Qinghe Li

Host cell interactome of PA protein of H5N1 influenza A virus in chicken cells.

UNLABELLED Influenza A virus (IAV) heavily depends on viral-host protein interactions in order to replicate and spread. Identification of host factors that interact with viral proteins plays crucial roles in understanding the mechanism of IAV infection. Here we report the interaction landscape of H5N1 IAV PA protein in chicken cells through the use of affinity purification and mass spectrometry. PA protein was expressed in chicken cells and PA interacting complexes were captured by co-immunoprecipitation and analyzed by mass spectrometry. A total of 134 proteins were identified as PA-host interacting factors. Protein complexes including the minichromosome maintenance complex (MCM), 26S proteasome and the coat protein I (COPI) complex associated with PA in chicken cells, indicating the essential roles of these functional protein complexes during the course of IAV infection. Gene Ontology and pathway enrichment analysis both showed strong enrichment of PA interacting proteins in the category of DNA replication, covering genes such as PCNA, MCM2, MCM3, MCM4, MCM5 and MCM7. This study has uncovered the comprehensive interactome of H5N1 IAV PA protein in its chicken host and helps to establish the foundation for further investigation into the newly identified viral-host interactions. BIOLOGICAL SIGNIFICANCE Influenza A virus (IAV) is a great threat to public health and avian production. However, the manner in which avian IAV recruits the host cellular machinery for replication and how the host antagonizes the IAV infection was previously poorly understood. Here we present the viral-host interactome of the H5N1 IAV PA protein and reveal the comprehensive association of host factors with PA.

Protein Profiles for Muscle Development and Intramuscular Fat Accumulation at Different Post-Hatching Ages in Chickens

Muscle development and growth influences the efficiency of poultry meat production, and is closely related to deposition of intramuscular fat (IMF), which is crucial in meat quality. To clarify the molecular mechanisms underlying muscle development and IMF deposition in chickens, protein expression profiles were examined in the breast muscle of Beijing-You chickens at ages 1, 56, 98 and 140 days, using isobaric tags for relative and absolute quantification (iTRAQ). Two hundred and four of 494 proteins were expressed differentially. The expression profile at day 1 differed greatly from those at day 56, 98 and 140. KEGG pathway analysis of differential protein expression from pair-wise comparisons (day 1 vs. 56; 56 vs. 98; 98 vs. 140), showed that the fatty acid degradation pathway was more active during the stage from day 1 to 56 than at other periods. This was consistent with the change in IMF content, which was highest at day 1 and declined dramatically thereafter. When muscle growth was most rapid (days 56–98), pathways involved in muscle development were dominant, including hypertrophic cardiomyopathy, dilated cardiomyopathy, cardiac muscle contraction, tight junctions and focal adhesion. In contrast with hatchlings, the fatty acid degradation pathway was downregulated from day 98 to 140, which was consistent with the period for IMF deposition following rapid muscle growth. Changes in some key specific proteins, including fast skeletal muscle troponin T isoform, aldehyde dehydrogenase 1A1 and apolipoprotein A1, were verified by Western blotting, and could be potential biomarkers for IMF deposition in chickens. Protein–protein interaction networks showed that ribosome-related functional modules were clustered in all three stages. However, the functional module involved in the metabolic pathway was only clustered in the first stage (day 1 vs. 56). This study improves our understanding of the molecular mechanisms underlying muscle development and IMF deposition in chickens.

Interactomic landscape of PA-X-chicken protein complexes of H5N1 influenza A virus.

UNLABELLED As a newly identified isoform generated from the PA segment and an essential factor for viral virulence, little is known about PA-X-host interactions. Here we present the interactomic landscape of PA-X protein of H5N1 influenza A virus (IAV), described using data generated from affinity purification and mass spectrometry (AP-MS). PA-X was exogenously expressed in chicken fibroblast cells and PA-X associated protein complexes were identified by AP-MS. Using a high confidence threshold for interaction 56 unique proteins were found to have physical interactions with PA-X. PA-X associated host factors showed strong enrichment for specific protein domains, including annexin and WD40 domains. Many proteins that have been described as pro or antiviral host proteins interact with PA-X, indicating the possible effect of these proteins on influenza A viral infections facilitated by interactions with PA-X. This study has uncovered the comprehensive interactomic landscape of PA-X and laid the foundation for further understanding of PA-X function in terms of viral-host protein interactions. BIOLOGICAL SIGNIFICANCE Identification of viral-host interacting proteins is vital for the comprehensive understanding of how virus recruits the host cellular machinery and how host antagonizes virus infection. Our study reveals the viral-host interactome of PA-X and uncovers interactions between host proteins and PA-X which might have crucial roles in viral infection.

Effects of caponization and ovariectomy on objective indices related to meat quality in chickens

&NA; Capons and ovariectomized chickens are birds that have been gonadectomized to improve the meat quality. This study investigated the effects of caponization and ovariectomy on physical, chemical, and fatty acid and amino acid profiles of meat from Beijing‐You chickens (a Chinese local breed) at market age (17 wk). All birds (20 capons, 20 ovariectomized, and 40 controls) were reared under the same conditions. Breast muscle fiber diameter and area were significantly smaller and the fiber density was higher in capons and ovariectomized chickens than in controls (P < 0.05). Compared with controls, caponization and ovariectomy significantly decreased breast muscle shear values and redness (a*), as well as increased yellowness (b*), hue (H*), and chroma (C*) (P < 0.05). There was significantly more intramuscular fat (IMF) in capons than in controls (P < 0.05), and there was a tendency for more inosine‐5′‐monophosphate (IMP) in capons than in controls (P = 0.10). The levels of IMF and IMP in ovariectomized chickens were significantly higher than those in controls (P < 0.05). Capons and ovariectomized chickens exhibited a significantly higher content of palmitic acid (C16:0), palmitoleic acid (C16:1) and oleic acid (C18:1), together with a lower content of stearic acid (C18:0), arachidonic acid (C20:4), and lignoceric acid (C24:0) compared to controls (P < 0.05). The total saturated, monounsaturated, and polyunsaturated fatty acids as well as amino acid composition were not affected by gonadectomy (P > 0.05). Overall, this study indicates that both caponization and ovariectomy likely improve the meat quality of the breast muscle based on the objective indices of IMF, appearance (color), texture, and minor change of the fatty acid profile; ovariectomy improves flavor‐related indices.

Allelic variation in TLR4 is linked to resistance to Salmonella Enteritidis infection in chickens.

&NA; Salmonella Enteritidis (SE) is a foodborne pathogen that negatively affects both animal and human health. Polymorphisms of the TLR4 gene may affect recognition by Toll‐like receptor 4 (TLR4) of bacterial lipopolysaccharide (LPS), leading to differences in host resistance to pathogenic infections. The present study has investigated polymorphic loci of chicken TLR4 (ChTLR4) in ten chicken breeds, electrostatic potentials of mutant structures of TLR4, and a linkage analysis between allelic variation and survival ratio to infection with SE in specific‐pathogen‐free (SPF) White Leghorns. A total of 19 Single Nucleotide Polymorphisms (SNPs), of which 10 were novel, were found in chicken breeds. Seven newly identified amino acid variants (C68G, G674A, G782A, A896T, T959G, T986A, and A1104C) and previously reported important mutations (G247A, G1028A, C1147T, and A1832G) were demonstrated in the extracellular domain of the ChTLR4 gene. Significant changes in surface electrostatic potential of the ectodomain of TLR4, built by homology modeling, were observed at the Glu83Lys (G247A), Arg298Ser (A896T), Ser368Arg (A1104C), and Gln611Arg (A1832G) substitutions. Linkage analysis showed that one polymorphic locus G247A of TLR4 gene, common in all breeds examined, was significantly associated with increased resistance to SE in SPF White Leghorns chicks (log‐rank P‐value = 0.04). The genotypes from A1832G SNPs did not show statistically significant survival differences. This study has provided the first direct evidence that G247A substitution in ChTLR4 is associated with increased resistance to Salmonella Enteritidis.

Splenic microRNA Expression Profiles and Integration Analyses Involved in Host Responses to Salmonella enteritidis Infection in Chickens

To understand the role of miRNAs in regulating genes involved in the host response to Salmonella enteritidis (SE) infection, next generation sequencing was applied to explore the altered splenic expression of microRNAs (miRNAs) and deregulated genes in specific-pathogen-free chickens. Birds were either infected or not (controls, C) and those challenged with SE were evaluated 24 h later and separated into two groups on the basis of the severity of clinical symptoms and blood load of SE: resistant (R, SE challenged-slight clinical symptoms and <105 cfu / 10 μL), and susceptible (S, SE challenged-severe clinical symptoms and >107 cfu/10 μL). Thirty-two differentially expressed (DE) miRNAs were identified in spleen, including 16 miRNAs between S and C, 13 between R and C, and 13 between S and R. Through integration analysis of DE miRNAs and mRNA, a total of 273 miRNA-target genes were identified. Functional annotation analysis showed that Apoptosis and NOD-like receptor signaling pathway and adaptive immune response were significantly enriched (P < 0.05). Interestingly, apoptosis pathway was significantly enriched in S vs. C, while NOD-like receptor pathway was enriched in R vs. C (P < 0.05). Two miRNAs, gga-miR-101-3p and gga-miR-155, in the hub positions of the miRNA-mRNA regulatory network, were identified as candidates potentially associated with SE infection. These 2 miRNAs directly repressed luciferase reporter gene activity via binding to 3′-untranslated regions of immune-related genes IRF4 and LRRC59; over-expressed gga-miR-155 and interference gga-miR-101-3p in chicken HD11 macrophage cells significantly altered expression of their target genes and decreased the production of pro-inflammatory cytokines. These findings facilitate better understanding of the mechanisms of host resistance and susceptibility to SE infection in chickens.

Follicle-stimulating hormone increases the intramuscular fat content and expression of lipid biosynthesis genes in chicken breast muscle

Intramuscular fat (IMF) is a crucial factor in the quality of chicken meat. The genetic basis underlying it is complex. Follicle-stimulating hormone (FSH), well-known as an effector in reproductive tissues, was recently discovered to stimulate abdominal fat accumulation in chicken. The effect of FSH on IMF accumulation and the underlying molecular regulatory mechanisms controlling both IMF and abdominal fat deposition in vivo are largely unknown. In this study, two groups of chickens were treated with chicken FSH or a placebo. The lipid content of breast muscle, abdominal fat volume, and serum concentrations of FSH were examined. Related genes implicated in breast muscle and abdominal fat accumulation were also investigated. Compared to the control group, the triglyceride (TG) content of breast muscle and the percentage of abdominal fat in FSH-treated chickens were significantly increased by 64.9% and 56.5% (P<0.01), respectively. The FSH content in the serum of FSH-treated chickens was 2.1 times than that of control chickens (P<0.01). Results from quantitative real-time polymerase chain reaction (qRT-PCR) assays showed that relative expression levels of fatty acid synthase (FAS), lipoprotein lipase (LPL), diacylglycerol acyltransferase 2 (DGAT2), adipocyte fatty acid binding protein (A-FABP), and peroxisome proliferator-activated receptor γ (PPARγ) were significantly upregulated in breast muscle following FSH treatment (P<0.01). Treatment with FSH also significantly increased relative expression levels of FAS, LPL, DGAT2, A-FABP, and PPARγ in abdominal fat tissue (P<0.05). The results of principal component analysis (PCA) for gene expression (breast muscle and abdominal fat) showed that the control and FSH treatment groups were well separated, which indicated the reliability of the data. This study demonstrates that FSH plays an important role in IMF accumulation in female chickens, which likely involves the regulation of biosynthesis genes related to lipid metabolism.概要目 的肌内脂肪 (IMF) 是衡量鸡肉品质的一个重要指标, 其遗传机理是复杂的。探讨卵泡刺激素 (FSH) 对 IMF 的调控作用, 有利于更好地理解鸡肉中 IMF 沉积的分子机制。创新点在鸡上证明 FSH 可促进胸肌中 IMF 的沉积, 且此作用与脂质合成基因表达上调有关。方 法将 7 日龄北京油鸡母鸡分为对照组 (皮下注射生理盐水) 和处理组 (皮下注射 4 mIU 鸡FSH)。 连续注射 7 天后采集血清和组织, 采用氯仿-甲醇法抽提脂肪, 测定胸肌甘油三酯含量; 采用鸡特异性酶联免疫吸附测定 (ELISA) 试剂盒检测血清 FSH 含量; 使用实时定量聚合酶链反应 (qRT-PCR) 检测胸肌和腹脂组织中 FAS、 LPL、 DGAT2、 A-FABP 和 PPARγ 基因的表达水平, 并对基因表达数据做主成份分析 (PCA) 以检测样本的重复性。结 论本实验结果显示外源 FSH 注射能显著提高鸡的胸肌甘油三酯含量和腹脂含量 (图 1); 同时能显著上调胸肌和腹脂组织中 FAS、 LPL、 DGAT2、 A-FABP 和 PPARγ 基因的表达 (图 2)。 本研究结果表明 FSH 可能通过调控脂类合成基因的表达来促进母鸡 IMF 沉积。

Alteration of Hepatic Gene Expression along with the Inherited Phenotype of Acquired Fatty Liver in Chicken

Fatty liver is a widespread disease in chickens that causes a decrease in egg production and even death. The characteristics of the inherited phenotype of acquired fatty liver and the molecular mechanisms underlying it, however, are largely unknown. In the current study, fatty liver was induced in 3 breeds by a high-fat (HF) diet and a methionine choline-deficient (MCD) diet. The results showed that the dwarf Jingxing-Huang (JXH) chicken was more susceptible to fatty liver compared with the layer White Leghorns (WL) and local Beijing-You (BJY) breeds. In addition, it was found that the paternal fatty livers induced by HF diet in JXH chickens were inherited. Compared to birds without fatty liver in the control group, both offsprings and their sires with fatty livers in the paternal group exhibited altered hepatic gene expression profiles, including upregulation of several key genes involved in fatty acid metabolism, lipid metabolism and glucose metabolism (ACACA, FASN, SCD, ACSL5, FADS2, FABP1, APOA4 and ME1). This study uniquely revealed that acquired fatty liver in cocks can be inherited. The hepatic gene expression profiles were altered in chickens with the inherited phenotype of acquired paternal fatty liver and several genes could be candidate biomarkers.

Host Interaction Analysis of PA-N155 and PA-N182 in Chicken Cells Reveals an Essential Role of UBA52 for Replication of H5N1 Avian Influenza Virus

PA-N155 and PA-N182 proteins were translated from the 11th and 13th start codon AUG of the RNA polymerase acidic protein (PA) mRNA of H5N1 influenza A virus (IAV), which plays an important role in viral replication. Little is known about the interactions between PA-N155 and PA-N182 and the host proteins. This study investigated the interaction landscape of PA-N155 and PA-N182 of H5N1 IAV in chicken cells while their interacting complexes were captured by immunoprecipitation and analyzed by mass spectrometry. A total of 491 (PA-N155) and 302 (PA-N182) interacting proteins were identified. Gene ontology and pathway enrichment analyses showed that proteins of the two interactomes were enriched in RNA processing, viral processing and protein transport, and proteins related to signaling pathways of proteasome, ribosome, and aminoacy1-tRNA biosynthesis were significantly enriched, suggesting their potential roles in H5N1 IAV infection. Comparative analysis of the interactome of PA, PA-N155, and PA-N182 identified UBA52 as a conserved host factor that interacted with all three viral proteins. UBA52 is a fusion protein consisting of ubiquitin at the N terminus and ribosomal protein L40 at the C terminus. Knockdown of UBA52 significantly decreased the titer of H5N1 IAV in chicken cells and was accompanied with attenuated production of proinflammatory cytokines. Our analyses of the influenza–host protein interactomes identified UBA52 as a PA interaction protein for virus replication.

Decreased testosterone levels after caponization leads to abdominal fat deposition in chickens

BackgroundCaponization results in reduced androgen levels, which leads to abdominal fat accumulation in capons. In this study, we sought to understand the molecular mechanisms behind this fat accumulation.ResultsAbdominal fat (AF) content increased significantly (P < 0.05) and serum and AF testosterone levels decreased significantly (P < 0.05 or P < 0.01) after caponization. In AF tissue, 90 differentially expressed genes related to lipid metabolism were screened by gene expression profiling in caponized and sham-treated chickens. Among these, six representative genes were significantly up-regulated (APOA1, SCD, FABP7, RXRG, and FADS2) or down-regulated (FABP3) (P < 0.05 or P < 0.01) and were strongly associated with the PPAR pathway. In addition, cell junction pathways were also enriched. In vitro, Fat content was significantly lower in cells treated with testosterone compared with control cells (P < 0.01), and mRNA levels of RXRG, FABP7, and FABP3 changed accordingly, confirming the effect of testosterone on fat deposition.ConclusionsThe results of this study indicate that testosterone reduction likely regulates gene expression through PPAR and cell junction pathways resulting in increased fat accumulation. These results provide increase our understanding of the biological mechanisms by which caponization induces greater fat accumulation.