Interaction between envelope protein of Jaagsiekte Sheep Retrovirus and Hippo signaling pathway is inferred from transcriptome analysis of naturally infected Ovine Pulmonary Adenomatosis

Background: Ovine pulmonary adenomatosis (OPA) is a contagious lung epithelial 2 tumor of sheep caused by jaagsiekte sheep retrovirus (JSRV), which causes severe 3 economic losses for the sheep industry in the world. The specific oncogenic 4 mechanism of JSRV is not yet clarified. 5 Methods: In this study, RNA was extracted from lung tissues of 3 naturally infected 6 OPA cases and 3 healthy individuals for transcriptome sequencing (RNA-Seq). 7 Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was used to 8 confirm the sequencing data. Immunohistochemistry (IHC) and western blot (WB) 9 were performed to confirm the signaling pathway enriched by DEGs that was 10 activated in naturally infected OPA cases. Cell viability, wound-healing, transwell and 11 colony formation assays were performed to assess the cell malignant transformation 12 of sheep trophoblast cells (STCs) transformed with JSRV- env lentivirus in vitro, and 13 then WB was performed to confirm the signaling pathway that had been validated in 14 the lung tissues. Results: A total of 366 DEGs (154 up-regulated and 212 down-regulated) were 16 identified by of lung of naturally infected OPA cases and healthy 17 individuals. GO analysis showed that 366 DEGs were significantly enriched in 178 18 GO terms, including 114 biological processes, 19 cellular components and 45 19 molecular functions. KEGG analysis showed that the DEGs mainly enriched in cell 20 proliferation, differentiation, and migration, such as PI3K/Akt/mTOR, 21 MAPK and Hippo and Hippo signaling pathway has never been 22 reported in naturally infected cases. qRT-PCR results of 10 DEGs which were selected randomly results. protein expression of Hippo signaling pathway up-regulated in Cell viability, wound-healing, transwell and colony formation assays confirmed that 26 JSRV- env lentivirus caused malignant transformation of STCs and JSRV Env 27 increased the protein expression of Hippo signaling pathway. 28 Conclusions: This research first identified the changes in the transcriptome level of 29 naturally infected OPA lung tissues. These data confirm that the Hippo signaling 30 pathway is involved in the mechanism of OPA, clarify the interaction between Hippo 31 signaling pathway and JSRV Env, provide further evidence for the tumorigenic 32 mechanism of JSRV. 33 Mitogen-activated AIDS: DEGs: differentially expressed genes; BSA: Bovine serum albumin; EMT: 575 epithelial-to-mesen-chymal transition; LUAD: adenocarcinoma; HCC: hepatocellular 576 carcinoma; AGR2: anterior gradient 2; EGF: epidermal growth factor; LTR: long 577 terminal repeats.

Additionally, advanced stage of naturally infected OPA cases can be regarded as a  tumorigenicity of JSRV depends on the envelope protein (Env) which is encoded by 54 oncogene-env, mediating malignant transformation of cells [5]. The malignant 55 transformation of cells is mainly regulated by cancer-related signaling pathways, 56 which are usually divided into receptor-dependent and non-receptor-dependent 57 mechanisms. The receptor-dependent mechanism is that the surface protein of JSRV 58 Env recognize and bind to the specific receptor hyaluronidase 2 (Hyal2) of cell 59 surface, then the transmembrane protein of JSRV Env is responsible for the fusion of found 1,971 differential transcripts (1,237 up-regulated, 734 down-regulated), but this 80 data was not applicable to clinically significant adult sheep [17]. 81 Up to now, RNA-Seq sequencing has not been used to reveal transcript 82 differences in naturally infected OPA cases. In order to further study the 83 tumorigenesis mechanism of JSRV, RNA-seq sequencing technology was used in this 84 study to analyze the naturally infected OPA cases and identify the differentially 85 expressed genes (DEGs). Through GO and KEGG pathway enrichment analysis, the 86 biological functions of DEGs were analyzed and new signaling pathways related to 87 DEGs enrichment were found. This study provides new research data for the 88 mechanism of JSRV tumorigenesis.   CA, USA) and the length of paired-end reads was 300 bp (±50 bp). 124 To ensure the quality and reliability of the data analysis, the quality of the 125 original data was controlled before the data analysis. Data processing prior to 126 assembly, low quality reads (including reads from sequencing adapters; reads with a 127 ratio of N>10; reads with all the A base; the number of bases of qvalue≤20 accounted 128 for more than 50% of the whole read) were removed [18]. The clean reads obtained 129 were compared and assembled with the sheep reference genome (version: To confirm the RNA-Seq data, 10 DEGs randomly selected were verified by 146 qRT-PCR. In this experiment, there were 3 biological replicates per group. Table 1 147 shows the primer sequences of selected mRNA transcripts and the reference gene    Bonferroni's post-hoc test. In addition, p < 0.05 (*), p < 0.01 (**) or p < 0.001 (***) 265 was considered as statistically significant.

RNA-Seq data and DEGs analysis 268
To further study the interaction mechanism of OPA, RNA-Seq was used to 269 analyze the transcriptome differences between naturally infected OPA cases and 270 healthy individuals. The RNA-Seq of each sample produced raw reads of 271 4.88±1.34×10 7 , and 4.81±0.75×10 7 of clean reads were obtained after quality control.

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More than 85.77% of the clean reads were mapped to the sheep reference genome.

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The detailed mapping output were summarized in Table 3. A total of 15,149 sheep  Firstly, we aimed to investigate whether JSRV Env could affect the proliferation of 370 STCs. MTT assay was performed to assess JSRV Env on the proliferation of STCs. As 371 a result, the growth rates of STCs with stable JSRV Env expression and A549 cells as 372 positive control were significantly higher when compared to negative control and 373 blank control (Fig.5C). Therefore, we concluded that JSRV Env promoted the 374 proliferation of STCs. Secondly, we aimed to investigate whether JSRV Env can play 375 a role in the migration and invasion of STCs, wound healing and transwell assays 376 were performed to assess the metastatic ability of JSRV Env-transformed STCs. As 377 shown in Fig.5D, JSRV Env enhanced STCs motility was observed using wound 378 healing assay, the scratch areas of JSRV Env-transformed STCs and A549 cells as  adenocarcinoma cells [61]. Due to the process that AREG is activated by AGR2 is 493 mediated by YAP1, and YAP1 is the nuclear effector of Hippo signaling pathway. It is 494 speculated that Hippo signaling pathway may be involved in the development of OPA.

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The IHC detection of Hippo signaling pathway showed that the core components    Availability of data and materials 595 The datasets supporting the conclusions of this article were included within the article 596 and its additional files.          (e to f) MST1/2; 3 (g to h) LATS1/2; (i to j) Total YAP1; (k to l) phosphorylated YAP1 (p-YAP1). Note that YAP1 was strongly positive in nucleus and cytoplasm of proliferative type II pneumocytes and nuclear labeling was more intense than the labeling of the cytoplasm (j). The phosphorylated YAP1 was predominantly detected in the cytoplasm of proliferative type II pneumocytes (l). Red arrows in panels j indicate YAP1 locating in the nucleus. (B) Results are the mean of four separate experiments performed in triplicate ± SD. p < 0.05 signi cantly different from healthy control groups. (C) Western blot analysis of Hippo signaling pathway core components in lung of naturally infected OPA cases and healthy sheep. (D)The data are presented as mean ± SD. value from the lungs of 3 healthy sheep and 3 naturally infected OPA cases (p < 0.05).