401 144 427 468 557 129 104 129 339 232 1786 Score Accession no Accession 1::GOAT_ENSBTAP00000015875-D2 1::GOAT_ENSP00000403265 1::GOAT_ENSBTAP00000028486 1::GOAT_ENSBTAP00000003340 1::goat_GLEAN_10019219 1::GOAT_ENSBTAP00000009803 1::GOAT_ENSBTAP00000001791-D2 1::GOAT_ENSBTAP00000013650 1::GOAT_ENSBTAP00000031070 1::goat_GLEAN_10019253 1::GOAT_ENSBTAP00000025691 1::GOAT_ENSP00000408907-D2 1::GOAT_ENSBTAP00000014585 1::GOAT_ENSBTAP00000006383 1::GOAT_ENSP00000381916 1::goat_GLEAN_10020553 1::GOAT_ENSBTAP00000037502 1::GOAT_ENSBTAP00000001309 1::GOAT_ENSP00000369421 1::GOAT_ENSBTAP00000053565 1::GOAT_ENSBTAP00000002642 (continued) 2 Table Group ID Group 169 170 171 172 173 174 173 177 176 175 178 179 180 181 182 183 184 183 185 186 187

1 3 2576 J. Hao et al. - family S9A family synthetases domain) lyltransferase (alpha) subunit lyltransferase (S12/L30/L7a) domain) (IF-1) COG function descriptionCOG - Heme oxygenase - - FOG: TPR repeat FOG: Prefoldin, chaperonin cofactor chaperonin Prefoldin, - Histone H2A Histone - Serine of the peptidase proteases - Ribosomal protein L6P/L9E Ribosomal protein Aspartyl/asparaginyl-tRNA Aspartyl/asparaginyl-tRNA mRNA capping enzyme, guany mRNA RNA-binding proteins (RRM proteins RNA-binding Ribosomal protein L5 Ribosomal protein - - Ribosomal protein HS6-type Ribosomal protein - RNA-binding proteins (RRM proteins RNA-binding Translation initiation factor 1 initiation factor Translation - protein 1 protein taining protein 9 taining protein interacting protein interacting subunit TOM70 teinase domain-containing 9 protein cytoplasmic tor PUF60 tor ase 1 complex subunit 2 complex nucleoprotein C nucleoprotein tion factor 4H tion factor - X-chromo 1A, tion factor somal Protein descriptionProtein Far upstream element-binding upstream Far Heme oxygenase 1 Heme oxygenase Ras association domain-con - Ras Myosin phosphatase Rho- Myosin Mitochondrial import receptor Prefoldin subunit 6 Prefoldin Disintegrin- and metallopro Core histone macro-H2A.2 histone Core Galectin-1 Prolyl endopeptidase Prolyl Zinc fnger protein 207 protein Zinc fnger 60S ribosomal L9 protein Asparagine–tRNA ligase, ligase, Asparagine–tRNA mRNA-capping enzyme mRNA-capping 60S ribosomal L11 protein - fac Poly(U)-binding-splicing Keratin, type II cytoskeletal 1 type II cytoskeletal Keratin, Poly [ADP-ribose] polymer [ADP-ribose] Poly H/ACA ribonucleoprotein H/ACA Heterogeneous nuclear ribo - Heterogeneous Eukaryotic translation initia - translation Eukaryotic Eukaryotic translation initia - translation Eukaryotic Protein Protein abbreviation FUBP1 HMOX1 RASSF9 MPRIP TOMM70A PFDN6 ADAM9 H2AFY2 LGALS1 PREP ZNF207 RPL9 NARS ​ RNGTT RPL11 PUF60 KRT1 PARP1 NHP2 HNRNPC EIF4H Eif1ax vorfM_116: CK_119 1.328 1.271 1.539 1.298 1.311 1.437 1.348 1.204 1.507 1.431 1.422 1.441 1.29 1.49 1.379 1.297 1.663 1.202 1.844 1.211 1.249 1.369 5 5 1 4 2 2 4 2 6 3 2 3 3 1 3 6 3 7 1 5 3 2 Unique Unique peptide 7 5 1 4 2 2 4 3 6 3 2 3 3 1 3 6 5 7 1 5 3 2 Peptide (95%) 1.9 4 3.8 7.6 6 5.2 6.2 2.1 9.1 11.4 24.3 15 11.3 62.3 18.8 %Cov (95%) 16.9 14 11.3 10.7 16.6 18.1 18 35 87 53 46 64 40 92 21 65 102 148 138 750 115 149 160 218 121 302 239 513 149 Score Accession no Accession 1::GOAT_ENSP00000389536 1::GOAT_ENSBTAP00000020701 1::GOAT_ENSP00000354884 1::GOAT_ENSP00000317786 1::GOAT_ENSBTAP00000019001 1::GOAT_ENSBTAP00000022382 1::GOAT_ENSBTAP00000023197 1::GOAT_ENSP00000362352 1::GOAT_ENSP00000215909 1::GOAT_ENSBTAP00000021658 1::GOAT_ENSP00000378172 1::GOAT_ENSP00000346022 1::GOAT_ENSBTAP00000025659 1::GOAT_ENSBTAP00000003532 1::GOAT_ENSP00000363676 1::GOAT_ENSP00000322016 1::GOAT_ENSP00000369317-D4 1::GOAT_ENSBTAP00000001113 1::GOAT_ENSBTAP00000008363 1::GOAT_ENSP00000338095 1::GOAT_ENSP00000265753 1::GOAT_ENSP00000368927 (continued) 2 Table ID Group 188 189 190 191 192 193 194 195 197 196 198 200 199 202 203 201 204 206 205 208 207 209

1 3 Inhibition of orf virus replication by HSPA1B 2577 dehydrogenase/erythrose- 4-phosphate dehydrogenase COG function descriptionCOG Seryl-tRNA synthetase Seryl-tRNA - - Isocitrate dehydrogenases Isocitrate Ribosomal protein L35AE/L33A Ribosomal protein - - Ribosomal protein S15P/S13E Ribosomal protein - Ribosomal protein L23 Ribosomal protein Glyceraldehyde-3-phosphate Glyceraldehyde-3-phosphate Ribosomal protein S10 Ribosomal protein - plasmic [NADP] cytoplasmic [NADP] factor 11 factor dehydrogenase (Fragment) dehydrogenase Protein descriptionProtein - cyto Serine–tRNA ligase, Scafold attachment factor B1 factor Scafold attachment RNA-binding protein Raly protein RNA-binding Isocitrate dehydrogenase dehydrogenase Isocitrate 60S ribosomal L35a protein Serotransferrin Serine/arginine-rich splicing 40S ribosomal S13 protein 60S ribosomal L28 protein 60S ribosomal L23a protein Glyceraldehyde-3-phosphate Glyceraldehyde-3-phosphate 40S ribosomal S20 protein Protein CDV3 homolog CDV3 Protein Protein Protein abbreviation SARS Safb Raly IDH1 RPL35A TF SRSF11 Rps13 RPL28 Rpl23a GAPDH RPS20 CDV3 vorfM_116: CK_119 1.266 1.502 1.243 1.296 1.917 1.368 1.312 1.437 1.546 1.405 1.677 1.34 2.097 6 4 3 5 1 2 4 2 3 9 2 2 10 Unique Unique peptide 6 4 3 6 1 2 4 2 3 9 2 2 10 Peptide (95%) 6.6 6.9 5.3 13.4 10.4 16.9 13.6 31.5 13.3 19.9 33.1 18.3 %Cov (95%) 14.9 76 30 77 45 99 74 119 130 126 334 213 111 1295 Score Accession no Accession 1::GOAT_ENSP00000358939 1::GOAT_ENSP00000404545 1::GOAT_ENSBTAP00000041837 1::GOAT_ENSBTAP00000027348 1::GOAT_ENSBTAP00000018888 1::GOAT_ENSBTAP00000009564 1::GOAT_ENSBTAP00000020564 1::GOAT_ENSBTAP00000052105 1::GOAT_ENSBTAP00000031700 1::GOAT_ENSBTAP00000037041 1::GOAT_ENSBTAP00000040484 1::GOAT_ENSBTAP00000025484 1::GOAT_ENSBTAP00000023484 (continued) 2 Table Group ID Group 210 211 212 213 214 215 216 217 218 220 219 221 222

1 3 2578 J. Hao et al. chromosome segregation chromosome racemase A) kinases typically periplasmic, containtypically C-terminal PDZ domain G proteins transport receptor - - - CAS/CSE protein involved in involved protein CAS/CSE - - Acyl-CoA dehydrogenases Acyl-CoA - - FOG: TPR repeat FOG: - - - FOG: WD40 repeat FOG: Enoyl-CoA hydratase/carnitine Enoyl-CoA Carboxypeptidase C (cathepsin Carboxypeptidase Adenylate kinase and related kinase and related Adenylate Activator of HSP90 ATPase Activator Trypsin-like serine Trypsin-like proteases, - - - - GTPase SAR1 and related small and related SAR1 GTPase Importin nuclear beta-related COG function descriptionCOG 0.8 relative to the to group at the control same time postinfection 0.8 relative < copeptide repeats 5 repeats copeptide G(I)/G(S)/G(O) subunit gamma-2 auxiliary subunit isomerase, mitochondrial isomerase, dase ATPase homolog 1 ATPase alpha subcomplex subunit 4 alpha subcomplex Interferon-induced protein with tetratri protein - Interferon-induced Proteasome activator complex subunit 2 complex activator Proteasome Mitochondrial fssion 1 protein Importin-9 Caveolin-1 Guanine nucleotide-binding protein protein Guanine nucleotide-binding Peroxisomal acyl-coenzyme A oxidase 3 A oxidase acyl-coenzyme Peroxisomal Golgi apparatus protein 1 protein Golgi apparatus Rho GDP-dissociation inhibitor 1 inhibitor Rho GDP-dissociation N-alpha-acetyltransferase 15, NatA 15, NatA N-alpha-acetyltransferase Phospholipid transfer protein Phospholipid transfer Glutathione S-transferase P Glutathione S-transferase Twinflin-2 Phospholipase A-2-activating protein Phospholipase A-2-activating Delta(3,5)-Delta(2,4)-dienoyl-CoA Delta(3,5)-Delta(2,4)-dienoyl-CoA - serine carboxypepti Retinoid-inducible UMP-CMP kinase UMP-CMP Activator of 90 kDa heat shock protein protein of 90 kDa heat shock Activator Serine HTRA1 protease NADH dehydrogenase [ubiquinone] 1 [ubiquinone] dehydrogenase NADH IST1 homolog IST1 Cellular retinoic acid-binding protein 2 acid-binding protein Cellular retinoic Collagen alpha-1(III) chain Collagen GTP-binding protein SAR1a protein GTP-binding Exportin-1 Protein descriptionProtein IFIT5 PSME2 FIS1 Ipo9 CAV1 GNG2 ACOX3 Glg1 ARHGDIA NAA15 PLTP GSTP1 TWF2 PLAA Ech1 SCPEP1 CMPK1 AHSA1 HTRA1 NDUFA4 IST1 CRABP2 COL3A1 SAR1A Xpo1 Protein Protein abbreviation 0.751 0.772 0.722 0.778 0.813 0.815 0.816 0.754 0.818 0.832 0.746 0.72 0.704 0.739 0.812 0.8 0.808 0.805 0.745 0.775 0.688 0.732 0.566 0.824 0.784 vorfM_116: CK_119 5 3 2 2 2 1 3 6 6 2 4 1 2 2 4 4 1 4 9 1 1 6 3 6 12 Unique Unique peptide 5 3 2 2 2 1 3 6 6 2 4 1 2 2 4 4 1 4 9 1 1 6 5 6 13 Peptide (95%) 12.9 14.6 15.1 3.7 15.1 22.5 6 7.5 41.7 3.3 13.6 11.9 8.5 2.6 17.3 18.8 6.2 17 39 14.6 5.1 51.1 14.4 30.8 7.2 Cov (95%) 73 80 56 91 84 87 87 66 96 72 102 102 122 106 446 183 167 111 199 386 125 451 833 203 173 Score ratios had expression GSF cells. These proteins of ORFV-infected analysis iTRAQ identifed by proteins Downregulated 1::GOAT_ENSP00000360860 1::GOAT_ENSP00000416650 1::GOAT_ENSBTAP00000010389 1::GOAT_ENSBTAP00000005837 1::GOAT_ENSBTAP00000043782 1::GOAT_ENSBTAP00000003959 1::GOAT_ENSP00000348775 1::GOAT_ENSP00000205061 1::GOAT_ENSP00000269321 1::GOAT_ENSBTAP00000045426 1::GOAT_ENSP00000361508 1::GOAT_ENSP00000381607 1::goat_GLEAN_10009864 1::GOAT_ENSBTAP00000017298 1::GOAT_ENSBTAP00000031937 1::GOAT_ENSP00000262288 1::GOAT_ENSP00000360939 1::GOAT_ENSP00000216479 1::GOAT_ENSP00000357980 1::GOAT_ENSBTAP00000014801 1::GOAT_ENSP00000414942 1::GOAT_ENSBTAP00000007515 1::GOAT_ENSP00000304408 1::GOAT_ENSP00000362335 1::GOAT_ENSP00000385942 Accession no Accession 25 24 23 22 21 20 19 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 3 Table Group ID

1 3 Inhibition of orf virus replication by HSPA1B 2579 related) (AMP-forming) activator ing a von Willebrand factor type factor Willebrand ing a von domain A (vWA) Predicted GTPases (dynamin- GTPases Predicted - - C4-type Zn-fnger protein C4-type Zn-fnger Long-chain acyl-CoA synthetases acyl-CoA Long-chain FOG: TPR repeat FOG: - Dihydroxyacetone kinase Dihydroxyacetone Phosphoglycerate mutase 1 Phosphoglycerate - Serine inhibitor protease - - Phosphotyrosyl phosphatase Phosphotyrosyl Serine inhibitor protease - - - - Beta-tubulin folding cofactor D cofactor Beta-tubulin folding - - Serine/threonine kinase protein - Uncharacterized protein contain - protein Uncharacterized COG function descriptionCOG - 2A 65 kDa regulatory subunit A alpha isoform droxyacetone kinase/FAD-AMP lyase lyase kinase/FAD-AMP droxyacetone (cyclizing) 2A activator subunit 3 chondrial taining protein 5A taining protein Dynamin-2 Protein NOXP20 Protein Alpha-parvin Zinc fnger protein ZPR1 protein Zinc fnger Long-chain-fatty-acid–CoA ligase 1 ligase Long-chain-fatty-acid–CoA Protein unc-45 homolog A Protein Serine/threonine-protein phosphatase Bifunctional ATP-dependent dihy Bifunctional ATP-dependent Phosphoglycerate mutase 1 Phosphoglycerate Upstream-binding protein 1 protein Upstream-binding Leukocyte elastase inhibitor Leukocyte Vinculin Nucleosome assembly protein 1-like 4 1-like protein assembly Nucleosome Serine/threonine-protein phosphatase Antithrombin-III Hemoglobin subunit alpha-1/2 Hemoglobin Synaptonemal complex protein SC65 protein complex Synaptonemal SUN domain-containing protein 2 SUN domain-containing protein Serum amyloid A protein Serum amyloid Tubulin-specifc chaperone D chaperone Tubulin-specifc Programmed cell death protein 6 Programmed cell death protein Actin-related protein 2/3 complex 2/3 complex protein Actin-related Mitogen-activated protein kinase 1 protein Mitogen-activated - 6, mito synthase-coupling factor ATP von Willebrand factor A domain-con - factor Willebrand von Protein descriptionProtein DNM2 FAM114A1 PARVA ZNF259 ACSL1 UNC45A PPP2R1A DAK PGAM1 UBP1 SERPINB1 Vcl NAP1L4 PPP2R4 SERPINC1 HBA LEPREL4 SUN2 SAA1 TBCD Pdcd6 ARPC3 MAPK1 ATP5J VWA5A Protein Protein abbreviation 0.806 0.795 0.761 0.826 0.825 0.813 0.825 0.795 0.755 0.823 0.744 0.827 0.761 0.779 0.803 0.777 0.796 0.805 0.706 0.8 0.789 0.71 0.816 0.812 0.676 vorfM_116: CK_119 4 3 4 4 3 7 7 5 2 1 3 2 7 2 5 7 3 8 3 3 4 2 9 11 39 Unique Unique peptide 4 3 4 4 3 7 7 5 7 1 4 2 7 2 5 7 3 8 3 3 5 2 9 11 39 Peptide (95%) 5.8 7.1 15.9 10.5 6.4 9.2 15.9 14.8 45.3 3.1 34.5 43.4 14.8 7.1 22.2 14.8 24.6 13.3 20 8.1 23.2 16.9 18.8 22.2 14.5 Cov (95%) 94 65 98 72 73 104 218 153 449 148 412 338 151 645 180 182 211 135 209 106 133 116 107 237 2353 Score (continued) 1::GOAT_ENSBTAP00000035984 1::GOAT_ENSP00000351740 1::GOAT_ENSP00000334008 1::GOAT_ENSP00000227322 1::GOAT_ENSP00000425006 1::GOAT_ENSP00000407487 1::GOAT_ENSBTAP00000026449 1::GOAT_ENSBTAP00000024227 1::GOAT_ENSBTAP00000032864 1::GOAT_ENSBTAP00000005181 1::GOAT_ENSBTAP00000024445-D2 1::GOAT_ENSBTAP00000053644 1::GOAT_ENSP00000382533 1::GOAT_ENSP00000394338 1::GOAT_ENSBTAP00000005713 1::GOAT_ENSP00000252951-D2 1::GOAT_ENSBTAP00000036255 1::GOAT_ENSP00000409204 1::GOAT_ENSBTAP00000030311 1::GOAT_ENSBTAP00000020484 1::GOAT_ENSP00000264933 1::GOAT_ENSBTAP00000007028 1::GOAT_ENSP00000381803 1::goat_GLEAN_10015090 1::GOAT_ENSP00000407726 Accession no Accession 50 49 48 47 46 45 44 43 42 41 40 39 38 37 36 35 34 33 32 31 30 29 28 27 26 3 Table Group Group ID

1 3 2580 J. Hao et al.

Fig. 3 Classifcation of the identifed proteins based on their func- tional annotations using Gene Ontology enrichment analysis. (A) Cel- lular components of the identifed proteins. (B) Molecular functions of the identifed proteins. (C) Biological processes of the identifed proteins. (D) Gene Ontology enrichment analysis of cellular compo- nents of diferentially expressed proteins. (E) Gene Ontology enrich- ment analysis of molecular functions of diferentially expressed pro- teins. (F) Gene Ontology enrichment analysis of biological processes of diferentially expressed proteins. (G) COG classifcation of difer- entially expressed proteins. P-values were calculated using MetaCore in the GeneGO package (https​://www.geneg​o.com/) protein subunit SEC24/subunit SFB2/ subunit SFB3 - - - Serine inhibitor protease Isopeptidase T Isopeptidase - Uncharacterized conserved conserved Uncharacterized FOG: WD40 repeat FOG: - Vesicle coat complex COPII, COPII, coat complex Vesicle COG function descriptionCOG

information complementary to the protein expression data obtained by proteomics analysis.

The subcellular localization of HSPA1B

GSF cells were transfected with the recombinant plasmid pEGFP-HSPA1B for transient expression. The pEGFP- HSPA1B protein was used to observe the subcellular locali- zation of HSPA1B using CLSM (Fig. 5A). Following DAPI staining, the nuclear excitation of the blue fuorescence was protein KIAA0913 protein Acid ceramidase Acid Collagen alpha-2(I) chain Collagen Niban-like protein 1 protein Niban-like Plasminogen activator inhibitor 2 inhibitor activator Plasminogen Ubiquitin carboxyl-terminal hydrolase 5 hydrolase carboxyl-terminal Ubiquitin Laminin subunit beta-2 Zinc fnger SWIM domain-containing SWIM Zinc fnger Coronin-1B Sulfotransferase 1A1 Sulfotransferase Protein transport Sec24D Protein protein Protein descriptionProtein monitored (Fig. 5B) while the subcellular localization of pEGFP-N1 was monitored (Fig. 5D and E). Compared with pEGFP-N1 (Fig. 5F), the overlap of fuorescence in Fig. 5A and B indicated that the HSPA1B protein was mainly dis- ASAH1 COL1A2 Fam129b SERPINB2 Usp5 LAMB2 K0913 CORO1B SULT1A1 SEC24D Protein Protein abbreviation tributed in the cytoplasm of GSF cells (Fig. 5C).

Inhibition of ORFV proliferation by HSPA1B 0.819 0.702 0.824 0.812 0.743 0.721 0.825 0.709 0.784 0.785 vorfM_116: CK_119 GSF cells were transfected with pEGFP-HSPA1B, cultured for 18 h, and infected with ORFV. Cell suspension sam- 3 9 5 8 3 2 3 8 3 25 Unique Unique peptide ples were collected at diferent time points after infection to measure virus proliferation by RT-qPCR. The results indicated that the diference in viral proliferation at 6 and 3 9 8 8 3 2 3 8 3 25 Peptide (95%) 15 h postinfection between HSPA1B overexpressing cells and the control cells was not statistically signifcant. How- 9.8 27.9 23.1 26 13.3 2.6 1.3 10.6 27.1 3.2 Cov (95%) ever, at 24 and 36 h postinfection, the viral genome copy number was considerably lower in cells overexpressing 45 41 57 162 186 116 122 188 1409 1622 Score HSPA1B, indicating that this protein inhibits proliferation of ORFV in GSF cells (Fig. 6A). Next, we examined ORFV replication in HSPA1B-downregulated cells. HSPA1B was knocked down in GSF cells using RNAi. Three HSPA1B small interfering RNAs (siRNAs) were designed and syn- thesized, and their silencing efciency was evaluated using a Western blot assay. SiRNA-517 was found to be the most efcient for decreasing HSPA1B expression (Fig. 6B). GSF cells were transfected with negative- control (NC) siRNA or siRNA-517 and then infected with equal amounts of ORFV. The siRNA knockdown efciency was confrmed by West- ern blotting, and the levels of viral RNA and viral proteins (continued) 1::GOAT_ENSBTAP00000014960 1::GOAT_ENSBTAP00000033771 1::GOAT_ENSBTAP00000017069 1::GOAT_ENSBTAP00000031243 1::GOAT_ENSP00000229268 1::GOAT_ENSBTAP00000051130 1::GOAT_ENSP00000395277 1::GOAT_ENSP00000340211 1::GOAT_ENSBTAP00000011388 1::GOAT_ENSP00000369059 Accession no Accession in the siRNA-517 cells were compared to NC siRNA cells at diferent time points after virus infection. The levels of ORFV replication were higher in HSPA1B siRNA cells than 60 59 58 57 56 55 54 53 52 51 3 Table Group ID

1 3 Inhibition of orf virus replication by HSPA1B 2581

1 3 2582 J. Hao et al.

Fig. 3 (continued) in control cells (Fig. 6C-E), suggesting that ORFV replica- Discussion tion was signifcantly enhanced in the HSPA1B knockdown cells. These data suggest that HSPA1B has an inhibitory ORFV is an epitheliotropic virus that infects damaged or efect on ORFV replication in GSF cells. scarified skin and replicates in regenerating epidermal

1 3 Inhibition of orf virus replication by HSPA1B 2583

Fig. 3 (continued) keratinocytes [27]. In a previous study, healthy OFTu cells proliferation rate was initially slow but increased from 12 were inoculated with a fltered viral suspension, and CPE to 72 h postinfection. A one-step growth curve of ORFV in was observed after 7–9 days. After fve blind passages of GSF cells showed that the intracellular viral level was the ORFV, approximately 80% of the cells exhibited rounding, highest at 36 h postinfection. By 60 h, the cells had detached detachment, and clustering, creating a net-like form [9]. In from the cell plate and the viral titer decreased, possibly the present study, the proliferation of GSF cells infected with due to the lysis of the infected cells. Since GSF cells are ORFV was studied for the frst time, using virus titration, derived from the primary host of ORF, they provide a good microscopic observations, and RT-qPCR. The data indi- model system for understanding the changes that occur in cated that GSF cells are susceptible to ORFV infection. The these cells following infection with ORFV. In the present

1 3 2584 J. Hao et al.

cytoplasmic membrane trafc or interact directly with the cytoskeletal transport machinery [8]. In the present study, the expression levels of two specifc proteins involved in cytoskeleton networks and cell communication were altered following ORFV infection. TBCD expression was down- regulated, and SPECC1L expression was upregulated. CD9 promotes adeno-associated virus type 2 infection of mammary carcinoma cells with low cell surface expres- sion of heparan sulfate proteoglycans [19]. Parseval et al. [6] showed that the monoclonal antibody MAb vpg15, which targets a determinant of the feline cell surface marker CD9, which may serve as a receptor or co-receptor for feline immunodefciency virus (FIV), markedly delayed infection Fig. 4 Confrmation of diferential expression by RT-qPCR. RT- qPCR was used to verify the diferences in the transcription levels of with that virus. In the present study, CD9 was found to be diferentially expressed proteins. The results indicated that the abun- upregulated in infected cells. dance of BCLF1, MAP4, 6PGD, G3P, ZN207, and CDV3 mRNA We also observed diferential expression of components increased, whereas the expression levels of SC24D, FIS1, COR1B, of several ubiquitin-mediated protein degradation pathways and PSME2 decreased. The expression level of the PARP1 gene was upregulated, although the results were not conclusive. The mRNA in ORFV-infected GSF cells. UBP5 is able to hydrolyze level of VMA5A was upregulated following viral infection, but RT- conjugates of the ubiquitin-like protein ISG15, as demon- qPCR and LC–MS/MS assays showed a decrease in the level of strated in previous experiments in which these conjugates the corresponding protein. This inconsistency could be due to post- were shown to bind to the suicide probe ISG15-VS, which in translational modifcation, including methylation, phosphorylation, or acetylation, or protein for unknown reasons turn inhibited protein degradation [36]. In the present study, UBP5 expression was downregulated, and PSMA7 expres- sion was upregulated in ORFV-infected GSF cells. PSMA7 study, iTRAQ LC − MS/MS was applied for the frst time to is a component of the 26S proteasome, participating in pro- identify diferentially expressed proteins in ORFV-infected tein degradation and cell apoptosis. However, PMSA6 and GSF cells. The data demonstrated that 282 proteins were PSME3, which belong to the same protein family, exhibited diferentially expressed, 222 of which were upregulated and downregulated expression in infectious bursal disease virus 60 of which were downregulated. Changes in mRNA levels (IBDV)-infected chicken embryo fbroblast (CEF) cells, sug- measured by RT-qPCR were in agreement with the changes gesting that diferent viruses may use diferent pathways to observed for the corresponding proteins by iTRAQ. These regulate protein degradation and cell apoptosis [43]. fndings may be helpful for elucidating the molecular mecha- It is noteworthy that ATP synthase-coupling factor 6 nisms by which target cells interact with the virus. (ATP5J), which is involved in ATP synthesis-coupled pro- The host cytoskeletal network participates in the transport ton transport [37], exhibited decreased expression in ORFV- of viral components, particularly during the stages of entry infected cells. A total of six proteins with receptor activity and exit of the virus [30]. Viral components either hijack the were identifed in ORFV-infected GSF cells. TR150, K2C1,

Fig. 5 The subcellular localiza- tion of HSPA1B. A monolayer of GSF cells was transfected by conventional methods using empty pEGFP-N1 as a control. Expression of the fusion protein in GSF cells was detected 18–24 h after transfection by confocal laser scanning microscopy. A. Green fuores- cence showing the location of pEGFP-HSPA1B. B. Nuclear staining of pEGFP-HSPA1B. C. Merged image of panels A and B. D. Green fuorescence show- ing the location of pEGFP-N1. E. Nuclear staining of pEGFP- N1. F. Merged image of panels D and E

1 3 Inhibition of orf virus replication by HSPA1B 2585

Fig. 6 HSPA1B inhibits ORFV replication. (A) Overexpression of 150 nM HSPA1B siRNA or NC, and the expression of HSPA1B HSPA1B suppresses ORFV replication. GSF cells were transfected mRNA or protein was detected by Western blotting. (C-E) Down- with pEGFP-HSPA1B and pEGFP-N1 and infected 18 h later with regulation of HSPA1B promotes ORFV replication. GSF cells were ORFV at an MOI of 0.1. Cultured cells were collected at 6, 15, 24, transfected with NC siRNA or HSPA1B siRNA and subsequently and 36 h postinfection, and the viral DNA content was measured by infected with equal amounts of ORFV at an MOI of 0.1. The expres- RT-qPCR. (B) Evaluation of the efciency of NC or HSPA1B siRNA sion of HSPA1B and viral mRNA or protein was detected by RT- in silencing HSPA1B expression. GSF cells were transfected with qPCR or Western blotting

SUCR1, MPRI, O10AG, and TKT were upregulated. Tran- complex [16, 20]. K2C1 may regulate the activity of kinases, sketolase (TK) catalyzes several reactions in the non-oxi- such as PKC and SRC via binding to integrin beta-1 (ITB1) dative branch of the pentose phosphate pathway (PPP) and and the receptor of activated protein kinase C (RACK1/ serves as a bridge between the oxidative part of the PPP and GNB2L1). Additionally, it can form a complex with C1QBP, the oxidative decarboxylation of glucose [17]. Recently, it which is a high-afnity receptor for kininogen-1/HMWK. has been reported that TK and its cofactor thiamine have a The functions of these proteins in infected host cells are not very high growth control coefcient. TR150 has been shown well understood. to be involved in pre-mRNA splicing and was previously HSPA1B is a member of the HSP70 family. The expres- believed to participate in transcriptional co-activation via sion level of HSP70 rapidly increases in response to cellu- its association with the TRAP complex. However, studies lar stresses (e.g., heat shock) or in response to certain viral have not shown TR150 to be a subunit of a stable mediator infections [4, 21, 24, 26]. Genetic variations in HSP70 have

1 3 2586 J. Hao et al. been found to be associated with individual susceptibility to 3. Carvalho RN, Lettieri T (2011) Proteomic analysis of the marine Thalassiosira pseudonana several diseases by alterations in protein expression and/or diatom upon exposure to benzo(a)pyr- ene. BMC Genom 12:159 function. Studies have shown that HSPs may play a signif- 4. Cheung RK, Dosch HM (1993) The growth transformation of cant role in virus-host cell interactions during viral infection human B cells involves superinduction of hsp70 and hsp90. Virol- in vivo and in vitro [1, 31]. HSP70 stabilizes proteins against ogy 193:700–708 aggregation and mediates the folding of newly translated 5. Chittur S, Parr B, Marcovici G (2011) Inhibition of infammatory gene expression in keratinocytes using a composition contain- polypeptides in the cytosol, as well as within organelles. It ing carnitine, thioctic Acid and saw palmetto extract. Evid Based can bind with nucleotides via an ATP-dependent process and Complement Alternat Med 2011:985345 is involved in the response to stress as well as in cell apop- 6. de Parseval A, Lerner DL, Borrow P, Willett BJ, Elder JH (1997) tosis. HSP70 is associated with membrane microdomains Blocking of feline immunodefciency virus infection by a mono- clonal antibody to CD9 is via inhibition of virus release rather (lipid rafts) in response to dengue virus infection and acts than interference with receptor binding. J Virol 71:5742–5749 as a receptor complex in human cell lines and in monocytes/ 7. Deane D, McInnes CJ, Percival A, Wood A, Thomson J, Lear A, macrophages that are susceptible to dengue virus (DENV) Gilray J, Fleming S, Mercer A, Haig D (2000) Orf virus encodes infection [32]. Therefore, we hypothesize that the increased a novel secreted protein inhibitor of granulocyte-macrophage colony-stimulating factor and interleukin-2. 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