Validation of Candidate Host Cell Entry Factors for Bovine Herpes Virus Type-1 Based on a Genome-Wide CRISPR Knockout Screen.

To identify host factors that affect Bovine Herpes Virus Type 1 (BoHV-1) infection we previously applied a genome wide CRISPR knockout screen targeting all bovine protein coding genes. By doing so we compiled a list of both pro-viral and anti-viral proteins involved in BoHV-1 replication. Here we provide further analysis of those that are potentially involved in viral entry into the host cell. We first generated single cell knockout clones deficient in some of the candidate genes for validation. We provide evidence that Polio Virus Receptor-related protein (PVRL2) serves as a receptor for BoHV-1, mediating more efficient entry than the previously identified Polio Virus Receptor (PVR). By knocking out two enzymes that catalyze HSPG chain elongation, HST2ST1 and GLCE, we further demonstrate the significance of HSPG in BoHV-1 entry. Another intriguing cluster of candidate genes, COG1, COG2 and COG4-7 encode six subunits of the Conserved Oligomeric Golgi (COG) complex. MDBK cells lacking COG6 produced fewer but bigger plaques compared to control cells, suggesting more efficient release of newly produced virions from these COG6 knockout cells, due to impaired HSPG biosynthesis. We further observed that viruses produced by the COG6 knockout cells consist of protein(s) with reduced N-glycosylation, potentially explaining their lower infectivity. To facilitate candidate validation, we also detailed a one-step multiplex CRISPR interference (CRISPRi) system, an orthogonal method to KO that enables quick and simultaneous deployment of three CRISPRs for efficient gene inactivation. Using CRISPR3i, we verified eight candidates that have been implicated in the synthesis of surface heparan sulfate proteoglycans (HSPGs). In summary, our experiments confirmed the two receptors PVR and PVRL2 for BoHV-1 entry into the host cell and other factors that affect this process, likely through the direct or indirect roles they play during HSPG synthesis and glycosylation of viral proteins.

GARP and EARP are required for efficient BoHV-1 replication as identified by a genome wide CRISPR knockout screen.

The advances in gene editing bring unprecedented opportunities in high throughput functional genomics to animal research. Here we describe a genome wide CRISPR knockout library, btCRISPRko.v1, targeting all protein coding genes in the cattle genome. Using it, we conducted genome wide screens during Bovine Herpes Virus type 1 (BoHV-1) replication and compiled a list of pro-viral and anti-viral candidates. These candidates might influence multiple aspects of BoHV-1 biology such as viral entry, genome replication and transcription, viral protein trafficking and virion maturation in the cytoplasm. Some of the most intriguing examples are VPS51, VPS52 and VPS53 that code for subunits of two membrane tethering complexes, the endosome-associated recycling protein (EARP) complex and the Golgi-associated retrograde protein (GARP) complex. These complexes mediate endosomal recycling and retrograde trafficking to the trans Golgi Network (TGN). Simultaneous loss of both complexes in MDBKs resulted in greatly reduced production of infectious BoHV-1 virions. We also found that viruses released by these deficient cells severely lack VP8, the most abundant tegument protein of BoHV-1 that are crucial for its virulence. In combination with previous reports, our data suggest vital roles GARP and EARP play during viral protein packaging and capsid re-envelopment in the cytoplasm. It also contributes to evidence that both the TGN and the recycling endosomes are recruited in this process, mediated by these complexes. The btCRISPRko.v1 library generated here has been controlled for quality and shown to be effective in host gene discovery. We hope it will facilitate efforts in the study of other pathogens and various aspects of cell biology in cattle.

Functional divergence of oligoadenylate synthetase 1 (OAS1) proteins in Tetrapods.

OASs play critical roles in immune response against virus infection by polymerizing ATP into 2-5As, which initiate the classical OAS/RNase L pathway and induce degradation of viral RNA. OAS members are functionally diverged in four known innate immune pathways (OAS/RNase L, OASL/IRF7, OASL/RIG-I, and OASL/cGAS), but how they functionally diverged is unclear. Here, we focus on evolutionary patterns and explore the link between evolutionary processes and functional divergence of Tetrapod OAS1. We show that Palaeognathae and Primate OAS1 genes are conserved in genomic and protein structures but differ in function. The former (i.e., ostrich) efficiently synthesized long 2-5A and activated RNase L, while the latter (i.e., human) synthesized short 2-5A and did not activate RNase L. We predicted and verified that two in-frame indels and one positively selected site in the active site pocket contributed to the functional divergence of Palaeognathae and Primate OAS1. Moreover, we discovered and validated that an in-frame indel in the C-terminus of Palaeognathae OAS1 affected the binding affinity of dsRNA and enzymatic activity, and contributed to the functional divergence of Palaeognathae OAS1 proteins. Our findings unravel the molecular mechanism for functional divergence and give insights into the emergence of novel functions in Tetrapod OAS1.

Functional Analysis of Sheep POU2F3 Isoforms.

POU domain class 2 transcription factor 3 (POU2F3) plays an important role in keratinocyte proliferation and differentiation. Our previous study identified four sheep POU2F3 transcript variants (POU2F3-1, POU2F3-2, POU2F3-3, and POU2F3-4), encoding three POU2F3 protein isoforms (POU2F3-1, POU2F3-2, and POU2F3-3). However, the functional differences among the three POU2F3 isoforms remain unknown. The objective of this study was to determine the tissue expression pattern of the four POU2F3 transcript variants in sheep and to investigate the functional differences in cell proliferation among the three POU2F3 isoforms. Quantitative RT-PCR analysis showed that the four POU2F3 transcripts were ubiquitously expressed in all tested adult sheep tissues, and POU2F3-1 exhibited higher expression level than the other three POU2F3 transcript variants in skin (P < 0.05). Cell proliferation assay showed that overexpression of any one of the three POU2F3 isoforms significantly inhibited the proliferation of sheep fetal fibroblasts and HaCaT cells at 48 and 72 h after transfection (P < 0.05). POU2F3-3 had less inhibitory effect on cell proliferation than POU2F3-1 and POU2F3-2 (P < 0.05), and POU2F3-1 and POU2F3-2 had similar inhibitory effects (P > 0.05). Dual luciferase reporter assays demonstrated that overexpression of any one of the three POU2F3 isoforms significantly inhibited the promoter activities of keratin 14 (KRT14) and matrix metalloproteinase 19 (MMP19) genes (P < 0.05). POU2F3-3 had less inhibitory effect on the promoter activities of KRT14 and MMP19 genes than POU2F3-1 and POU2F3-2 (P < 0.05), and POU2F3-1 and POU2F3-2 had similar inhibitory effects (P > 0.05). These results suggest three sheep POU2F3 isoforms have similar functional effects, but to a different extent.

Origin and development of oligoadenylate synthetase immune system.

BACKGROUND: Oligoadenylate synthetases (OASs) are widely distributed in Metazoa including sponges, fish, reptiles, birds and mammals and show large variation, with one to twelve members in any given species. Upon double-stranded RNA (dsRNA) binding, avian and mammalian OASs generate the second messenger 2'-5'-linked oligoadenylate (2-5A), which activates ribonuclease L (RNaseL) and blocks viral replication. However, how Metazoa shape their OAS repertoires to keep evolutionary balance to virus infection is largely unknown. We performed comprehensive phylogenetic and functional analyses of OAS genes from evolutionarily lower to higher Metazoa to demonstrate how the OAS repertoires have developed anti-viral activity and diversified their functions. RESULTS: Ancient Metazoa harbor OAS genes, but lack both upstream and downstream genes of the OAS-related pathways, indicating that ancient OASs are not interferon-induced genes involved in the innate immune system. Compared to OASs of ancient Metazoa (i.e. sponge), the corresponding ones of higher Metazoa present an increasing number of basic residues on the OAS/dsRNA interaction interface. Such an increase of basic residues might improve their binding affinity to dsRNA. Moreover, mutations of functional residues in the active pocket might lead to the fact that higher Metazoan OASs lose the ability to produce 3'-5'-linked oligoadenylate (3-5A) and turn into specific 2-5A synthetases. In addition, we found that multiple rounds of gene duplication and domain coupling events occurred in the OAS family and mutations at functionally critical sites were observed in most new OAS members. CONCLUSIONS: We propose a model for the expansion of OAS members and provide comprehensive evidence of subsequent neo-functionalization and sub-functionalization. Our observations lay the foundation for interrogating the evolutionary transition of ancient OAS genes to host defense genes and provide important information for exploring the unknown function of the OAS gene family.

Heteroplasmy Detection of Mitochondrial DNA A3243G Mutation Using Quantitative Real-Time PCR Assay Based on TaqMan-MGB Probes.

A point mutation of mitochondrial DNA (mtDNA) at nucleotide position 3243 A to G (mt.3243A>G) is involved in many common diseases, including maternally inherited diabetes and deafness (MIDD) and mitochondrial encephalomyopathy, lactic acidosis with stroke-like episodes (MELAS). However, the mutant level of mt.3243A>G varies both among individuals and in different organs, tissues, and even cells of single individuals. For detection of this mutation, current methods have limited universality and sensitivity and may be not adequate for a routine clinical test. Here, we develop and evaluate a rapid TaqMan-MGB quantitative real-time PCR (qPCR) method for detecting and quantifying the heteroplasmy level of mt.3243A>G in single-tube analysis. With our method, the sensitivity of detection was as low as 0.1%, but the accuracy of quantification was reliable, down to 4%. All positives could be correctly identified, and the heteroplasmy levels determined by qPCR correlated well with the results from restriction fragment length polymorphism (RFLP) and pyrosequencing assays (r = 0.921~0.973 and 0.972~0.984). In addition, we demonstrated that the urinary sediments, leukocytes, or hair follicles might be ideal templates to detect and quantify the heteroplasmy of mt.3243A>G mutation; however, they should be optimized or retreated for further accurate quantification. Our study should allow rapid and high throughput diagnostic testing and can potentially be used to clarify the association between clinical phenotype and pathogenic mitochondrial mutations derived from various tissues.

Molecular Mechanisms for the Adaptive Switching Between the OAS/RNase L and OASL/RIG-I Pathways in Birds and Mammals.

Host cells develop the OAS/RNase L [2'-5'-oligoadenylate synthetase (OAS)/ribonuclease L] system to degrade cellular and viral RNA, and/or the OASL/RIG-I (2'-5'-OAS like/retinoic acid inducible protein I) system to enhance RIG-I-mediated IFN induction, thus providing the first line of defense against viral infection. The 2'-5'-OAS-like (OASL) protein may activate the OAS/RNase L system using its typical OAS-like domain (OLD) or mimic the K63-linked pUb to enhance antiviral activity of the OASL/RIG-I system using its two tandem ubiquitin-like domains (UBLs). We first describe that divergent avian (duck and ostrich) OASL inhibit the replication of a broad range of RNA viruses by activating and magnifying the OAS/RNase L pathway in a UBL-dependent manner. This is in sharp contrast to mammalian enzymatic OASL, which activates and magnifies the OAS/RNase L pathway in a UBL-independent manner, similar to 2'-5'-oligoadenylate synthetase 1 (OAS1). We further show that both avian and mammalian OASL can reversibly exchange to activate and magnify the OAS/RNase L and OASL/RIG-I system by introducing only three key residues, suggesting that ancient OASL possess 2-5A [px5'A(2'p5'A)n; x = 1-3; n ≥ 2] activity and has functionally switched to the OASL/RIG-I pathway recently. Our findings indicate the molecular mechanisms involved in the switching of avian and mammalian OASL molecules to activate and enhance the OAS/RNase L and OASL/RIG-I pathways in response to infection by RNA viruses.

Broad-spectrum antiviral functions of duck interferon-induced protein with tetratricopeptide repeats (AvIFIT).

Mammalian interferon-induced proteins with tetratricopeptide repeats (IFITs) play important roles in many cellular processes and host innate immune response to viruses. However, the functions of IFIT proteins in birds are largely unknown. Here, we first describe that the only one avian IFIT protein is orthologous to ancestor of mammalian IFITs. We find that the predicted structure of duck AvIFIT protein is similar to that of human IFIT5. We also find that duck AvIFIT protein shows antiviral activity to a broad range of specific RNA and DNA viruses like mammalian IFIT proteins. Further analysis indicates that overexpression of duck AvIFIT protein in DF1 cells leads to a remarkable accumulation of cells at G1/S transition associated with growth arrest and may promote apoptosis. Moreover, duck AvIFIT binds to nucleoprotein (NP) of H5N1 influenza virus and upregulates the expression of genes involving the IFN pathway in DF1 cells. In summary, our findings support that duck AvIFIT protein plays critical role in host immune response to viruses, at least H5N1 virus, through affecting function of viral NP protein, magnifying the IFN signaling and arresting cell growth.

Structural Characterization and Association of Ovine Dickkopf-1 Gene with Wool Production and Quality Traits in Chinese Merino.

Dickkopf-1 (DKK1) is an inhibitor of canonical Wnt signaling pathway and regulates hair follicle morphogenesis and cycling. To investigate the potential involvement of DKK1 in wool production and quality traits, we characterized the genomic structure of ovine DKK1, performed polymorphism detection and association analysis of ovine DKK1 with wool production and quality traits in Chinese Merino. Our results showed that ovine DKK1 consists of four exons and three introns, which encodes a protein of 262 amino acids. The coding sequence of ovine DKK1 and its deduced amino acid sequence were highly conserved in mammals. Eleven single nucleotide polymorphisms (SNPs) were identified within the ovine DKK1 genomic region. Gene-wide association analysis showed that SNP5 was significantly associated with mean fiber diameter (MFD) in the B (selected for long wool fiber and high-quality wool), PW (selected for high reproductive capacity, high clean wool yield and high-quality wool) and U (selected for long wool fiber with good uniformity, high wool yield and lower fiber diameter) strains (p < 4.55 × 10-3 = 0.05/11). Single Nucleotide Polymorphisms wide association analysis showed that SNP8 was significantly associated with MFD in A strain and fleece weight in A (selected for large body size), PM (selected for large body size, high reproductive capacity and high meat yield) and SF (selected for mean fiber diameter less than 18 µm and wool fiber length between 5 and 9 cm) strains (p < 0.05), SNP9 was significantly associated with curvature in B and U strains (p < 0.05) and SNP10 was significantly associated with coefficient of variation of fiber diameter in A, PW and PM strains and standard deviation of fiber diameter in A and PM strains (p < 0.05). The haplotypes derived from these 11 identified SNPs were significantly associated with MFD (p < 0.05). In conclusion, our results suggest that DKK1 may be a major gene controlling wool production and quality traits, also the identified SNPs (SNPs5, 8, 9 and 10) might be used as potential molecular markers for improving sheep wool production and quality in sheep breeding.

Polymorphisms of FST gene and their association with wool quality traits in Chinese Merino sheep.

Follistatin (FST) is involved in hair follicle morphogenesis. However, its effects on hair traits are not clear. This study was designed to investigate the effects of FST gene single nucleotide polymorphisms (SNP) on wool quality traits in Chinese Merino sheep (Junken Type). We performed gene expression analysis, SNP detection, and association analysis of FST gene with sheep wool quality traits. The real-time RT-PCR analysis showed that FST gene was differentially expressed in adult skin between Chinese Merino sheep (Junken Type) and Suffolk sheep. Immunostaining showed that FST was localized in inner root sheath (IRS) and matrix of hair follicle (HF) in both SF and Suffolk sheep. Sequencing analysis identified a total of seven SNPs (termed SNPs 1-7) in the FST gene in Chinese Merino sheep (Junken Type). Association analysis showed that SNP2 (Chr 16. 25,633,662 G>A) was significantly associated with average wool fiber diameter, wool fineness SD, and wool crimp (P < 0.05). SNP4 (Chr 16. 25,633,569 C>T) was significantly associated with wool fineness SD and CV of fiber diameter (P < 0.05). Similarly, the haplotypes derived from these seven identified SNPs were also significantly associated with average wool fiber diameter, wool fineness SD, CV of fiber diameter, and wool crimp (P < 0.05). Our results suggest that FST influences wool quality traits and its SNPs 2 and 4 might be useful markers for marker-assisted selection and sheep breeding.

Functional Characterization of a Single Nucleotide Polymorphism in the 3' Untranslated Region of Sheep DLX3 Gene.

The Distal-less 3 (homeobox protein DLX-3), a transcription factor, is critical for the development of hair follicle and hair formation and regeneration. We previously identified and found that four SNPs (c. *118T>C, c. *228T>C, c. *688A>G and c. *1,038_1,039 insC) in 3' untranslated region (UTR) of sheep DLX3 were in high linkage disequilibrium with each other and significantly associated with wool crimp (P<0.05), however, the underlying mechanisms by which these SNPs affect the wool crimp remains unknown. In the present study, we performed association analysis between these four identified SNPs and DLX3 gene expression in sheep skin using quantitative real-time RT-PCR. The results showed that these SNPs were significantly associated with sheep skin DLX3 mRNA expression levels. Then, we constructed DLX3 3'UTR luciferase reporters and validated the association. The reporter assays showed that the three major haplotypes, derived from the four SNPs, had significantly different effects on luciferase reporter activity and the four SNPs also had significantly different allelic effects on the luciferase reporter activity (p < 0.05). Bioinformatics analysis showed that the SNP (c. *1,038_1,039 insC) was located within a potential miR-188 binding site of the 3'UTR of sheep DLX3 mRNA. This SNP may affect miR-188-mediated DLX3 gene expression and result in phenotypic variation. To test the hypothesis, we investigated the effects of miR-188 mimic and inhibitor on the activity of the DLX3 3'UTR luciferase reporter with different SNP alleles. The results showed that in both sheep fetal fibroblasts (SFFs) and human HaCaT cells, miR-188 mimic could significantly decrease the allele D (deletion) luciferase reporter activity (p < 0.05), but miR-188 inhibitor could increased the reporter activitiy. However, neither miR-188 mimc nor inhibitor could influence the allele I (insertion) reporter activity. In addition, transfection of miR-188 mimic dramatically decreased the endogenous expression of DLX3 in SFFs (p < 0.05). Taken together, we demonstrated that DLX3 is a target gene of miR-188 and the SNP (c. *1,038_1,039 insC) is a functional SNP, and affects miR-188-mediated gene regulation of sheep DLX3. Our finding may in part explain allelic difference in gene expression and wool crimp in our tested sheep population.

Genome-wide association study for wool production traits in a Chinese Merino sheep population.

Genome-wide association studies (GWAS) provide a powerful approach for identifying quantitative trait loci without prior knowledge of location or function. To identify loci associated with wool production traits, we performed a genome-wide association study on a total of 765 Chinese Merino sheep (JunKen type) genotyped with 50 K single nucleotide polymorphisms (SNPs). In the present study, five wool production traits were examined: fiber diameter, fiber diameter coefficient of variation, fineness dispersion, staple length and crimp. We detected 28 genome-wide significant SNPs for fiber diameter, fiber diameter coefficient of variation, fineness dispersion, and crimp trait in the Chinese Merino sheep. About 43% of the significant SNP markers were located within known or predicted genes, including YWHAZ, KRTCAP3, TSPEAR, PIK3R4, KIF16B, PTPN3, GPRC5A, DDX47, TCF9, TPTE2, EPHA5 and NBEA genes. Our results not only confirm the results of previous reports, but also provide a suite of novel SNP markers and candidate genes associated with wool traits. Our findings will be useful for exploring the genetic control of wool traits in sheep.