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Swinepox virus vector-based vaccines: attenuation and biosafety assessments following subcutaneous prick inoculation

Swinepox virus (SPV) has several advantages as a potential clinical vector for a live vector vaccine. In this study, to obtain a safer and more efficient SPV vector, three SPV mutants, Δ003, Δ010, and ΔTK were successfully constructed. A virus replication experiment showed that these SPV mutants had lower replication abilities compared to wtSPV in 10 different host-derived cell lines. Animal experiments with mouse and rabbit models demonstrate that these three mutants and wtSPV did not cause any clinical signs of dermatitis. No fatalities were observed during a peritoneal challenge assay with these mutants and wtSPV in a mouse model. Additionally, the three mutants and wtSPV were not infectious at 60 h after vaccination in rabbit models. Furthermore, we evaluated biosafety, immunogenicity and effectiveness of the three mutants in 65 1-month-old piglets. The results show that there were no clinical signs of dermatitis in the Δ003 and ΔTK vaccination groups. However, mild signs were observed in the Δ010 vaccination groups when virus titres were high, and apparent clinical signs were observed at the sites of inoculation. Samples from all experimental pig groups were assessed by qPCR, and no SPV genomic DNA was found in five organs, faeces or blood. This suggests that the infectious abilities of wtSPV and the SPV mutants were poor and limited. In summary, this study indicates that two mutants of SPV, Δ003 and ΔTK, may be promising candidates for an attenuated viral vector in veterinary medicine.

Introduction
Swinepox virus is a dsDNA virus that naturally infects only swine and is a member of the poxvirus family. With the advent of recombinant DNA techniques in the 1980s, Swinepox virus (SPV) was first considered as a recombinant vaccine vector almost 20 years ago [1,2]. Subsequently, recombinant SPV (rSPV) was tested as a vaccine delivery vector for various swine infectious diseases, most notably Streptococcus suis and swine influenza, because it is non-pathogenic and possesses the ability to carry and express large amounts of foreign genetic material [3][4][5][6][7][8].
Given the interest in developing more effective rSPV vaccines and expression vectors, we sequenced and analysed the genome of a pathogenic SPV. SPV genome sequences were assembled into a contiguous sequence of approximately 146 kb, as in a previous report [9]. SPV has a compact gene arrangement with almost no overlapping ORF and no evidence of introns or large regions of noncoding DNA. SPV contains 150 putative genes that encode proteins of 53-1959 amino acids, of which 146 are poxvirus homologues [10]. Many swinepox virus gene functions are unknown. However, functions may be inferred based on the functions of other chordo poxviruses (ChPV). Since the first demonstration in 1982 of the ability of vaccinia virus to express an inserted herpes simplex virus thymidine kinase (TK) gene [11], a large variety of foreign genes have been expressed by recombinant vaccinia viruses. To date, Streptococcus suis, porcine circovirus, swine influenza virus, swine infectious gastroenteritis virus and other pathogenic protective antigen genes have been successfully expressed in swinepox virus [3][4][5][6][7][8]. Swinepox virus has broad application potential as a live vector of the swine vaccine.
Various delivery routes employed for viral vaccine vectors induce a diverse range of host immune responses to vaccine antigens [12][13][14]. Plasmodium berghei malaria and Mycobacterium tuberculosis antigens inserted into rFPV/rMVA and administered in a prime-boost regimen induce strong antigen-specific T cell responses [14,15]. Furthermore, the co-expression of certain chemokines and cytokines has been demonstrated to enhance host immune responses to vaccine antigens [16][17][18][19][20][21][22][23]. These studies showed that rational combinations of vectors, delivery routes and co-stimulatory molecules play crucial roles in regulating immune responses to vaccine antigens.
For live viral vectors such as vaccina vectors, biosafety assessments have been deemed essential before clinical applications. A large number of studies have sought to reduce the risk of the vector itself to avoid adverse reactions [24]. Modified vaccinia virus Ankara (MVA) was one of the first reported VACV strains shown to be highly attenuated in animal models and safe in human trials [25,26]. MVA was isolated in the 1960s from the chorioallantoic vaccinia virus Ankara strain (CVA) via extensive consecutive passaging in chicken embryo fibroblasts, a process that resulted in six major deletions within 122 of the 195 open reading frames (ORF) [27][28][29]. The best property of MVA is its failure to multiply in the vast majority of mammalian cells, particularly human cells. Three strategies have been commonly used to investigate the attenuation and biosafety of pox virus strains. (i) A novel attenuated virus vector constructed by gene engineering. The current method for VACV modification is based on homologous recombination in mammalian cells [30]. (ii) An attenuated virus obtained using routine methods. A viral vector is extensively serially passaged in established cell lines, resulting in the mutation of major pathogenesis-associated genes and the creation of a milder strain [27][28][29]. (iii) A novel virus strain newly found in nature. Some novel strains are naturally mild and attenuated, and these strains may be vaccine vector candidates.
Due to the development of gene editing technology, attenuated viruses obtained by genetic engineering are a hot spot in vaccine research. These vectors not only exert stronger immune effects and demonstrate higher safety but also cost less than those obtained by conventional methods. The use of swinepox virus as a viral vaccine vector has drawn widespread attention, but only a few studies have reported the optimization of swinepox virus attenuation. In a previous study, our group isolated and identified a novel strain of swinepox virus. The newly isolated SPV strain displayed mild and attenuated characteristics, and it was not fatal to either the host or non-host animals. However, swinepox virus also possesses a certain degree of virulence. We expect to perform biosafety assessments on this newly isolated attenuated SPV strain before it can be used in the veterinary clinic.
We have shown that rSPV is an excellent novel vaccine vector compared to traditional vaccines and that vaccinevector combinations generate vastly different immune outcomes in a prime-boost setting. In particular, SPV has been considered a high biosafety standard live vaccine vector [3][4][5][6]8]. These studies have further confirmed that SPV can be optimized to become a veterinary live vector with high biosafety.

Facility and ethics statements
All experiments were conducted in the Key Laboratory of Animal Bacteriology, Ministry of Agricultural, Education Department of the College of Veterinary Medicine, Nanjing Agricultural University. Animal studies were approved by the Laboratory Animal Monitoring Committee of Jiangsu Province and by the Ministry of Agriculture of the College of Veterinary Medicine, Nanjing Agricultural University. The study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals. Protocols for the animal studies were approved by the Committee on the Ethics of Animal Experiments of the College of Veterinary Medicine, Nanjing Agricultural University (No. SYXK2016-0006).

Virus strains, cells and medium
Wild-type swinepox virus (wtSPV [NT1501 strain]) was newly isolated and stored in our laboratory. The following cells were stored in our laboratory: Vero cells, African green monkey kidney cells; PK-15, porcine kidney cells; DF-1, chicken fibroblast cells: LLC, Lewis lung cancer cells; BHK-21, baby hamster Syrian kidney cells; MDCK, Madin-Darby canine kidney cells; MDBK, Madin-Darby bovine kidney cells; RK-13, rabbit kidney cells; F81, feline kidney cell cells; and HeLa cells. These cells were cultured in Dulbecco's modified essential medium (DMEM), and all cells were maintained at 37 °C in a 5% (v/v) CO 2 atmosphere.

Construction of the deletion mutant vector
The primers used in this study are listed in Table 1. Using the swine pox virus genome as a template, primers L1/ L2 and R1/R2 amplified the corresponding 003, 010, TK gene LF and RF. The synthetic plasmid pEGFP N1-Pdx1 PCR primer was used as a template with EGFP-P11-1 (5′-GCGGCCGCTTTACTTGTACA-3′) and EGFP-P11-2 ( 5 ′ -G T C G A G ATATA G TA G A AT T T C AT T T T-GTTTTTTTCTATGCTATAAATGAA-3′). The fragment contains the SPV specific promoter P11. The overlapping PCR technique was used to generate targeted constructs for LF, EGFP-P11 and RF (pUS-DE vector containing only LF ad RF). PCR amplified products were purified from a 1% agarose gel using Gel PCR DNA Fragments Extraction Kit (Geneaid, Taiwan) and then ligated into the pMD-19T vector. The resulting vectors were designated pUSG-003/010/TK and pUS-003/010/TK ( Figure 1).

Generation and screening of swinepox virus deletion mutants
A sub-confluent culture of PK-15 cells was infected with wtSPV (0.02 multiplicity of infection [MOI]) for 2 h. Then, the cells were transfected with 10 µg of the pSPVG-003, pSPVG-010 or pSPVG-TK plasmid using Exfect ™ Transfection Reagent (Vazyme Biotech Co., Ltd). After 72 h in culture, the PK-15 cells were harvested and lysed via five rounds of freezing and thawing. The lysate was then used to infect PK-15 cells in a 12-well plate to plaque-purify virus deletion mutants. Briefly, for plaque purification with fluorescence microscopy (Zeiss, Germany), 1.5 mL of medium containing 1% LMP agarose (DingGuo, Beijing, China) was added to each well of the 12-well plate, and the infected cells were incubated for 5 days until plaques became visible under a light microscope. The plaques were resuspended in 0.3 mL of medium containing 2% foetal bovine serum. Plaque purification was repeated for 3-5 rounds until all plaques in a given well showed green fluorescence. Deletion strains expressing green fluorescent protein were designated Δ003-G, Δ010-G and ΔTK-G.
The deletion viruses labelled with green fluorescence (Δ003-G, Δ010-G and ΔTK-G) were then re-plated into a 6-well culture plate containing PK-15 cells and the second transfection was performed with pUS-003, pUS-010 and pUS-TK. The culture medium was collected on the fifth day after transfection and then incubated twice with new PK-15 cells in a 12-well plate. After incubation at 37 °C for 2 h, medium was added to the semi-solid medium containing 1% methylcellulose of DMEM. The plaques were resuspended in 0.3 mL of medium containing 2% FBS. Plaque purification was repeated for 3-5 rounds using an ordinary microscope and a fluorescence microscope, therefore screening the deletion mutant without GFP. The obtained virus strains were the traceless deletion strains Δ003, Δ010 and ΔTK.

Virus purification and concentration
SPV was propagated in PK-15 cells and purified using a sucrose density gradient separation. Briefly, the virus was released by three rounds of freezing and thawing of the cell cultures, and then was concentrated from the supernatant via ultracentrifugation at 120 000 × g for 1.5 h at 4 °C. The viral pellet was then resuspended in phosphate buffered saline (PBS), layered on 30 and 60% (w/v)

Vector
Target Primer Primers for plasmid construction (5′-3′) sucrose gradients, and centrifuged at 100 000 × g for 2 h and 4 °C. The purified virus band was collected and resuspended in PBS, and centrifuged at 120 000 × g for 1.5 h at 4 °C to pellet the purified virus. Finally, the purified-SPV was resuspended in PBS and stored at −80 °C.

Growth characterization of SPV deletion mutants in vitro
To analyse the growth characteristics of SPV virus in vitro, a growth curve was constructed in PK-15 cells.

Virus replication characteristics in cells
The replication capacity and genetic stability of deletion mutants were evaluated using ten different cell lines. PFU calculation was performed using an evaluation method for deletion mutants and wild SPV replication, yielding differences in different cells. The subculture of viruses in different cell lines was performed to evaluate their genetic stability. Viral loads in cell samples were detected for virusinfected PK-15 cells as previously described [31][32][33]. Confluent PK-15 cell monolayers in 12-well cell culture plates were inoculated with virus at a multiplicity of infection (MOI) of 0.01. Δ003, Δ010, ΔTK or wtSPV infected cells were collected at different time points (0, 12, 24, 36, 48, 60, and 72 h). The supernatants were collected to extract viral DNA with a MiniBEST Viral RNA/DNA Extraction Kit ver. 5.0 (Takara). A pair of SPV genomespecific primers (5′-AAACATCATCATCGCTAC-3′/5′-TACCAGGAGAATGAAAAG-3′) was designed. A 100-bp gene fragment was amplified by PCR from SPV genomic DNA using these two primers and inserted into the Simple 19T vector (Takara) to construct the 19T-V plasmid. After confirmation, 19T-V was transformed into competent Escherichia coli DH5α to prepare large amounts of 19T-V plasmid DNA to establish a standard curve for real-time PCR.

Virulence in mice
To determine the safety of these viral vectors in mice, we evaluated six virus titres (1 × 10 1 , 1 × 10 2 , 1 × 10 3 , 1 × 10 5 , 1 × 10 6 and 1 × 10 7 PFU/mL). (i) Four groups of 4-week-old mice (ICR, BALB/C and nude) were used as animal models. Ten randomly selected mice from each group were infected with Δ003, Δ010, ΔTK or wtSPV, via intradermal injection (100 μL of viral medium, per os), and observed for 30 days. (ii) Then, 2-week-old and 6-week-old BAB/C female mice were subjected to abdominal challenge (100 μL viral medium, 1 × 10 3 PFU/ mL). Each group of 10 was divided into four groups (Δ003, Δ010, ΔTK or wtSPV) and observed for 30 days. (iii) Forty new-born BALB/C mice were divided into 4 groups (Δ003, Δ010, ΔTK or wtSPV) of 10. Vaccine solution was orally administered (50 μL viral medium) via oral gavage using a 5-mL syringe; the sharp syringe head was removed to insert the syringe into the mouth of the animal, slowly pushing the liquid in the syringe into the animal's oesophagus.
Another group of rabbits was used to assess virus clearance. Each of three rabbits was inoculated intradermally at multiple sites with 0.1 mL of PBS containing 10 3 PFU/ mL of each test virus or PBS alone. Indurations and ulcerations were measured and recorded. After 60 h, the rabbits were euthanised, and skin biopsy specimens collected from each of the inoculation sites were aseptically prepared via mechanical disruption and indirect sonication for virus recovery. Infectious viruses were assayed by plaque titration on CEF monolayers.

Evaluation of lesions in swine
To test the efficacy of the SPV deletion in piglets, sixtyone-month-old piglets (verified as SPV negative) were randomly divided into four groups (n = 15/group, five one-month-old piglets as the blank control group). The four virus groups were divided into three dilutions (10 3 , 10 2 and 10 1 PFU/mL) and were skin pricked for inoculation as follows: (1) Δ003; (2) Δ010; (3) ΔTK; and (4) wtSPV. Five pigs were inoculated with PBS as negative controls. The inoculation sites were in the right front legs. After the first inoculation, we collected the serum, faeces, mosquitoes, and water source within one kilometre of the detected pathogenic microorganisms every 3 days. All pigs were observed daily for skin and mental state and for general signs of disease. The mean weight change was calculated from the body weights of five pigs recorded daily after infection with 10 3 PFU/mL relative to normally growing pigs. All observations described in this study were conducted by five people in our laboratory in a blinded and randomised manner.

Indirect ELISA to determine antibody titres
Serum was collected at 0, 7, 14, 21, 28, 35, 42 and 49 days post-primary immunisation from the vaccinated pigs, and an indirect ELISA was used to detect SPV-specific antibodies. We used purified SPV in 100 μL of PBS (pH 7.2) at an empirically optimised dilution to coat 96-well plates. The virus was added to the plates and then incubated overnight at 4 °C. The following morning, the plates were washed three times with PBST and then blocked with 5% skim milk (in PBST) at 37 °C for 2 h. Serum samples were serially diluted and incubated at 37 °C for 1 h. The samples were set up at the same time and divided into the following five groups: (1) Δ003 positive serum, (2) Δ010 serum, (3) ΔTK serum, (4) wtSPV positive serum, and (5) blank control. After incubation, the plates were washed three times with PBST, and then horseradish peroxidase (HRP)-conjugated goat anti-SPA IgG (1:10 000 diluted in PBST, Signalway Antibody) was added to each well. The plates were incubated at room temperature in the dark for 30 min and then washed three times with PBST. The TMB microwell peroxidase substrate system (TIANGEN) was used to develop the reaction. Samples were developed for 20 min, and the reaction was stopped with 2.0 M sulfuric acid. All assays were performed in duplicate. A micro plate reader (Bio-Rad) was used to measure the reaction products at an absorbance of 450 nm [34].

Quantitation of viral loads
Quantitative assays were carried out to measure viral loads in the blood and faeces as previous described [31,33]. Serum and faeces were collected from all experimental pigs at 0, 7, 14, 21, 28, 35, 42 and 49 days postinoculation. Tissue samples from five organs (heart, liver, spleen, lung, and kidney) were collected from pigs at 28 and 49 days. SPV genomic copies were detected by real-time PCR. Serum, faeces and tissue samples were homogenised in PBS at a ratio of 1:5 (g/mL) and centrifuged at 10 000 × g for 30 min. The supernatants were collected to extract viral DNA with a MiniBEST Viral RNA/DNA Extraction Kit ver. 5.0 (Takara, Japan). The sensitivity and specificity of the real-time PCR assay were then determined, referring to the viral load test of the cells above.

Cytokine assay
A cytokine assay was performed as described previously [35]. The cellular immune response type was indirectly assessed by measuring IFN-γ and IL-4 levels in the serum. The pigs were sacrificed at 30 days after the first immunisation with wtSPV, Δ003, Δ010, ΔTK or PBS. Cytokines were detected using ELISA kits (ExCell Bio, China) according to the manufacturer's instructions. Standard curves were generated using control IFN-γ and IL-4 serially diluted twofold in PBS and coated onto ELISA plates overnight at 37 °C. Serum IFN-γ and IL-4 were calculated according to their corresponding standard curves.

Neutralising antibody assay
Assays to detect neutralising antibodies were performed as described previously [6,7]. Serum was collected at 0, 7, 14, 21, 35, and 42 days post-primary immunisation from pigs immunised with wtSPV, Δ003, Δ010, ΔTK or PBS. The serum was diluted from 1:4-1:1024 in a 100-μL volume. The sera were mixed with an equal volume of 70 PFU/mL SPV at 37 °C. After incubation for 1.5 h, the antibody-virus mixtures were used to infect PK-15 cells in 96-well plates, and the cells were overlaid with agar. The cells were grown for 3 days at 37 °C in a 5% CO2 atmosphere to detect SPV-specific CPE.

Statistical analysis
All data were analysed using one-way ANOVA. The threshold for significance was P < 0.05.

Characterisation of SPV deletion mutants
To confirm the construction of SPV deletion mutants, PCR was used to amplify the deletion genes. The Δ003, Δ010, and ΔTK mutants contained 1020, 258, and 543 bp fragment deletions, respectively. The titre of the Δ010 mutant was approximately 1 × 10 5 PFU/mL. The titres of the Δ003 and ΔTK mutants were lower than those of the Δ010 mutant and wtSPV, at approximately 1 × 10 3 PFU/ mL. Plaque assays were used to confirm the presence of deletion mutants, as indicated by green fluorescence (Figure 2A). The growth characteristics of deletion mutants and wtSPV are shown in Figure 2B. After the deletion mutants and wild type virus were purified and concentrated, the following virus titres were determined in PK-15 cells: 1 × 10 7.2 PFU/mL (Δ003), 1 × 10 7.4 PFU/ mL (Δ010), 1 × 10 7.1 PFU/mL (ΔTK) and 1 × 10 8.1 PFU/ mL (wtSPV).

Characterisation in cells
Following evaluation in porcine cell lines and non-porcine-derived cell lines, we determined that the titres of wtSPV and deletion mutant viruses were higher in PK-15 and Vero cells than in non-porcine cell lines. In the majority of non-swine cells, virus replication did not survive passage for more than three generations. Virus titres in the experimental group cells were lower than those in the wild-type SPV and the Δ010 groups in the non-porcine cell line but higher than those in the Δ003 and ΔTK groups. The ΔTK group titres were minimal ( Table 2).

Assessment of attenuation of the deletion mutants in mice
When we tested six virus titres via mouse inoculation, we obtained the following results: (i) the intradermal injection of different strains of SPV, Δ003, Δ010, ΔTK and wtSPV into 4-week-old mice did not lead to pox virus-related disorders in mice at either high or low doses of virus. Genomic copies of SPV were not detected in any serum and faeces samples after 15 days of challenge. Mice infected with Δ003, Δ010, ΔTK and wtSPV did not show weight loss ( Figure 2D). (ii) Twoweek-old and 6-week-old BALB/C mice were injected intraperitoneally. The results show that both the deletion mutants and wtSPV did not cause death. (iii) Oneday-old BALB/C mice were orally administered deletion mutants and wtSPV and had no adverse reactions, indicating that the oral administration of swinepox virus is safe.

Evaluation in rabbit models
Intramuscular injection showed that the injection of wtSPV and ∆010 at 10 7 PFU/mL and 10 6 PFU/mL into rabbits resulted in a 0.1-cm diameter pox rash and a slight increase in weight at 8 dpi, followed by recovery at 20 dpi. No adverse effects were observed in rabbits vaccinated with Δ003 and ΔTK individually via the intradermal route. The skin and tissue at the vaccine administration position were surgically removed and homogenised 60 h after vaccination, and applied to the PK-15 cell based plaque assay. No induration or ulcerative lesions were observed at the inoculation sites for Δ003, Δ010, ΔTK and wild type SPV. These results indicate that the viruses were present at undetectable levels 60 h after vaccination.

Evaluation in pigs
At 5 days post-immunisation, pigs that received wtSPV had pox marks approximately 0.2 cm in diameter around the infection sites. At 10 days post-immunisation, the pox marks were significantly reduced ( Figure 3D), and all pigs recovered after 15 days. High-virus-titre groups administered Δ010 also exhibited slight skin inflammation. As virus titres decreased, swinepox virusrelated skin clinical signs were reduced. The deletion mutant dilutions administered to pigs resulted in no skin inflammation or other adverse reactions. The weight loss curves for pigs inoculated with a virus titre of 10 3 PFU/mL are shown in Figure 3E. Indirect ELISA results for serum SPV antibody levels are shown in Figure 3A, and cytokine levels are shown in Figure 3B. Wild-type and deletion virus infections in experimental group pigs yielded undetectable levels of SPV virus. Neutralisation test results are shown in Figure 3C, 35 days after inoculation, wtSPV and Δ010 stimulated the host to produce neutralising antibody titres of 1:80, and Δ003 and ΔTK produced titres of 1:64. There were no indications of abnormal body temperature, appetite, or mental state in any of the vaccinated animals. On the 15th day after challenge, two pigs were randomly selected from each experimental group and sacrificed for biopsy. Internal organs, including heart, spleen, lung, kidney and intestine, were extracted and analysed for SPV viral load. Genomic copies of SPV were not detected in any serum and faeces samples at 0, 7, 14, 21, 28, 35, 42 and 49 days post-inoculation in any pigs.
An environmental release test showed that SPV was not present in mosquitoes, water sources and faeces collected every 3 days within a 1 km radius of the experimental pig. All pigs on the pig farms were SPV-negative by PCR at different time points.

Discussion
Here, we engineered three gene knockout swinepox viruses, Δ003, Δ010 and ΔTK, and tested their replication in ten types of host cells, their virulence in ICR, BALB/C and nude mouse models, their induction of specific immune responses in New Zealand rabbits, and their vaccine efficacy in a pig model, and we also assessed their biosafety as potential live vectors. When the 003 and TK genes were knocked out, the replication capacity of these viruses in non-target animal cells decreased significantly (with the exception of Vero cells), and virus subculture did not exceed more than three generations. Neither wtSPV nor gene-deleted SPV infected non-target animals. The biosecurity of the three gene-deleted SPV was similar to that of the SPV, but the reduced virulence of these three mutant SPV was significant. In swine model experiments, the deletion mutants only caused dermatology related signs and did not spread to other organs. In the Δ003 and ΔTK challenge group, the inflammatory responses of inoculated pig skin completely disappeared, indicating that the safety of Δ003 and ΔTK was higher than that of wtSPV. However, a live vector itself is often immunogenic and affects the host immunity. Neutralising antibody levels against these two deletion mutant SPV were decreased compared to that of wtSPV. Additionally, the TH1 and TH2 mediated immune response, e.g., interferon-γ and IL-4 levels, also decreased, the most highly significant decrease in these immune factors were observed for ΔTK-SPV among the three mutant deletion SPV. Intraspecific and interspecific transmission infection cases were not found in our study. A virus release experiment showed that the SPV deletion mutant did not cause large-scale virus spread, indicating that live viral vectors may be associated with notably low spreading. The TK gene of SPV is located in the middle of the viral genome, encoding a peptide that contains 175 amino acids. This SPV TK peptide shares 64% amino acid identity with the peptide encoded by the J2R gene of vaccina virus. An analysis showed that the homology between the pox virus TK gene and the herpes simplex virus TK gene was low. Even among TK genes from different pox viruses, the homology was also low. Nevertheless, there are several homologous regions. The TK gene encodes an enzyme that exists in both eukaryotes and pox viruses. This product phosphorylate thymidine riboside into mono-phosphoryl thymidine riboside, which is subsequently phosphorylated by an in vivo phosphatase into di-and tri-phosphoryl thymidine riboside, which then participates in DNA synthesis [36,37]. Therefore, deletion of the TK gene would jeopardy the viral DNA replication, leading to decreased SPV virulence, and explains the dermatitis signs that appeared in wild type SPV inoculated pigs that were not observed in ΔTK SPV inoculated pigs.
The SPV 010 gene encodes the repressor of eIF2-like protein kinase R. PKR is an enzyme encoded by the EIF2AK2 gene that protects against viral infections. PKR is central to the cellular response to different stress signals, such as pathogens, lack of nutrients, cytokines, irradiation, mechanical stress, or ER stress. The PKR pathway leads to the stress response through the activation of other stress pathways, such as JNK, p38, NF-kB, PP2A and phosphorylation of eIF2α. ER stress caused by an excess of unfolded proteins leads to inflammatory responses. PKR contributes to this response by interacting with several inflammatory kinases, such as IKK, JNK, ElF2α, insulin receptor and others. This metabolically activated inflammatory complex is called the metabolic inflammasome or metaflammasome [38,39]. After deletion of the SPV 010 gene, only a slight decrease in SPV virulence was detected; therefore, we propose that the SPV 010 gene may not be a key virulence gene.
The SPV 003 gene encodes the MHC class I alpha chainlike protein. The MHC class I proteins are a set of surface proteins in vertebrates and are involved in the endogenous antigen presenting process. Indeed, the ~9-aminoacid antigenic peptide generated from endogenous antigens bind to MHC class I, forming an antigenic peptide-MHC class I complex, which is subsequently presented to CD8+ cytotoxic T cells [40]. Here, we suggest that the MHC class I alpha chain-like protein encoded by the SPV 003 gene competitively inhibit the MHC class I protein-mediated antigen presenting process, intervening in the immune system and leading to immune-escape [10]. Therefore, deletion of the SPV 003 gene contributes to the significant decrease in SPV virulence and mild clinical dermatitis signs observed in our experiment, indicating that this gene is highly related to SPV virulence.
In this study, we show that (i) SPV is an excellent skin prick inoculation vector. Both scratching/penetrating the skin surface and oral administration achieve desirable immune effects, resulting in animal protection. (ii) SPV demonstrates strict host specificity. Both deletion mutants and wtSPV are highly specific hosts. (iii) Used as a priming vaccine, SPV induces significantly increased cellular and humoural immune responses in pig models. Based on this study, gene deletion of the swinepox virus may generate additional innovative and efficient vaccine vector candidates.


extfieldsofstudy: Medicine Biology

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Oncoprotein AEG-1 is an endoplasmic reticulum RNA-binding protein whose interactome is enriched in organelle resident protein-encoding mRNAs

Astrocyte elevated gene-1 (AEG-1), an oncogene whose overexpression promotes tumor cell proliferation, angiogenesis, invasion, and enhanced chemoresistance, is thought to function primarily as a scaffolding protein, regulating PI3K/Akt and Wnt/β-catenin signaling pathways. Here we report that AEG-1 is an endoplasmic reticulum (ER) resident integral membrane RNA-binding protein (RBP). Examination of the AEG-1 RNA interactome by HITS-CLIP and PAR-CLIP methodologies revealed a high enrichment for endomembrane organelle-encoding transcripts, most prominently those encoding ER resident proteins, and within this cohort, for integral membrane protein-encoding RNAs. Cluster mapping of the AEG-1/RNA interaction sites demonstrated a normalized rank order interaction of coding sequence >5′ untranslated region, with 3′ untranslated region interactions only weakly represented. Intriguingly, AEG-1/membrane protein mRNA interaction sites clustered downstream from encoded transmembrane domains, suggestive of a role in membrane protein biogenesis. Secretory and cytosolic protein-encoding mRNAs were also represented in the AEG-1 RNA interactome, with the latter category notably enriched in genes functioning in mRNA localization, translational regulation, and RNA quality control. Bioinformatic analyses of RNA-binding motifs and predicted secondary structure characteristics indicate that AEG-1 lacks established RNA-binding sites though shares the property of high intrinsic disorder commonly seen in RBPs. These data implicate AEG-1 in the localization and regulation of secretory and membrane protein-encoding mRNAs and provide a framework for understanding AEG-1 function in health and disease.

INTRODUCTION
RNA-binding proteins (RBPs) serve diverse and often pleiotropic roles in the post-transcriptional regulation of gene expression. In addition to prominent nuclear functions in transcription, splicing and mRNA export, RBPs function in the cytosol to regulate RNA localization, translation, and stability (Keene 2007;Glisovic et al. 2008;Halbeisen et al. 2008). Although the primary biological principles of RBP function in RNA localization, translation and turnover are now established, little is known regarding the subcellular distributions and potential compartment-specific functions of RBPs.
Studies into the mechanism(s) of RNA localization to the endoplasmic reticulum (ER), combined with recent land-mark surveys of protein-mRNA interactomes, have provided intriguing evidence for an RBP function in mRNA localization to the ER (Chen et al. 2011;Baltz et al. 2012;Castello et al. 2012Castello et al. , 2016aKwon et al. 2013;Jagannathan et al. 2014a;Reid and Nicchitta 2015). In regard to the former, biochemical studies of mRNA-ER interactions, conducted in the classical pancreas rough microsome model as well as tissue culture cells, have demonstrated that organelle resident protein-encoding mRNAs are bound to the ER via a direct, i.e., ribosome-independent, mechanism (Chen et al. 2011;Jagannathan et al. 2014a;Reid and Nicchitta 2015). Secretory protein-encoding mRNAs, in contrast, are largely ER-associated via their translation on ER-bound ribosomes and are efficiently released in high salt/EDTA buffers, a finding consistent with current models of signal recognition particle (SRP) pathway-mediated RNA localization to the ER (Blobel and Dobberstein 1975a,b;Shields 1979;Chen et al. 2011;Jagannathan et al. 2014a;Reid and Nicchitta 2015). These findings point to a mRNA selective direct membrane anchoring interaction, presumably mediated by integral membrane RNA-binding protein(s), and which might function to efficiently constrain translation of the associated mRNAs to the ER membrane.
The biochemical and RNA interactome studies noted above identified an unexpected diversity of candidate ER membrane RBPs. Although RBP functions for this cohort of RBPs are largely unvalidated, one candidate, the ribosome receptor protein p180 (RRBP1), was previously identified as a general enhancer of poly(A) mRNA association with the ER and specifically implicated in the ribosome-independent anchoring of placental alkaline phosphatase-and calreticulinencoding mRNAs to the ER (Cui et al. 2012. A direct proteomic polyribosome interactor screen identified numerous candidate ER resident RNA-binding proteins, including p180, components of the protein translocation machinery, subunits of the N-linked oligosaccharyl transferase enzyme complex, reticulons, as well as other ER resident membrane proteins (Jagannathan et al. 2014a). Similarly, proteomic screens for the mRNA interactomes of HeLa, HEK293, and mouse embryonic stem cells (mESCs) identified a diversity of candidate ER RNA-binding membrane proteins (Baltz et al. 2012;Castello et al. 2012;Kwon et al. 2013), also including components of the ER protein translocation machinery, the N-linked oligosaccharyl transferase complex, and other previously identified ER membrane proteins, as candidate ER RNA-binding proteins. Given the previously established biochemical functions for many of these resident ER proteins in protein translocation and secretory/membrane protein biogenesis, candidate functions for these proteins in RNA binding to the ER membrane were not anticipated.
RNA-binding proteins (RBPs) typically possess canonical RNA-binding domains, such as the RNA recognition motif (RRM), the K homology domain (KH), the Pumilio homology domain (PUM-HD), or the double-stranded RNA-binding domain (dsRBD), and frequently such RNA-binding motifs are present as multidomain/tandem RNA interaction sites (Lunde et al. 2007;Glisovic et al. 2008;Gerstberger et al. 2014). The candidate ER RNA-binding membrane proteins noted above lack known RNA-binding domains and thus, as proposed for other RBPs lacking canonical RNA-binding motifs, alternative mechanisms of protein-RNA recognition likely contribute to potential functions in mRNA localization, anchoring, and/or translational regulation on the ER (Castello et al. 2012Beckmann et al. 2015). Little is known regarding the mechanism(s) of RNA recognition by any of this newly discovered class of noncanonical RNAbinding proteins, though a recent proteomic analysis of RNA-protein interaction sites, captured via UV crosslinking, represents a substantial advance toward answering this fundamental question (Baltz et al. 2012;Castello et al. 2012Castello et al. , 2016aGerstberger et al. 2013;Kwon et al. 2013). Viewed from the perspective of emerging evidence that many previously identified RNA-binding proteins are multifunctional, a role for nonconventional modes of RNA localization and anchoring to the ER membrane can now be reasonably considered (Mangus et al. 2003;Sawicka et al. 2008;Markus and Morris 2009;Turner and Hodson 2012).
Here we report that AEG-1 (LYRIC, metadherin), an ER resident integral membrane protein previously identified as an oncoprotein Hu et al. 2009;Yoo et al. 2011a) and identified in recent RNA interactome screens, functions as a selective ER mRNA-binding protein (Chen et al. 2011;Baltz et al. 2012;Castello et al. 2012Castello et al. , 2016aKwon et al. 2013;Jagannathan et al. 2014a;Reid and Nicchitta 2015). Genome-scale analysis of the AEG-1 RNA interactome by HITS-CLIP (high-throughput sequencing of RNA isolated by crosslinking immunoprecipitation) and PAR-CLIP (photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation) revealed that the AEG-1 RNA interactome is enriched in organelle protein-encoding transcripts. AEG-1-RNA interaction sites were enriched in coding regions, present to a lesser degree in the 5 ′ untranslated region (UTR), and largely absent from the 3 ′ UTR. In summary, these data identify AEG-1 as an ER resident RNA binding protein whose interactome is enriched for organelle resident protein-encoding mRNAs, most notably those encoding membrane proteins. In addition to membrane protein-encoding mRNAs, we also report that AEG-1 binds a subset of secretory protein-and cytosolic protein-encoding mRNAs, suggestive of a broad role for AEG-1 in the coordinate regulation of translation on the ER. These data provide a useful framework for examining how AEG-1 upregulation in the 8q22 genomic gain characteristic of aggressive cancers contributes to enhanced proliferation, metastasis, angiogenesis, and chemoresistance phenotypes.

Identification of high confidence candidate endoplasmic reticulum (ER) RNA-binding proteins
Recent poly(A) RNA interactome screens have identified numerous candidate ER integral membrane RNA-binding proteins (RBPs) including, unexpectedly, components of the protein translocation machinery (e.g., Sec61α, Sec61β, Sec63), the N-linked protein glycosylation machinery (e.g., ribophorin I) and ER-associated microtubule-binding proteins (e.g., CKAP4) (Baltz et al. 2012;Castello et al. 2012Castello et al. , 2016aKwon et al. 2013). At present, validation of RBP function for these candidate proteins is lacking, and the larger question of RBP function in mRNA localization, translation and/or stability on the ER has only recently come under investigation. Recent studies have implicated RBP(s) in the membrane association of mRNAs, both with respect to specific cohorts of mRNAs (Chen et al. 2011;Jagannathan et al. 2014a), and more broadly in the localization and association of poly(A) RNA to the ER (Cui et al. 2012. Complementing the global poly(A) RNA interactome screens, a recent proteomic interactor screen for ER poly(A) RNA-as-sociated RBPs also revealed a diversity of candidate RBPs, including proteins with previously established functions in ER-associated processes, such as ribosome binding, protein translocation, and/or secretory/membrane protein processing ( Fig. 1A; Jagannathan et al. 2014a).
The Venn diagram depicted in Figure 1B depicts the common ER RBP candidates identified in the ER poly(A) RNA FIGURE 1. RNA interactome screens identify candidate endoplasmic reticulum RNA-binding proteins (RBPs). (A) Proteomic interactor screens identify 170 poly(A) RNA-binding proteins (mRNPs) in canine pancreas rough microsomes and 21 candidate ER integral membrane mRNPs (Jagannathan et al. 2014a). A tabulated listing of 14 of the 21 candidates is provided. (B) Venn diagrams of ER-associated integral membrane mRNA-binding proteins identified in HeLa cells, HEK293 cells, and mouse embryonic stem cells (mESC) (Baltz et al. 2012;Castello et al. 2012;Kwon et al. 2013). The candidate mRBPs identified in the canine rough microsome screen in A are underlined. (C ) Schematic of experimental protocol for identifying candidate RNA-binding activity in living cells by UV cross-linking and immunoprecipitation (CLIP). A representative phosphorimage depicting GFP-Sec61β and GFP-AEG-1 RNA complexes' formation by UV cross-linking is depicted. The empty vector transfection condition is included to depict background labeling. interactor screen of Jagannathan et al. (2014a), and three poly(A) RNA interactomes, conducted in HeLa, HEK293, or mouse embryonic stem cells (mESC), with AEG-1 and CKAP4 as the two candidate ER membrane RBPs common to the four studies (Baltz et al. 2012;Castello et al. 2012;Kwon et al. 2013;Jagannathan et al. 2014a). AEG-1 was previously reported to function as a scaffolding protein, interacting with PLZF, NF-κB, RXR, and the RNA-binding protein SND1 (Sarkar et al. 2008;Thirkettle et al. 2009;Yoo et al. 2011b;Meng et al. 2013;Srivastava et al. 2014) and demonstrated to associate with RBPs and ribosomal proteins in a nuclease-sensitive manner (Meng et al. 2013), a finding consistent with a direct or indirect mRNA binding function. CKAP4 is a microtubule binding protein functioning in the regulation of ER morphology and has also been implicated in the regulation of mRNA localization to the ER (Cui et al. 2012;Sandoz and van der Goot 2015). In this report, we focus on AEG-1.
To distinguish between a direct vs. indirect RNA-binding function for AEG-1, CLIP (crosslinking followed by immunoprecipitation) experiments were performed in tissue cell cultures transfected with constructs encoding FLAG epitope-tagged, cytosolic domains of AEG-1 or Sec61β, the latter of which was identified in three of the four RNA interactome screens noted here (Fig. 1A,B). In these experiments, tissue culture cells were UV-irradiated to introduce covalent protein-RNA cross-links, detergent-lysed, and cross-linked complexes isolated under stringent conditions. Following digestion with RNase I, the RNA moiety was radiolabeled with T4 polynucleotide kinase/[γ-32 P]ATP (Fig. 1C). Mock transfection and (−) UV irradiation control conditions were included, to assess immunoprecipitation background and direct protein radiolabeling via protein kinase contaminants, respectively. As shown, prominent radiolabeled products were recovered in both the Sec61β and AEG-1 anti-FLAG immunoprecipitates, and in the transfected but not mock transfected samples (Fig. 1C). For both candidate RBPs, PNK labeling was largely UV irradiation-dependent, consistent with an RNA-binding function. Note that the extensive RNase digestion preceding PNK labeling eliminates the heterogeneity of RNA lengths in protein-RNA com-plexes to yield uniform protein-RNA complex mobility in SDS-PAGE (Konig et al. 2010). As previously reported, AEG-1 displays a lower than predicted mobility, a consequence of its highly basic pI (Lee et al. 2013).
To determine if AEG-1 binds actively translating (i.e., ribosome-associated) mRNAs, cells were detergent-solubilized and the polyribosome fraction assayed for associated AEG-1 (Fig. 2). In one set of experiments, we sought to distinguish between mRNA-associated vs. ribosome-associated AEG-1. To this end, detergent extracts were treated with ribonuclease, subjected to ultracentrifugation, and AEG-1 levels in FIGURE 2. AEG-1 associates with actively translating mRNAs. (A) Schematic of experimental protocol for examining AEG-1 RNA-binding activity by ultracentrifugation analysis. Cell lysates were ultracentrifuged subsequent to RNase or control digestion. The polysome fraction (ribosome pellet) was analyzed by immunoblot. (B) Immunoblot analysis of AEG-1, poly(A)-binding protein (PABP), and ribosomal protein (RPL17) in the pellet fraction with/without RNase digestion. (C) Schematic of experimental protocol for the analysis of mRNA-binding activity of AEG-1 by polyribosome profiling. (D) Immunoblot analysis of AEG-1 and ribosomal protein (RPL17) in sucrose density gradient fractions. (E) Mobility of AEG-1 in sucrose density gradient velocity sedimentation in control (−EDTA) or after polyribosome disassembly (+EDTA). Immunoblot analysis of the lysosomal membrane protein NPC1 is illustrated as a membrane protein control. the ribosome pellet fractions determined by immunoblot ( Fig. 2A,B). As shown in Figure 2B, in control samples lacking ribonuclease, AEG-1 was largely recovered in the ribosome pellet, which we confirmed by immunoblot analysis for RPL17 and poly(A)-binding protein (PABP). Following ribonuclease digestion, only background levels of AEG-1 and PABP were recovered in the ribosome pellet, indicating that AEG-1 stably interacts with mRNAs, but not ribosomes. To further examine this conclusion, sucrose density gradient sedimentation analyses were performed ( Fig. 2C-E), and demonstrated that AEG-1 is present in the polysome fraction ( Fig. 2D), with EDTA-elicited polysome resulting in a shift in AEG-1 migration to slower migrating fractions (Fig. 2E). As a further control, we also examined the distribution of NPC1, a lysosomal membrane protein. As a lysosomal membrane protein, NPC1 would not be expected to migrate with polysomes, which was confirmed by immunoblot analysis of the gradient fractions in the control and EDTA-treatment conditions (Fig. 2E). Combined, the data in Figures 1 and 2 identify AEG-1 as a poly(A) RNA-binding protein that can bind actively translating mRNAs.

AEG-1 RNA-binding activity maps to the central disordered region
Poly(A) RNA interactome screens have identified over 1000 mRNA-binding proteins (RBPs), of which approximately 300 are common to the mammalian cell-derived data sets referenced above (Baltz et al. 2012;Castello et al. 2012;Kwon et al. 2013;Gerstberger et al. 2014). Approximately two-thirds of the candidate RBPs contain established RNA-binding motifs, e.g., RRM, DEAD, KH, zinc finger domain, frequently in self-tandem repeats or in combination (Gerstberger et al. 2014). AEG-1, however, lacks established RNA-binding domain motifs, and BLASTP searches against the cytosolic domain of the protein (aa70-582) did not reveal homologies with any proteins other than AEG-1 species variants (data not shown). To further validate a direct RNA-binding function for AEG-1, we first performed CLIP experiments on the native, endogenous AEG-1. As shown in Figure 3A, and similar to that depicted above for the FLAG-tagged AEG-1 cytosolic domain (Fig. 1C), UV-dependent RNA-protein crosslinks were readily identified with endogenous AEG-1. Having confirmed an apparent RNAbinding function for the native protein, we used HA-tagged AEG-1 constructs to map RNA-binding sites, using a nested deletion approach. AEG-1 is a Type I single transmembrane protein with a positively charged cytosolic domain (Britt et al. 2004;Sutherland et al. 2004;Kang et al. 2005). We prepared epitope-tagged C-terminal deletion mutants, guided by the RNA-binding prediction algorithm BindN+, AEG-1 FIGURE 3. Mutagenic domain mapping of AEG-1-RNA interaction. (A) Endogenous AEG-1 can be crosslinked to RNA in living cells. Cells were UV-irradiated and processed as described in the legend to Figure 1, using antibodies directed against native AEG-1. Depicted is a phosphorimage of the radiolabeled anti-AEG-1 immunoprecipitates and paired immunoblots in control (−UV) and experimental (+UV) conditions. (B) Schematic illustration of AEG-1 deletion mutants. HA-tagged full length (FL) and four C-terminal deletion mutants are shown. (TM) Transmembrane domain. (C) Immunoblot analysis of HA-tagged AEG-1 mutants, poly(A)binding protein (PABP), and ribosomal protein (RPL17) in pellet fractions derived from ultracentrifugation analyses with/without RNase digestion. (D) Phosphorimager images depicting radiolabeled protein-RNA complex formation of AEG-1 mutants +/− UV crosslinking (left panels). Immunoblot analysis of HA-tagged AEG-1 truncation mutants +/− UV crosslinking, demonstrating recovery of HA-tagged AEG-1 forms in both fractions. (E) Immunofluorescence micrographs of full length (AEG-1) and truncation mutants illustrating ER localization of C-terminal truncated forms. Scale bar, 25 µm.
RNA-peptide crosslink sites as reported in a recent global proteomic screen for RNA interaction sites (Castello et al. 2016b), and algorithmic analyses of AEG-1 structural disorder ( Fig. 4; Wang et al. 2010). Individual members of the truncation series of epitope-tagged C-terminal deletion mutants ( Fig. 3B) were expressed by transient transfection, ER localization confirmed by immunofluorescence imaging (Fig. 3E), and RNA-binding activity assessed by the polysome association/centrifugation assay described above (Figs. 2, 3D). We first confirmed that tagged, full-length AEG-1 bound to translating mRNAs and that its recovery in the polysome pellet was abolished by RNase-treatment ( Fig.  3C, FL). Deletion mutants containing the first 350 or 462 amino acids of AEG-1 (AEG-1 [aa1-350] and AEG-1 [aa1-462]) retained RNA-binding activity and localized to the ER. Depicted in Figure 3E are representative micrographs depicting the ER localization of HA-tagged full length AEG-1 and AEG-1 (aa1-350), where the individual channels are depicted in gray scale and the merged channels in color, where white represents colocalization of AEG-1/AEG-1 (aa1-350) and the ER marker TRAPα. Two AEG-1 deletion mutants (AEG-1 [aa1-69] and AEG-1 [aa1-138]) were expressed but did not associate with translating mRNAs (Fig. 3C). To further substantiate the RNA-binding activity of the deletion mutants, we performed companion CLIP analyses (Fig. 3D). As shown, AEG-1 (aa1-350) and AEG-1 (aa1-462) formed protein-RNA complexes upon UV irradiation, whereas AEG-1 (aa1-138) lacked detectable RNA-binding activity. Though lacking primary sequence and RNA-binding domain homology with established RBPs, AEG-1 does share with other RBPs the structural property of high intrinsic disorder with these deletion mutant studies mapping the (a) AEG-1-RNA-binding region to the central disordered region (Fig. 4;Varadi et al. 2015;Järvelin et al. 2016). A more precise functional mapping of the AEG-1 RNA-binding domain(s) is currently in progress.

AEG-1 CLIP-seq mapping reveals a high enrichment in coding sequence interactions
Genome-wide CLIP-seq experimental approaches have been recently applied to the study of RBP interactomes in living cells (Ule et al. 2003;Hafner et al. 2010;Konig et al. 2010). By these methods, bound RNA identities and singlenucleotide resolution mapping of target RNA interaction sequences can be obtained (Ule et al. 2003;Hafner et al. 2010;Konig et al. 2010). To identify the AEG-1 RNA interactome, we used two variations of this technique, high-throughput sequencing-CLIP (HITS-CLIP) and photoactivatable ribonucleoside-enhanced-CLIP (PAR-CLIP) (Darnell 2010;Hafner et al. 2010). The experimental approach is illustrated in Figure 5A. Cells expressing HA epitope-tagged AEG-1, were cultured in the absence (HITS-CLIP) or presence (PAR-CLIP) of 4SU and UV-irradiated to cross-link AEG-1-RNA complexes in vivo. The complexes were then partially RNase-digested, purified by immunoprecipitation, covalently associated RNA radiolabeled, resolved by gel electrophoresis, and transferred to nitrocellulose membranes. A representative autoradiograph of a transferred membrane is depicted in Figure 5B and shows prominent UV irradiation-dependent radiolabeled complexes at 90-100 kDa. These complexes have a molecular weight 10-20 kDa larger than that of AEG-1 protein (80 kDa), corresponding to AEG-1 protein cross-linked to small RNA fragments (Huppertz et al. 2014). The RNA fragments were released from the nitrocellulose membranes by proteinase K digestion, reverse transcribed for cDNA library construction (Fig. 5C), and both control (−UV irradiation) and experimental (+UV irradiation) cDNA libraries deep sequenced. As previously reported, proteinase K digestion leaves a small amino acid/peptide residue at the cross-linked site, resulting in a diagnostic point mutation during reverse transcription (Zhang and Darnell 2011;Sievers et al. 2012). One hundred to 120 million reads, with approximately 4 (HITS-CLIP) to 16 (PAR-CLIP) million reads uniquely mapped to the human transcriptome were obtained. Overall, sequence read distributions were highly enriched in mRNAs: 84%-93% mapped to mRNA, 3%-7% to snRNA, 3%-6% to snoRNA, 1%-2% to tRNA, and 1% to miRNA (Fig. 5D).
The AEG-1 RNA interactome is enriched in transmembrane protein-encoding mRNAs The AEG-1-bound clusters distributed over the 1234 mRNAs common to the two HITS-CLIP and single PAR-CLIP data sets were defined as AEG-1 bound mRNAs (Fig. 6A). In addition to a high shared mRNA composition, the HITS-CLIP replicates displayed a high cluster score reproducibility (Pearson correlation coefficient = 0.947) (Fig. 6B). The correlation between the HITS-CLIP and PAR-CLIP data sets was less robust. The approximately fourfold higher cluster depth in the PAR-CLIP data set however, compromises a FIGURE 4. AEG-1 primary amino acid sequence alignment, predicted RNA interaction sites, and candidate RNA interaction sites determined by proteomic mapping. Sequence alignment of AEG-1 homologs. Amino acid sequences of AEG-1 of human (NP_848927), bovine (NP_001039503), mouse (NP_080278), rat (NP_596889), and Xenopus tropicalis (NP_989164) were aligned by MegAlign Pro. A consensus sequence is shown as the uppermost sequence. The human AEG-1 C-terminal deletion mutants depicted in Figure 3B are indicated. Also indicated are candidate RNA-binding regions from the BindN(+) algorithm (blue bars), the location of a high-confidence RNA-binding region (peptide) identified in a proteomic screen for RNA-binding domains in HeLa cells (red bar) (Castello et al. 2016a), and two lower confidence RNA-binding regions (yellow bar). (TMD) Transmembrane domain. sound statistical comparison (Fig. 6A,B). This data set was further analyzed for topology (integral membrane, secretory, or cytosolic), predicted subcellular localization, and enriched gene ontology (GO) terms (Huang et al. 2009a,b). Depicted in Figure 6C are the summary subcellular localization data, demonstrating that the majority (51%) of the clusters map to organelle resident protein-encoding mRNAs, with the remaining fraction to secretory (20%) and, intriguingly, cytosolic protein-encoding mRNAs (29%). The most abundant categories are proteins resident to the ER and the plasma membrane, with lysosome, Golgi apparatus, mitochondrial, and nuclear envelope mRNAs also represented, with ER enrichment being the most significant (P = 3.7 × 10 −67 ) (Fig. 6C,D). In this mRNA cohort, GO analysis also demonstrated significant enrichments for mRNAs encoding lysosomal proteins (P = 4.8 × 10 −17 ) (Fig. 6D). Intriguingly, the AEG-1 mRNA interactome displayed a very significant enrichment in transmembrane protein mRNAs (P = 3.5 × 10 −57 ) (Fig. 6D). This cluster enrichment was evident in both the HITS-CLIP and PAR-CLIP data sets and was distinct from RNA-seq read distributions, indicating that the TMD downstream cluster enrichments were independent of library bias or related artifacts. This enrichment is further highlighted in comparisons of the RNA read distributions in the RNA-seq and the CLIP-seq data sets. Whereas genes encoding transmembrane proteins comprise approximately 26% of the human protein-coding genes (Almén et al. 2009;Fagerberg et al. 2010), only a small fraction of the RNA-seq reads map to transmembrane proteins mRNA (8.9%), indicating that expression levels of this gene superfamily are relatively low. Yet, more than 50% of the cluster values from the HITS-CLIP studies mapped to transmembrane protein mRNAs (data not shown).
Further examination of the endomembrane resident protein GO categories scoring as highly significant identified numerous categories linked to ER function, membrane biogenesis, and ER proteostatic stress response pathways (Fig. 6E). Also of interest, the AEG-1 cluster data sets included significant enrichments in cytosolic protein gene subsets, specifically the SRP pathway, nonsense-mediated decay (NMD), translational initiation, and poly(A) RNA binding (Fig.  6E). With recent studies having demonstrated that miRNA-mediated translational silencing occurs on the ER, that NMD is functionally linked to ER stress regulation and may occur on ER/nuclear envelope-associated ribosomes, and that ER-bound ribosomes participate in de novo translation initiation, these data suggest functional links between AEG-1 and ER-localized synthesis of cytosolic proteins, and in particular those linking to mRNA export, localization, quality control, and translation (Sakaki et al.  Barman and Bhattacharyya 2015). These data corroborate recent reports that ER-bound ribosomes contribute significantly to cytosolic protein synthesis, and suggest a mechanism by which such noncanonical localization and translation of cytosolic proteinencoding RNAs could be mediated (Lerner et al. 2003;Pyhtila et al. 2008;Nicchitta 2012, 2015;Jagannathan et al. 2014b).
We used RNA-immunoprecipitation and qPCR (RIP-qPCR) as a complementary biochemical approach to validate AEG-1-mRNA interactions identified in the CLIP experiments. Here, HA-tagged AEG-1 and its bound mRNAs were immunoprecipitated from AEG-1-14 cells and the associated mRNAs interrogated by qPCR for mRNAs identified in the CLIP-seq study (Fig. 7A,B). Mocktransfected cells (PC-4) were used as negative controls. The RIP-qPCR data reveal significant enrichments of the AEG-1 bound mRNAs, MDR1, ATP1A1, HSPA5, NPC1, NPC2, and NPC1L1, confirming the specific association of AEG-1 with mRNAs encoding integral membrane and cytosolic proteins (Fig.  7B). Also illustrated are the cluster score enrichments from the two HITS-CLIP data sets, the single PAR-CLIP data set, and the control data sets, where it is evident that the overall rank order of enrichment seen in the RIP-qPCR data is evident in the cluster score enrichments. It was not possible to compare the foldenrichment rank order for the two approaches because the majority of the genes assayed were absent from the controls. In the one case (ATP1A1) where CLIP clusters were identified in the controls, the fold-enrichment is similar to that seen by RIP-qPCR. These data further strengthen the validity of the primary finding in this study, that AEG-1 is an integral ER RNA binding protein whose interactome is enriched for transmembrane-encoding organelle resident protein mRNAs.
The location (UTR vs. CDS) of RBPbinding sites mirrors RBP function, be it mRNA processing, transport, degradation and/or translation. Thus, translation initiation factors bind to the 5 ′ UTR, regulating translation initiation and translation efficiency (Wilkie et al. 2003;Araujo et al. 2012); splicing factors recognize the intron-exon junctions (Glisovic et al. 2008;Gerstberger et al. 2014); and many mRBPs bind sites in the 3 ′ UTR functioning as regulatory signals for mRNA stability, transport, and degradation (Dreyfuss et al. 2002;Glisovic et al. 2008;Gerstberger et al. 2014). To gain insight into the possible molecular consequences of the AEG-1 mRNA interaction, we further (C) Subcellular locales of AEG-1 HITS-CLIP genes, depicting endomembrane organelle, cytosolic and secreted protein-encoding genes on the left. The composition of the category "endomembrane" is depicted on the right, including plasma membrane, endoplasmic reticulum, lysosome, Golgi apparatus, mitochondrion, and nuclear envelope. (D) Gene ontology analysis of AEG-1 associated mRNAs. The mRNAs were subjected to gene ontology analysis using DAVID (Huang et al. 2009a,b). Endomembrane organelle distributions of proteins encoded in the AEG-1 bound mRNAs are illustrated. (E) GO category enrichments of the AEG-1 RNA interactome in the categories "endomembrane proteins" and "cytosolic proteins." analyzed intratranscript distributions of AEG-1 clusters in the integral membrane protein cohort, which is the most enriched in the data sets. Since AEG-1 cluster sites enrich in the CDS of actively translated mRNAs (Fig. 5F), we speculated that AEG-1 regulates translation initiation and/or termination by interacting with sequences flanking translation start/stop sites. We examined AEG-1 clusters within 200 nt flanking the start and stop codons; no significant over-representations of cluster density at either position were, however, observed ( Fig. 8A-D). In contrast, we observed a significant over-representation of AEG-1 clusters downstream from encoded transmembrane domains, with total read densities increasing near encoded transmembrane domains and peaking from 100 to 250 codons downstream (Fig. 8E). This cluster enrichment was evident in both the HITS-CLIP and PAR-CLIP data sets and was distinct from RNA-seq read distributions, indicating that the TMD downstream cluster enrichments were independent of library bias or related artifacts. We also examined AEG-1 clusters proximal to the transmembrane domains of Type I membrane proteins as well as the transmembrane domains occurring subsequent to the first encoded transmembrane domain and found very similar read cluster distribution patterns, where AEG-1 binds proximal to transmembrane domains regardless of their processive order in the mRNA (Fig. 8E-G). Since AEG-1 interactions are enriched in the CDS of translating mRNAs, this transmembrane domain proximal association pattern suggests that AEG-1 may regulate the translation of transmembrane proteins, perhaps in a manner coupled to membrane insertion of transmembrane domains and/or topological assembly.
In summary, the data included here demonstrate that AEG-1 functions in the association of select cohorts of mRNAs to the ER membrane. In the case of integral membrane protein mRNAs, the cluster enrichment downstream from encoded transmembrane domains suggests that AEG-1 may contribute to the dynamic regulation of transmembrane domain insertion, integration, and/or topological orientation. In the case of cytosolic protein-encoding mRNAs, interactions with AEG-1 may serve to localize translation to the ER and/or nuclear envelope membrane. More informed insights into AEG-1 function as an ER RNA anchoring protein await further study. In this regard, it should be noted that AEG-1 knockout mice are viable and thus AEG-1 is likely a member of the family of ER RNA-binding proteins that contribute redundant functionality to the localization and translational regulation of mRNAs on the ER (Fig. 1C; Lee et al. 2013;Robertson et al. 2015).

DISCUSSION
Here we report that AEG-1, an oncogene linked to the upregulated proliferation, metastasis, angiogenesis, and chemoresistance phenotypes of aggressive cancers, is an ER resident RNA binding protein. In vivo genome-scale mapping of AEG-1/RNA interaction sites by HITS-CLIP and PAR-CLIP methods provided complementary experimental evidence that the RNA interactome of AEG-1 is highly enriched for organelle resident protein mRNAs, and in particular those encoding transmembrane proteins. Intriguingly, AEG-1/ RNA-binding interactions are enriched in the coding sequence and although a clear consensus motif was not evident, a signature read cluster enrichment downstream from encoded transmembrane domains was identified. Also of significant interest, the AEG-1 interactome includes a cohort of cytosolic protein-encoding mRNAs, suggesting that AEG-1 contributes broadly to ER-localized mRNA translation.
RNA-binding proteins (RBPs) comprise a substantial fraction of the human proteome (approximately 7.5% of protein coding genes), with current estimates numbering close to 1000 RBPs binding the mRNA transcriptome (Baltz et al. 2012;Castello et al. 2012Castello et al. , 2016aKwon et al. 2013) Generally speaking, RBPs can be binned into two broad groups: canonical RBPs, which contain established RNA-binding motifs such as RRM, KH, dsRBD, and zinc finger, and whose binding sites are enriched in the untranslated regions (UTRs) of mRNAs, and noncanonical RBPs, generally multifunctional proteins which lack established RNA-binding motifs, and Immunoblots of RIP-qPCR for AEG-1 and ribophorin I (RPN1). (B) RIP-qPCR fold enrichments for MDR1, ATP1A1, NPC1, NPC2, NPC1L1, HSPA5 mRNAs from AEG-1-14 over PC-4 cells. GAPDH mRNA was used for normalization. ( * ) P < 0.05, ( * * ) P < 0.01. Included also are the cluster score enrichments for the indicated genes for the HITS-CLIP, PAR-CLIP, and control data sets, demonstrating generally similar rank order enrichments. whose binding enrichments remain largely unknown. The latter category has recently gained considerable interest, in large part because of the diversity of candidate RNA-binding proteins revealed in recent large-scale RNA interactome studies (Baltz et al. 2012;Castello et al. 2012;Kwon et al. 2013). Particularly surprising has been the identification of housekeeping proteins, such as glycolytic enzymes, as RNA-binding proteins (Castello et al. 2012). The intriguing ramification of this broadening view of RNA-binding protein identity and functionality is the opportunity for higher order spatial organization of RNA localization and translation, and thus additional mechanisms for the coordinate regulation of related genes (Keene 2007;Kishore et al. 2010;Gebauer et al. 2012).
In contrast to many canonical RBPs, which display bioinformatically discernible and experimentally defined target recognition motifs, primarily UTR-enriched, AEG-1 binding sites are almost wholly located in the CDS of its mRNA interactome. This coding sequence enrichment likely precludes a distinct nucleotide level AEG-1 interaction motif, as that would also yield a primary amino acid sequence motif, which is not evident. Consistent with this view, analyses of the combined HITS-CLIP and PAR-CLIP cluster data sets did not reveal a consensus motif (data not shown). In the absence of discrete interaction sequences, we suggest that AEG-1 affects ER localization via low affinity (sequence diverse), high avidity interactions within the CDS. Such an aviditybased mechanism would be expected to yield stable membrane association. We speculate that an abundance of low affinity CDS-binding sites may help constrain the secondary structural space of the target mRNAs, thereby favoring a largely unstructured CDS that would serve as a more optimal translation substrate. Though there is extensive evidence for the broad utilization of low affinity, high avidity RNA-protein interactions (Williamson 2000;Leulliot and Varani 2001;Van Roey and Davey 2015;Varadi et al. 2015), the selectivity and enrichment for organelle protein-encoding mRNAs does imply the recognition of common features of this cohort of mRNAs, which we have yet to identify. Compounding this challenge, RBPs are noteworthy for their abundance of intrinsically disordered domains, as well as a diversity of potential induced fit interactions with RNA substrates (Williamson 2000;Leulliot and Varani 2001;Varadi et al. 2015;Järvelin et al. 2016). AEG-1 is marked by an extensive abundance of intrinsically disordered domains. Primary sequence analysis of intrinsically disordered domains in AEG-1, using the DISOPRED algorithm (Ward et al. 2004a,b), indicates that almost the entirety of the cytoplasmic domain of AEG-1 is of low complexity/highly disordered. The challenge ahead is to identify mechanistic and structural links between the intrinsically disordered AEG-1 cytoplasmic domain and its selective association with organelle resident membrane protein-encoding mRNAs. Deletion analyses (Fig. 4) highlight the region aa138-aa350, which includes a prominent region of low complexity/high disorder and which displays RNA binding activity in the cell, as a focus area for future study.
AEG-1 is highly conserved in vertebrates ( Fig. 4; Lee et al. 2013), and so we hypothesized that the protein region(s) functioning in RNA binding would also be highly conserved. As illustrated in Figure 4, the region aa139 to aa350 is highly positively charged (27 Lys, 9 Arg residues) and the basic amino acid residues are both highly conserved and clustered in the region aa157 to aa209. Intriguingly, the primary sequence in this region is highly conserved from Xenopus to human (75.5% identity in 53 residues) although the overall sequence homology between Xenopus and human AEG-1 is significantly lower (46.3% identity in 587 residues). Also of high interest, a high-confidence RNA interaction site was identified in the region aa188-198, in a recent RNA interactome/ binding site proteomic screen (Castello et al. 2016b). Such regionally high sequence conservation is consistent with an important cellular function for the central domain of the protein, which we propose to be RNA binding. These findings are also consistent with proposals regarding the role of disordered domains in RNA binding, in particular the view that such interactions may enable multiple binding interactions without the need for the constraints imposed by discrete, defined sequence motifs, both in coding and structural terms (Järvelin et al. 2016).
An important clue to a specificity in AEG-1/RNA interactions was revealed in the HITS-CLIP and PAR-CLIP data sets, where a signature high read cluster enrichment was identified in the region surrounding and downstream from encoded transmembrane domains (Fig. 8). Such enhanced interactions may be functionally linked to protein biogenesis, perhaps by enabling translational stalling, and thereby contributing to the coupling of protein synthesis and folding. We note that studies of transmembrane domain integration have demonstrated that both the primary and secondary structural characteristics of transmembrane domains within the ribosomal exit tunnel can serve as a signal regulating the assembly/insertion of upstream transmembrane domains in the translocon (Do et al. 1996;Daniel et al. 2008). The findings reported here may represent an additional molecular component of this ordered protein synthesis, signaling and translocation process (Cross and High 2009;Skach 2009;Shao and Hegde 2011;Mandon et al. 2013). Alternatively, such cluster read enrichments may be a consequence of the binding interactions between SRP and the nascent transmembrane domains, though how such long range interactions might influence AEG-1/mRNA interactions remains to be determined.
A particularly intriguing element of the data sets presented here is the identification of cytosolic protein-encoding mRNAs in the AEG-1 RNA interactome. GO analysis of this mRNA cohort revealed significant enrichments for genes functioning in translation, translational regulation, and RNA localization to the ER (Fig. 6C,E). These data corroborate prior studies reporting the localization and translation of cytoplasmic protein mRNAs on ER-bound ribosomes and provide further evidence for the emerging view that ER-localized translation contributes broadly to cellular proteome expression (Lerner et al. 2003;Pyhtila et al. 2008;Nicchitta 2012, 2015;Jagannathan et al. 2014b). These data, and specifically the GO analysis findings, link to recent studies demonstrating de novo translation initiation on the ER (Jagannathan et al. 2014b), as well as reports demonstrating that translational regulatory processes presumed to occur in the cytosol compartment are, at least in part, ER-localized (Sakaki et al. 2012;Li et al. 2013;Stalder et al. 2013;Barman and Bhattacharyya 2015). The functional significance of such noncanonical mRNA localization warrants further study, because such data reopen investigations into the mechanisms governing mRNA partitioning between the cytosol and the ER, and the contributions of ER-localized translation to post-transcriptional gene regulation.
In summary, we report that AEG-1, a resident ER membrane protein with a positively charged and largely disordered cytoplasmic domain, is an RNA-binding protein functioning in the selective localization and anchoring of mRNAs to the ER. This selective RNA binding/anchoring functionality expands the mechanisms used by eukaryotic cells to selectively localize translation to the ER and by virtue of the direct binding mechanism, serves to "imprint" the ER membrane with the information necessary for the synthesis of resident organelle proteins. From existing global RNA interactome studies, as well as prior reports, we speculate that AEG-1 is one of many ER membrane proteins that contribute to the ER-mRNA transcriptome and its composite regulation (Chen et al. 2011;Cui et al. 2012;Jagannathan et al. 2014a;Reid and Nicchitta 2015). Defining how these composite mRNA localization processes regulate proteome expression and secretory pathway functionality represents an intriguing new direction of research into the primary organizational principles governing eukaryotic cell structure and function.

Sucrose cushion and density gradient polyribosome profiling
Cells grown to 80%-90% confluence were treated with 50 µg/mL cycloheximide (CHX) at 37°C for 10 min, washed with ice-cold DPBS, incubated with 50 µg/mL CHX/DPBS at 4°C for 20 min, and lysed by adding sucrose cushion lysis buffer (200 mM KCl, 25 mM KHEPES, pH 7.2, 50 mM EDTA, 1 mM DTT, 2% n-dodecyl-β-D-maltoside [DDM], and protease inhibitor cocktail) on ice for 15 min. The lysates were centrifuged at 14,000 rpm for 15 min to remove cell debris. The supernatants were incubated with either RNaseOUT or RNase Cocktail (4 U/mL of RNase A and 80 U/mL of RNase T1; Thermo Fisher Scientific) on ice for 30 min. The samples were loaded onto a sucrose cushion (500 mM sucrose, 200 mM KCl, 25 mM KHEPES, pH 7.2, 2 mM EDTA, 1 mM DTT). Ribosomes were collected by centrifugation at 90,000 rpm at 4°C for 15 min (TLA 100.2). Ribosome pellets were resuspended in SDS sample buffer and analyzed by SDS-PAGE and immunoblot analysis. For polysome profiling, cells were treated with CHX as above, washed in ice-cold DPBS, and lysed by addition of polysome lysis buffer (200 mM KCl, 25 mM KHEPES, pH 7.2, 10 mM MgCl 2 , 1 mM DTT, 2% DDM, RNaseOUT, and protease inhibitor cocktail) on ice for 15 min. Cell lysates were centrifuged at 14,000 rpm for 15 min to remove cell debris. Polyribosomes were resolved on 15%-50% sucrose gradients and fractionated as described previously Nicchitta 2007, 2008). The fractions were TCA precipitated, resuspended in SDS sample buffer, and analyzed by SDS-PAGE and immunoblotting.

Immunofluorescence microscopy
AEG-1-14 and PC-4 cell monolayers were fixed in 10% formalin for 10 minutes at room temperature. After permeabilization in 0.1% Triton X-100/PBS and blocking with 1% bovine serum albumin in PBS, the cells were incubated in a primary antibody solution for 1 h, extensively washed and subsequently incubated in secondary antibody solution for 45 min. Cell monolayers were again washed and the nuclei stained using DAPI. Coverslips were then mounted and imaged on a Deltavision Elite deconvolution microscope, using the 60×/1.42 oil PLAPON60XO WD 150 micron DIC ∞/0.17/ FN26.5, UIS2, BFP1 objective. Light micrographs were uniformly size and contrast adjusted in Adobe Photoshop CS6.

CLIP-seq protocols
Cells were grown overnight with (PAR-CLIP), or without (HITS-CLIP), 100 µM 4-thiouridine. AEG-1-14 and PC-4 cells were UV cross-linked, nuclease-digested, and immunoprecipitated as described above. For RNA dephosphorylaton, samples were incubated with 0.5 U/µL of calf intestinal alkaline phosphatase (New England Biolabs) in dephosphorylation buffer (50 mM Tris-HCl, pH 8, 100 mM NaCl, 10 mM MgCl 2 , 1 mM DTT) at 37°C for 25 min. For 5 ′ -end radioisotope-labeling, the samples were incubated with 0.5 U/µL of polynucleotide kinase (New England Biolabs) and 0.5 µCi/µL [γ-32 P]ATP in PNK buffer supplemented with 1 mM DTT. Protein-RNA complexes were eluted by 2× sample buffer at 95°C for 5 min, separated by SDS-PAGE, transferred to nitrocellulose membranes, and analyzed by phosphorimaging. RNA fragments were extracted from the nitrocellulose membranes by proteinase K digestion and purified by phenol/chloroform extraction. cDNA library construction was performed according to the manufacturer's instructions (New England Biolabs; E7330). cDNA libraries were analyzed by the Bioanalyzer DNA 1000 Kit (Agilent Technologies), Sanger sequencing, and Illumina HiSeq for 50nucleotide single-end read runs.

Read mapping
A transcriptome was generated using RNA-seq data from HepG2 cells from the ENCODE project (GEO GSM958740). Tophat and Cufflinks were used to establish transcript abundance, and the most abundant isoform from each gene was selected for further analysis. PAR-CLIP and HITS-CLIP reads were trimmed using Cutadapt, then mapped to the reference transcriptome using Bowtie, allowing for one mismatch and one assigned location per read.

Cluster identification
Clusters were defined as an overlapping set of reads that contained at least one mismatch (T→C for PAR-CLIP, any mutation for HITS-CLIP) and contained at least six unique read starts or stops. A score for each cluster was also defined that favored clusters that had many unique reads and many mutations. The score was defined as log 2 [1000 * (1-D) * M/R], where M is the number of mutations in the cluster and R is the abundance of the RNA from RNA-seq. D represents the breadth of read distribution, defined as (1/N) N , where N is the number of unique read starts or stops in the cluster. Clusters with scores of less than 0 were discarded.

Cluster analysis
For cluster positional analysis, total cluster score was summed relative to each landmark. For coding sequence starts and stops, cluster scores were summed relative to the Refseq annotations. For TM domain analysis, the cluster scores were summed relative to the first nucleotide of the first codon of a predicted point TM domain.
TM domains and signal sequences were identified algorithmically, using TMHMM and SignalP, respectively (Krogh et al. 2001). For analysis of the distribution of RNAs and clusters between different cohorts of mRNAs, cytosolic mRNAs were defined as lacking both TM domains and signal sequences, secretory mRNAs encoded a signal sequence but lacked a TM domain, and TM protein mRNAs encoded a TM domain with or without a signal sequence. Gene Ontology analyses were performed with the DAVID tools (Huang et al. 2009a,b).

RNA immunoprecipitation (RIP) and quantitative PCR
RIP was performed as described with minor modifications (Keene et al. 2006). PC-4 and AEG-1-14 cells were washed and harvested in ice-cold PBS and lysed in RIP lysis buffer (100 mM KCl, 10 mM KHEPES, pH 7.2, 5 mM MgCl 2 , 1 mM DTT, 400 µM VRC, 1 mM PMSF, 10 mM NaF, 2 mM sodium orthovanadate, 2 mM β-glycerophosphate, 0.5% IGEPAL CA-630, RNaseOUT, and protease inhibitor cocktail). The lysates were incubated on ice for 10 min and centrifuged at 14,000 rpm for 15 min to clear cell debris. The supernatants were supplemented with NT2 buffer (150 mM NaCl, 1 mM MgCl 2 , 50 mM Tris-HCl, pH 7.4, 0.05% IGEPAL CA-630), EDTA to 15 mM, DTT to 1 mM, RNaseOUT and VRC, and then immunoprecipitated with anti-HA antibody prebound Dynabeads Protein G beads (Thermo Fisher Scientific) at 4°C overnight. The samples were washed five times with NT2 buffer supplemented with 15 mM EDTA. RNA was eluted with TRIsure (Bioline) according to the manufacturer's instructions. The isolated RNA from RIP was analyzed by quantitative PCR. The results were shown as the relative fold enrichment of AEG-1-14 cell line over the PC-4 control signal.

DATA DEPOSITION
The sequencing data discussed in this publication have been deposited in NCBI's Gene Expression Omnibus (Hsu et al. 2018) and are accessible through GEO Series accession number GSE110260 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE110260).

SUPPLEMENTAL MATERIAL
Supplemental material is available for this article.


extfieldsofstudy: Biology Medicine

text:
Blockade of ONECUT2 expression in ovarian cancer inhibited tumor cell proliferation, migration, invasion and angiogenesis

One cut homeobox 2 (ONECUT2 or OC‐2) is a newly discovered transcription factor. Aberrant expression of OC‐2 is closely related to cell proliferation, migration, invasion, and angiogenesis. In this study, we found that OC‐2 expression was upregulated in ovarian adenocarcinoma cells, by Western blot analysis. The results of immunohistochemistry showed that the expression of OC‐2 was also increased in malignant ovarian cancer tissue. In order to explore the role of OC‐2 in the development of ovarian cancer, siRNAs that specifically targets OC‐2 were designed. The siRNA targeting OC‐2 could effectively inhibit the vascular endothelial growth factor A (VEGFA) expression, but silence and overexpression of VEGFA did not affect OC‐2 expression. In addition, OC2‐siRNA could block the proliferation, migration, and invasion, and inhibit epithelial–mesenchymal transition and the AKT/ERK signaling pathway, of human ovarian cancer cells in vitro. In a mouse model of ovarian cancer xenograft tumors, OC2‐siRNA could significantly inhibit tumor cell growth and the tumor inhibition rate reached approximately 73%. The results of immunohistochemistry showed that the densities of microvessels stained with CD31, the expression of OC‐2 and VEGFA were significantly decreased in tumors. These data indicated that OC‐2 was an upstream regulator of VEGFA and silencing OC‐2 could inhibit ovarian cancer angiogenesis and tumor growth.

cell precursors, which can be derived from adjacent vessels or from incorporation of bone marrow-derived endothelial precursor cells. 9 However, tumor neovascularization, consisting only of vascular endothelial cells, has been challenged by vasculogenic mimicry (VM). 10 Tang et al 11 indicated that vasculogenic mimicry was an alternative way to supply adequate blood for ovarian cancer. Research on tumor angiogenesis had progressed slowly until important discoveries of the major molecular drivers of tumor angiogenesis: the VEGF family and its homologous receptors. 12 In addition to VEGF, other important pathways also contribute to tumor angiogenesis. 8 One cut homeobox 2 (ONECUT2 or OC-2) was first reported by Patrick et al 13 as the second member of the ONECUT family, whose prototype was hepatocyte nuclear factor-6, characterized by the presence of a single cut domain and by a specific homeodomain. The expression status of OC-2 could be used for bladder cancer epigenetic analysis and the results had high sensitivity and specificity. 14 As an angiogenic and EMT-related transcription factor, OC-2 plays a key role in tumor cell invasion, metastasis, EMT, and oncogenesis. 15 FBS. All these cells were cultured in an incubator with 95% humidity and 5% CO 2 at 37°C.
Ovarian cancer cells (SKOV3 and COV-504, 2 9 10 5 cells/well) were added to 6-well plates for 24 hours in DMEM with 10% FBS before transfection. SKOV3 and COV-504 cells were transfected with siRNAs or pAAV-VEGFA in ViaFect Transfection Reagent (Promega, Fitchburg, WI, USA) according to the manufacturer's instructions. After transfection, the cells were cultured in a 37°C incubator for 24 hours for the further experiments.

| Cell proliferation assay
The cell viability was measured by CCK-8 assay (Dojindo Molecular Technologies

| Western blot analysis
The ovarian cancer cells were washed with cold PBS and lysed in RIPA buffer (Beyotime, Suzhou, China). The lysates were centrifuged at 12 000 g for 8 minutes at 4°C and the total protein in the supernatant were quantified by Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Rockford, IL, USA). Total proteins were separated by SDS-PAGE and transferred to PVDF membranes (Millipore, USA).

| Real-time quantitative PCR analysis
The total RNAs were extracted from cells with TRIzol reagent (Invitrogen, China). The RT reactions were carried out using a SYBR green-containing PCR kit (Qingke, China). The primers for real-time PCR were used to detect the mRNA expression of hypoxia-inducible factor-1a (HIF-1a), VEGFC, hepatocyte growth factor (HGF), fibroblast growth factor 2 (FGF2), Snai1, Snai2, transforming growth factor-b1 (TGF-b1), and Twist1. The sequences of real-time PCR primers are listed in Table 3. The primers were synthesized and purified by Beijing Qingke Biotechnology. All real-time PCR procedures were carried out on the CFX96 detection system (Bio-Rad, USA). 15

| Wound healing assay
SKOV3 and COV-504 cells (4.0 9 10 5 cells/well) transfected with OC2-siRNA were transferred to 6-well plates and cultured in DMEM with 10% FBS. The control groups were transfected with scramble siRNA. After incubation overnight, the cell scratches were made. The plates were washed with serum-free DMEM and the medium was changed to DMEM with 0.5% FBS. After incubation for 24 hours, the migrated cells were imaged with a computerized imaging system.
The cell migration rate of each group was calculated. 19 The cell migration inhibition rate = (1 À OC2-siRNA group migration distance/siRNA NC group migration distance) 9 100%.

| Migration and invasion assays
Migration and invasion assays of ovarian cancer cells were evaluated

| Tube formation assay
The 96-well plates were maintained at 4°C for 2 hours and coated with 60 lL Matrigel matrix per well. The plates were incubated for 30 minutes at 37°C. Then HUVECs (5 9 10 3 cells/well) suspended in M199 medium with Low Serum Growth Supplement (Gibco) were transferred to each well. Conditioned medium (70 lL) of SKOV3 and COV-504 cells transfected with OC2-siRNAs was added in the wells for 6 hours at 37°C in a 5% CO 2 incubator. The tube formation was observed under a microscope (IX71; Olympus, Japan) and the tube numbers were counted in five random high-power fields.  19 At the end of 21 days, nude mice were euthanized and tumors were stripped for immunohistochemistry assays.

| Immunohistochemistry
Immunohistochemistry was carried out as previously described. 19,20 The tumor tissue samples of ovarian cancer patients were provided  Technology), and rabbit anti-mouse ONECUT2 polyclonal antibody (1:500 dilution, ab28466; Abcam). The biotinylated HRP-conjugated secondary antibody was a goat anti-rabbit IgG.

| Statistical analysis
Statistical analyses were carried out by one-way ANOVA followed by the least significant difference test using the statistical software and P < .01 (**) were considered statistically significant.

| Ethics
The hospital ethics committee approved the study protocol, and all patients provided gave consent for the utilization of their tissue samples in this study.

| Overexpression of OC-2 in some ovarian cancer cells and tissues
Previous studies have indicated that OC-2 was expressed highly in liver cancer and colon cancer. 15,16 To explore the expression and sig-

| Silencing OC-2 expression in ovarian cancer cells inhibits endothelial angiogenesis
Anti-angiogenic therapy is used as a primary treatment for ovarian cancer. 21 To test the role of OC-2 in ovarian cancer cells stimulating angiogenesis, the conditioned media were collected from the COV-

| Silencing of OC-2 inhibits phosphorylation of AKT, ERK1/2, and EMT
The pathway of AKT/ERK has been verified to correlate with tumor growth and angiogenesis. [22][23][24] ONECUT-2 decreased the proliferation rate of SKOV3 and COV-504 cells, and showed a time-dependent inhibition of cell growth ( Figure 2D,E). To further assess whether OC-2 affected the downstream signaling pathway involved in cell proliferation and angiogenesis, p-Akt and p-Erk were investigated by Western blot assay. The results showed that silencing OC-2 could inhibit the phosphorylation of Akt and Erk ( Figure 6A).
ONECUT-2 is a transcription factor associated with EMT, and Ncadherin and E-cadherin are important markers of EMT development. 15,25 As a feature of aggressive tumors, EMT is characterized by reduced E-cadherin and increased N-cadherin expression, contributing to a stroma-oriented cellular adhesion profile with increased tumor cell motility and invasive properties. [26][27][28] To explore the relationship between OC-2 and EMT-related markers, N-cadherin and E-cadherin expression were detected after silencing OC-2 by Western blot, and the mRNA expression of Snai1, Snai2, TGF-b1, and Twist1 were detected after silencing OC-2 by real-time PCR.
Our results showed that E-cadherin expression increased and Ncadherin decreased after OC-2 silencing on SKOV3 and COV-504 cells (Figure 6Ba-e). In addition, the real-time PCR results showed that the expression levels of TGF-b1 in COV-504 and SKOV3 cells transfected with siRNA#1 reduced by approximately 56% and 48%, respectively (P < .05). The Snai2 expression levels were downregulated by approximately 45% and 40%, respectively (P < .05). The Twist1 expression levels were decreased by nearly 51% and 53%, respectively (P < .05). In contrast, the Snai1 expression levels declined by 25% and 20%, respectively, but the results did not reach a significant level (Figure 6Bf).
These results implied that OC-2 might play a role through the Akt/Erk signaling pathway to promote ovarian cancer cell proliferation and activate EMT to improve migration and invasion in ovarian cancer cells.

| Silencing OC-2 inhibits ovarian tumor growth and angiogenesis
In the mice were euthanized and the tumors stripped ( Figure 7A). The OC-2 silenced group showed a significant inhibition in tumor growth (P < .05) from the day 7, whereas the siRNA NC group showed a continuous increase ( Figure 7B). In accordance with this observation, the average weight of tumors in the OC-2 silenced group was significantly reduced compared to the control group (P < .01) ( Figure 7C).
These data suggest that OC-2 could be an important regulator of angiogenesis and tumor growth in ovarian cancer.

| DISCUSSION
Previous studies regarding OC-2 focused on the regulatory role of the ONECUT family in the differentiation and growth of liver tissues F I G U R E 7 ONECUT-2 (OC-2) silencing inhibits tumor growth and angiogenesis in an ovarian cancer xenograft nude mice model. A, COV-504 ovarian cancer cells transfected with siRNA#1/scrambled siRNA (siRNA NC) were injected into the right shoulder of BALB/c nude mice (n = 10). Mice were euthanized and the tumors were excised after 21 d. B, Growth curves of tumors. C, Quantitative analysis of tumor weight. D, Paraffin sections of tumors were stained with OC-2, CD31, VEGFA, and HIF-1a antibody. The positive rates were statistically analyzed. E, Tumors were stained with CD31 antibody. Microvessel densities were quantified. The numbers of blood vessels at five high-power fields (9200) per section were counted. F, Positive expression rates of vascular endothelial growth factor A (VEGFA), OC-2, and hypoxia-inducible factor-1a (HIF-1a) in tumors. Data were represented as the means AE SD. *P < .05, **P < .01 and visual cells, as well as the specific diagnosis of bladder cancer. [29][30][31] It was reported that OC-2 existed in the nucleus and its expression was significantly increased in colorectal carcinoma relative to paracancerous normal tissues. 15 Zhang et al 16  These results indicated that high expression of OC-2 in ovarian cancer cells could stimulate cell migration and invasion and break through the epithelial basement membrane. This is particularly relevant to the strong metastatic capacity of highly differentiated malignant ovarian cancer cells. 38 Subsequently, we used siRNA to silence OC-2 expression to investigate its effect on angiogenesis in vitro and in vivo. In vitro, the conditioned medium after silencing OC-2 in ovarian cancer cells significantly inhibited the tube formation numbers of HUVECs. Western blot assay showed that VEGFA expression was decreased after OC-2 silencing, but silencing and overexpression of VEGFA did not significantly affect OC-2 expression. These results indicated that OC-2 was an upstream regulator of VEGFA. In addition, we found that HIF-1a expression was significantly reduced after silencing OC-2 by realtime PCR and the mouse tumor immunohistochemistry experiments.
The mRNA expression levels of VEGFC, HGF, and FGF2 also decreased in varying degrees after silencing OC-2 expression in ovarian cancer cells by RT-PCR. Hypoxia stimulates new blood vessel formation in coronary artery disease, tumor angiogenesis, and diabetic neovascularization. [39][40][41] Hypoxia-inducible factor-1a increase of VEGF expression is due to both transcriptional activation and increased mRNA stability. 42 Tacchini et al 43  naling pathway. We hypothesized that OC-2 might also act through other angiogenic signaling pathways, such as STAT3/GIV, rather than just the Akt/Erk and HIF-1 signaling pathways that we revealed here.
Therefore, silencing OC-2 might affect one or several signaling pathways, inhibiting the expression and secretion of angiogenesis-related factors and ultimately affecting angiogenesis.
In addition, nude mice bearing COV-504 ovarian cancer transfected with OC2-siRNA (siRNA#1) showed significant tumor growth inhibition. The siRNA#1-treated tumors showed a significant reduction in CD31 stained blood vessels, OC-2, VEGFA, and HIF-1a expression compared to the siRNA NC group. These data indicated that inhibition of OC-2 expression could affect tumor growth and angiogenesis.
Our results provided proof of targeting OC-2 for anti-angiogenesis therapy in ovarian cancer; OC-2 might be a marker of ovarian cancer-specific detection.

ACKNOWLEDG MENTS
This work was supported by grants from the State Natural Science

CONF LICT OF I NTEREST
The authors have no conflict of interest.