Optimized production of full-length PCV2d virus-like particles in Escherichia coli: A cost-effective and high-yield approach for potential vaccine antigen development.

Porcine circovirus type 2 (PCV2) is a globally prevalent infectious pathogen affecting swine, with its capsid protein (Cap) being the sole structural protein critical for vaccine development. Prior research has demonstrated that PCV2 Cap proteins produced in Escherichia coli (E. coli) can form virus-like particles (VLPs) in vitro, and nuclear localization signal peptides (NLS) play a pivotal role in stabilizing PCV2 VLPs. Recently, PCV2d has emerged as an important strain within the PCV2 epidemic. In this study, we systematically optimized the PCV2d Cap protein and successfully produced intact PCV2d VLPs containing NLS using E. coli. The recombinant PCV2d Cap protein was purified through affinity chromatography, yielding 7.5 mg of recombinant protein per 100 ml of bacterial culture. We augmented the conventional buffer system with various substances such as arginine, ß-mercaptoethanol, glycerol, polyethylene glycol, and glutathione to promote VLP assembly. The recombinant PCV2d Cap self-assembled into VLPs approximately 20 nm in diameter, featuring uniform distribution and exceptional stability in the optimized buffer. We developed the vaccine and immunized pigs and mice, evaluating the immunogenicity of the PCV2d VLPs vaccine by measuring PCV2-IgG, IL-4, TNF-α, and IFN-γ levels, comparing them to commercial vaccines utilizing truncated PCV2 Cap antigens. The HE staining and immunohistochemical tests confirmed that the PCV2 VLPs vaccine offered robust protection. The results revealed that animals vaccinated with the PCV2d VLPs vaccine exhibited high levels of PCV2 antibodies, with TNF-α and IFN-γ levels rapidly increasing at 14 days post-immunization, which were higher than those observed in commercially available vaccines, particularly in the mouse trial. This could be due to the fact that full-length Cap proteins can assemble into more stable PCV2d VLPs in the assembling buffer. In conclusion, our produced PCV2d VLPs vaccine elicited stronger immune responses in pigs and mice compared to commercial vaccines. The PCV2d VLPs from this study serve as an excellent candidate vaccine antigen, providing insights for PCV2d vaccine research.

METTL14 and FTO mediated m6A modification regulate PCV2 replication by affecting miR-30a-5p maturity.

The epigenetic modification of the N6-methyladenosine (m6A) methylation plays an important role in virus infection and replication. However, its role in Porcine circovirus type 2 (PCV2) replication has not been well studied. Here, we demonstrated that m6A modifications are increased in PK-15 cells after PCV2 infection. In particular, PCV2 infection could increase the expression of methyltransferase METTL14 and demethylase FTO. Moreover, interfering with METTL14 accumulation reduced the m6A methylation level and virus reproduction, whereas depleting the FTO demethylase enhanced the m6A methylation level and stimulated virus reproduction. Besides, we showed that METTL14 and FTO regulate PCV2 replication by affecting the process of miRNA maturity, especially the miRNA-30a-5p. Taken together, our results demonstrated that the m6A modification positively affects PCV2 replication and the role of m6A modification in the replication mechanism of the PCV2 virus provides a new idea for the prevention and control of the PCV2.

A chimeric PCV rescued virus with the immunogenic cap gene of PCV3 cloned into the genomic backbone of the nonpathogenic PCV1 induces specific antibodies but with no pathogenic in pigs.

A chimeric PCV called PCV1-3 with the immunogenic Cap gene of pathogenic PCV type 3(PCV3) cloned into the genomic skeleton of the nonpathogenic PCV1 was rescued and inoculated into PCV3 negative piglets. The results of fluorescence quantitative PCR showed that the PCV1-3 DNA detected in serum and tissues was negative. The pathogenicity of piglets showed that PCV 1-3 did not cause the clinical characteristics and pathological changes. The viral neutralization assay revealed that infected pigs could produce antibodies and neutralize the viral activity. All results showed that chimeric virus induced specific antibodies but with no pathogenic in pigs, which provided new candidate strains for the development of PCV3 vaccine.

CRISPR/Cas9-Mediated Knockout of the Dicer and Ago2 Genes in BHK-21 Cell Promoted Seneca Virus A Replication and Enhanced Autophagy.

RNA interference (RNAi) is a major form of antiviral defense in host cells, and Ago2 and Dicer are the major proteins of RNAi. The Senecavirus A (SVA) is a reemerging virus, resulting in vesicular lesions in sows and a sharp decline in neonatal piglet production. In this study, CRISPR/Cas9 technology was used to knock out Ago2 and Dicer genes in BHK-21 cell lines used for SVA vaccine production. Cell clones with homozygous frameshift mutations of Ago2 and Dicer genes were successfully identified. The two knockout cell lines were named BHK-DicerΔ- and BHK-Ago2Δ-. Results showed that the two genes' knockout cell lines were capable of stable passage and the cell growth rate did not change significantly. The replication rate and virus titers of SVA were significantly increased in knockout cell lines, indicating that RNAi could inhibit SVA replication. In addition, compared with normal cells, autophagy was significantly enhanced after SVA-infected knockout cell lines, while there was no significant difference in autophagy between the knockout and normal cell lines without SVA. The results confirmed that SVA could enhance the autophagy in knockout cells and promote viral replication. The two knockout cell lines can obtain viruses with high viral titers and have good application prospects in the production of SVA vaccine. At the same time, the RNAi knockout cell lines provide convenience for further studies on RNAi and SVA resistance to RNAi, and it lays a foundation for further study of SVA infection characteristics and screening of new therapeutic drugs and drug targets.

Treatment of Preschool Children With Autism Spectrum Disorder: A Trial to Evaluate a Learning Style Profile Intervention Program in China.

Objective: To investigate whether the provision of learning style profile (LSP) training improves development in preschool children with autism spectrum disorder (ASD) in China and to describe the characteristics of children who benefit from the intervention. Methods: Eighty-one children aged 36 to 72 months who were diagnosed with ASD for the first time were recruited for the intervention group. All of them received 24 weeks of LSP training, consisting of hospital- and home-based training. Twenty-one children with ASD of the same age in the control group had never received any intervention after diagnosis but underwent an assessment. Assessments were conducted at baseline and 24 weeks later. Differences in the developmental level and severity of ASD symptoms over time and between groups were analyzed by repeated standardized measures. Secondary analyses examined age effects among the 36- 48-, 48- 60-, and 60-72-month age groups. Results: Within-group comparison of the intervention group revealed significant treatment effects after the intervention, according to: language, social and adaptive developmental quotients (DQs) of the China Developmental Scale; total Childhood Autism Rating Scale (CARS) score; and hyperactivity, peer problems, total difficulties, and prosocial behavior scores of the Strengths and Difficulties Questionnaire (SDQ). Similar gains were observed in gross and fine motor DQs of the China Developmental Scale and emotional symptoms and conduct problems scores of the SDQ; however, the differences between these pre- and postintervention scores did not reach statistical significance. Comparisons among the three age groups in the intervention groups demonstrated a significant age effect on adaptive DQs of the China Developmental Scale; total CARS score; hyperactivity, peer problems and total difficulties scores of the SDQ. Comparison between the intervention and control groups revealed significant treatment effects on language, social and adaptive DQs of the China Developmental Scale; total CARS score; and emotional symptoms, conduct problems, hyperactivity, peer problems, total difficulties, and prosocial behavior scores of the SDQ after the intervention. Similar gains were observed in gross and fine motor DQs of the China Developmental Scale, although differences between the two groups did not reach statistical significance. Conclusion: Our findings suggest that LSP training can effectively improve social behavior and reduce the severity of ASD symptoms in children with ASD. Our data also highlight the importance of early intervention.

Complete genome and pathogenesis of a novel recombinant Senecavirus A isolate in P.R. China.

Senecavirus A (SVA), formerly called Seneca Valley virus (SVV) was first isolated from the USA in 2002. This study isolated an SVA strain from a pig herd in Shandong Province, PR China and designated it SVA-CH-SDGT-2017. The full-length genome, excluding the poly(A) tails of the SVA isolates, was 7280 nucleotides long, with the genomic organization resembling and sharing high nucleotide identities of 90.7-96.9 % with other previously reported SVA isolates. To investigate the pathogenicity of the SVA isolates, experimental infections of pigs were performed. The SVA strains successfully infected the pigs, as evidenced by the presence of virus shedding and robust serum neutralizing antibody responses. In addition, the contact-exposed experiment showed that the virus shedding of the contact-exposed pigs was approximately a 100-fold reduced compared to that of the inoculated group, indicating that the virus is capable of transmission to pigs. Our findings provide useful data for studying the pathogenesis and transmission of SVA in pigs.

Mutation of the sixth amino acid of the Rep protein has no effect on porcine circovirus 2b but enhances porcine circovirus 2d replication in vitro.

Porcine circovirus type 2 (PCV2) is the etiological agent of post-weaning multisystemic wasting syndrome (PMWS). The original prevalent genotype, PCV2a, has been replaced by genotypes 2b and 2d in the swine population worldwide. The Rep protein is critical for viral replication. Comparison of a large number of Rep protein amino acid (aa) sequences showed that three sites distinguish genotype 2b from genotype 2d. In order to analyze the effect of exchanging the amino acids (asparagine and serine) at position 6 in the Rep proteins of PCV2b and PCV2d, two wild-type and two mutant viruses were rescued. Real-time quantitative PCR and a one-step growth curve were used to determine the viral load to assess the replication ability of the rescued viruses. The results showed that there was no significant difference in in vitro performance between the wild-type PCV2b and the mutated virus, while the mutation of PCV2d enhanced viral replication.

PCV2 Triggers PK-15 Cell Apoptosis Through the PLC-IP3R-Ca2+ Signaling Pathway.

The endoplasmic reticulum (ER) plays an essential role in Ca2+ concentration balance and protein biosynthesis. During infection, the virus needs to complete its life process with the help of ER. At the same time, ER also produces ER stress (ERS), which induces apoptosis to resist virus infection. Our study explored the Ca2+ concentration, ERS, and the apoptosis mechanism after porcine circovirus 2 (PCV2) infection. We show here that PCV2 infection induces the increased cytoplasmic Ca2+ level and PK-15 cell ER swelling. The colocalization of phospholipase C (PLC) and inositol 1,4,5-trisphosphate receptor (IP3R) in the cytoplasm was observed by laser confocal microscopy. Western blot and quantitative polymerase chain reaction experiments confirmed that PLC and IP3R expression levels increased after PCV2 infection, and Ca2+ concentration in the cytoplasm increased after virus infection. These results suggest that PCV2 infection triggers ERS of PK-15 cells via the PLC-IP3R-Ca2+ signaling pathway to promote the release of intracellular Ca2+ and led to cell apoptosis.

Efficient influence of ssDNA virus PCV2 replication by CRISPR/Cas9 targeting of the viral genome.

Porcine circovirus type 2 (PCV2), a ubiquitous pathogen that primary cause of postweaning multisystemic wasting syndrome (PMWS), had caused significant morbidity and mortality in swine populations with huge economic losses in the worldwide swine industry. Currently, looking for effective antiviral drugs for PCV2 infection remains an important works. In our study, CRISPR/Cas9 system was used to further detected the key sites of PCV2 replication. We designed 8 single guide RNAs (sgRNA) by targeting essential genes across the genome of PCV2. Western-blot(WB), Cell counting kit-8 for high-throughput sgRNA screening were applied to detect PCV2 replication levels. The results showed that Oc8, O13, O134, NQT and NPS sgRNAs can edit the PCV2 genome efficiently and inhibit PCV2 replication in PK-15 cell; H3 sgRNA cannot edit the PCV2 genome successfully; NAT sgRNA can edit the PCV2 genome efficiently to improve the PCV2 replication in PK-15 cell; O26 sgRNA can edit the PCV2 genome successfully but it is not known yet of its effect on PCV2 replication, besides the Cas9 expression had no effect on cell viability. These data suggest that CRISPR/Cas9 system targeting PCV2 essential genes may serve as a novel therapeutic agent against PCV2 infection in the future.

Notch3 Modulates Cardiac Fibroblast Proliferation, Apoptosis, and Fibroblast to Myofibroblast Transition via Negative Regulation of the RhoA/ROCK/Hif1α Axis.

Cardiac fibrosis is a common pathological process in multiple cardiovascular diseases, including myocardial infarction (MI). Abnormal cardiac fibroblast (CF) activity is a key event in cardiac fibrosis. Although the Notch signaling pathway has been reported to play a vital role in protection from cardiac fibrosis, the exact mechanisms underlying cardiac fibrosis and protection from it have not yet been elucidated. Similarly, Hif1α and the RhoA/ROCK signaling pathway have been shown to participate in cardiac fibrosis. The RhoA/ROCK signaling pathway has been reported to be an upstream pathway of Hif1α in several pathophysiological processes. In the present study, we aimed to determine the effects of notch3 on CF activity and its relationship with the RhoA/ROCK/Hif1α signaling pathway. Using in vitro experiments, we demonstrated that notch3 inhibited CF proliferation and fibroblast to myofibroblast transition (FMT) and promoted CF apoptosis. A knockdown of notch3 using siRNAs had the exact opposite effect. Next, we found that notch3 regulated CF activity by negative regulation of the RhoA/ROCK/Hif1α signaling pathway. Extending CF-based studies to an in vivo rat MI model, we showed that overexpression of notch3 by the Ad-N3ICD injection attenuated the increase of RhoA, ROCK1, ROCK2, and Hif1α levels following MI and further prevented MI-induced cardiac fibrosis. On the basis of these results, we conclude that notch3 is involved in the regulation of several aspects of CF activity, including proliferation, FMT, and apoptosis, by inhibiting the RhoA/ROCK/Hif1α signaling pathway. These findings are significant to further our understanding of the pathogenesis of cardiac fibrosis and to ultimately identify new therapeutic targets for cardiac fibrosis, potentially based on the RhoA/ROCK/Hif1α signaling pathway.

MicroRNA-21-3p modulates FGF2 to facilitate influenza A virus H5N1 replication by refraining type I interferon response.

BACKGROUND: Influenza A virus (IAV) has greatly affected public health in recent decades. Accumulating data indicated that host microRNAs (miRNAs) were related to IAV replication. The present study mainly focused on the effects of microRNA-21-3p (miR-21-3p) on H5N1 replication. METHODS: The levels of miR-21-3p, virus structural factors (matrix 1 (M1), nucleoprotein (NP)), type I interferon (IFN) response markers (IFN-ß, IFN-α), IFN-stimulated genes (protein kinase R (PKR), myxovirus resistance A (MxA), 2'-5'-oligoadenylate synthetase 2 (OAS)), and fibroblast growth factor 2 (FGF2) were measured by quantitative real-time polymerase chain reaction (qRT-PCR). The protein levels of M1, NP, and FGF2 were tested by Western blot assay. The virus titer was assessed by tissue culture infective dose 50% (TCID50) assay. The dual-luciferase reporter assay and ribonucleic acid (RNA) immunoprecipitation (RIP) assay were used to verify the interaction between miR-21-3p and FGF2. RESULTS: MiR-21-3p was reduced in H5N1-infected patients and A549 cells. MiR-21-3p overexpression facilitated the levels of M1, NP, TCID50 value, and reduced the levels of IFN-ß, IFN-α, PKR, MxA, and OAS in H5N1-infected A549 cells. FGF2 was verified as a direct target of miR-21-3p. The introduction of FGF2 counteracted miR-21-3p-mediated decrease in the levels of M1, NP, and TCID50 value, as well as reduction in the levels of IFN-ß, IFN-α, PKR, MxA, and OAS in H5N1-infected A549 cells. CONCLUSION: MiR-21-3p down-regulated FGF2 expression to accelerate H5N1 replication and confine IFN response.

Genetic characterization of Porcine circovirus type 2 from 2013 to 2018 in Shandong Province, China.

PCV2 belongs to the genus Circovirus, family Circoviridae, who is recognized as the causative agents of postweaning multisystemic wasting syndrome. Since being found to China in 2000, it has caused serious damage to the pig industry. In this study, we downloaded 40 PCV2 genome-wide sequences uploaded to GenBank from 2013 to 2018 in Shandong Province, including 23 uploaded by our laboratory. Construction of a genome-wide evolution tree using MEGA V5.0 software. Phylogenetic tree analysis indicated that the genotype of PCV2 in Shandong Province was: three genotypes coexisted (2a, 2b, 2d); among them, PCV2d has become the main genotype in the province due to its number and spread range. Amino acid sequence analysis of different genotypes of ORF2 showed that specific amino acid sites exist in different genotypes, with the most significant range of 81-160; different genotypes of PCV2 can be distinguished at the molecular level. This study found that due to the increase in infections of the PCV2d genotype in recent years, it may replace PCV2b as the dominant base in Shandong.

Simultaneous detection of classical PRRSV, highly pathogenic PRRSV and NADC30-like PRRSV by TaqMan probe real-time PCR.

Porcine Reproductive and Respiratory Syndrome (PRRS), an acute infectious disease caused by the porcine reproductive and respiratory syndrome virus (PRRSV), is one of the most devastating diseases affecting the global swine industry. In order to establish a multiplex real-time PCR method for the simultaneous detection of the classical PRRSV (C-PRRSV) strain, the highly pathogenic PRRSV (HP-PRRSV) strain and NADC30-like PRRSV (NL-PRRSV) strain, we designed specific primers and TaqMan fluorescent probes based on the Nsp2 target gene sequence of these three different PRRSV strains, and designed American-type PRRSV (PRRSV-U) special primers and probes based on the relatively conserved target gene sequence of ORF7. The method established in this study can quickly and accurately detect and differentiate three types of strains of clinical tissue samples, respectively. This method plays a key role in the rapid diagnosis and determination of PRRSV.

Associations of leptin and leptin receptor genetic variants with coronary artery disease: a meta-analysis.

Background: Some pilot studies already tried to investigate potential associations of leptin (LEP) and LEP receptor (LEPR) variants with coronary artery disease (CAD). However, the results of these studies were not consistent. Thus, we performed the present meta-analysis to explore associations between LEP/LEPR variants and CAD in a larger pooled population.Methods: Systematic literature research of PubMed, Web of Science, Embase and CNKI was performed to identify eligible case-control studies on associations between LEP/LEPR variants and CAD. The initial search was conducted in September 2018 and the latest update was performed in December 2018. Q test and I2 statistic were employed to assess between-study heterogeneities. If probability value(P-value) of Q test was less than 0.1 or I2 was greater than 50%, random-effect models (REMs) would be used to pool the data. Otherwise, fixed-effect models (FEMs) would be applied for synthetic analyses.Results: A total of ten studies published between 2006 and 2018 were eligible for analyses (1989 cases and 2601 controls). Pooled analyses suggested that LEP rs7799039 variant was significantly associated with CAD under over-dominant model (P=0.0007, odds ratio (OR) = 1.36, 95% confidence interval (CI): 1.14-1.63, I2 = 41%, FEM) in overall population, and this significant finding was further confirmed in East Asians in subsequent subgroup analyses. However, no positive findings were observed for LEPR rs1137100 and rs1137101 variants in overall and subgroup analyses.Conclusions: Our meta-analysis suggested that LEP rs7799039 variant might affect individual susceptibility to CAD.

Associations between common ion channel single nucleotide polymorphisms and sudden cardiac death in adults: A MOOSE-compliant meta-analysis.

BACKGROUND: We sought to identify common ion channel single nucleotide polymorphisms (SNPs) associated with the occurrence of sudden cardiac death (SCD) to predict the incidence of SCD in clinical settings. METHODS: This study involved a systematic review and meta-analysis of ion channel SNPs and risk of SCD in adults. We searched public databases for studies published up to September 19, 2017. We examined relationships between SNPs in common ion channel genes and the incidence of SCD. RESULTS: We collected data for 22 trials that included a total of 4149 patients who experienced SCD or had a high risk of SCD and assessed these data in our meta-analysis. An allelic model showed that rs11720524 in SCN5A clearly protected against SCD (odds ratio [OR]: 0.76; 95% confidence interval [95% CI]: 0.67-0.85; P < .001). Subgroup analysis showed that rs11720524 in SCN5A protected against SCD in Europeans and Caucasians but not in Koreans. The allelic model indicated that rs12296050 in KCNQ1 also had significant protective effects against SCD (OR: 0.85; 95% CI: 0.76-0.96; P = .007). Moreover, this model demonstrated that rs2283222 in KCNQ1 had a significant negative relationship with SCD (OR: 0.73; 95% CI: 0.62-0.85; P < .001). Rs12296050 in KCNQ1 protected against SCD in Koreans and Americans. Our results also showed that rs790896 in RYR2 was negatively associated with SCD in a dominant model (OR: 0.66; 95% CI: 0.45-0.97; P = .033). CONCLUSIONS: Rs11720524 in SCN5A is negatively related to SCD in Europeans and Caucasians, and rs12296050 and rs2283222 in KCNQ1 and rs790896 in RYR2 clearly have protective effects against SCD.

Publisher Correction: Emergence of Different Recombinant Porcine Reproductive and Respiratory Syndrome Viruses, China.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

The correlation between the mutual deletions of amino acids within porcine circovirus rep protein and the discrepancy of replication.

Porcine circovirus (PCV) has two potential open reading frames, ORF1 and ORF2. ORF1 is predicted to encode a replication-associated protein (Rep) essential for replication of viral DNA. In some studies, PCV1 replicated more efficiently in PK-15 cells than PCV2 was elucidated. PCV1 compared with PCV2; there is some amino acids' deficiency on Rep protein. To identify whether the above amino acids deletion affects the replication of PCV1 and PCV2, we constructed three double copy clones by overlap extension PCR. The 2PCV2(vV) clone deleted the valine of Rep protein in the backbone of PCV2 genome. The 2PCV2(dSGR) clone inserted serine, glycine and arginine of Rep protein successively in the backbone of PCV2 genome. The 2PCV2(dSGR&vV) clone inserted serine, glycine and arginine as well as deleted the valine of Rep protein in the backbone of PCV2 genome. These clones we constructed with amino acid mutations and parental DNA clones were all transfected in PK-15 cells that free of PCV contamination, and their growth characteristics in vitro were determined and compared, to evaluating the replication of the mutant infectious DNA clones. Our results showed that the double copy infectious clones with amino acid mutations could be rescued in vitro. The 2PCV2(vV) replicated more efficiently than parental viruses 2PCV2 and 2PCV1 but the replicated ability of 2PCV2(dSGR) and 2PCV2(dSGR&vV) is attenuated than parental viruses 2PCV2 and 2PCV1. We can determine the valine is the important amino acid that cause PCV1 replicated more efficiently in PK-15 cells than PCV2 primarily. These findings are benefit for exploring the mechanisms of viral replication in pigs and important implications for PCV2 vaccine development.

Emergence of Different Recombinant Porcine Reproductive and Respiratory Syndrome Viruses, China.

Epidemiological investigations were conducted on recently emerging porcine reproductive and respiratory syndrome virus (PRRSV) strains in Shandong province in 2014-2015. The proportion of the NADC30 strain identified by ORF7 sequence alignment has been gradually increasing. Three emerging PRRSV strains were successfully isolated, and the complete genomic sequences were determined. Our results indicate the importance of recombinant strains in Shandong province, China. There was a varied degree of recombination of two or three strains (classical, HP-PRRSV and/or NADC30). Moreover, the recombination strains affected the pathogenicity of newly emerged strains.

Mometasone furoate inhibits growth of acute leukemia cells in childhood by regulating PI3K signaling pathway.

OBJECTIVES: Acute lymphoblastic leukemia (ALL) is the most common cancer before the age of 15 years, seriously endangering the health of children. The main treatment for Childhood ALL was pharmacotherapy. But these drugs have many side effects and some of them could develop drug resistance quickly. Mometasone furoate (MF) is an efficient glucocorticoid for topical treatment of inflammation on the skin, lung and nose. METHODS: In this study, we investigated whether the MF had effects on ALL cells proliferation and migration. RESULTS: The CCK-8 proliferation test showed that the cell viability was the lowest at 25 nM MF treatment and the increased OD value was time-dependent. In transwell assay, the number of CCRF-CEM cells was reduced in MF treated group. We found the expression of anti-apoptotic protein bcl-2 decreased the expression of pro-apoptotic protein caspase3 and bax increased in CCRF-CEM cell line treated with MF. The expression of p-AKT, p-mTOR, p70S6 K, vascular endothelial growth factor and CyclinD1 were decreased in MF treated group. CONCLUSION: This study reveals that MF can inhibit proliferation and invasion/migration and induce apoptosis in Childhood ALL cells, which may be regulated by Phosphatidylinositol 3-kinase signaling pathway. These results suggest MF may be a potential new drug target for clinical ALL treatment.

TSPAN12 Is a Norrin Co-receptor that Amplifies Frizzled4 Ligand Selectivity and Signaling.

Accessory proteins in Frizzled (FZD) receptor complexes are thought to determine ligand selectivity and signaling amplitude. Genetic evidence indicates that specific combinations of accessory proteins and ligands mediate vascular ß-catenin signaling in different CNS structures. In the retina, the tetraspanin TSPAN12 and the ligand norrin (NDP) mediate angiogenesis, and both genes are linked to familial exudative vitreoretinopathy (FEVR), yet the molecular function of TSPAN12 remains poorly understood. Here, we report that TSPAN12 is an essential component of the NDP receptor complex and interacts with FZD4 and NDP via its extracellular loops, consistent with an action as co-receptor that enhances FZD4 ligand selectivity for NDP. FEVR-linked mutations in TSPAN12 prevent the incorporation of TSPAN12 into the NDP receptor complex. In vitro and in Xenopus embryos, TSPAN12 alleviates defects of FZD4 M105V, a mutation that destabilizes the NDP/FZD4 interaction. This study sheds light on the poorly understood function of accessory proteins in FZD signaling.