Xuming Hu

Both MicroRNA-155 and Virus-Encoded MiR-155 Ortholog Regulate TLR3 Expression.

MicroRNA-155 (miR-155) has been as an important controller of TLR3 signalling. However, the interactions between miR-155 and TLR3 are poorly understood. Here, we focused on the regulation of the relationship between miR-155 and TLR3. Sequence analyses and firefly luciferase reporter assay revealed that miR-155 target were present in the coding sequences (CDS) of TLR3. And the expression of the TLR3 protein could be inhibited by a miR-155 mimic or by a virally encoded orthologue in chick embryo fibroblast cells. Notably, endogenous miR-155 induction emerged a negative regulation on TLR3 expression in TLR2, 4 and 7 ligands stimulated HD11 cells, an avian macrophage cell line. Moreover, treatment with the miR-155 antagomir increased TLR3 levels while significantly decreased the abundance of TLR3 with miR-155 agomir. In addition, our data showed that miR-155 could inhibit IFN-β production possibly though TLR3 signal pathway. All these findings might reveal a new mechanism by which miR-155 can regulate the TLR3 immune response.

Identification of novel viral receptors with cell line expressing viral receptor-binding protein

The viral cell receptors and infection can be blocked by the expression of the viral receptor-binding protein. Thus, the viral cell receptor is an attractive target for anti-viral strategies, and the identification of viral cell receptor is critical for better understanding and controlling viral disease. As a model system for viral entry and anti-retroviral approaches, avian sarcoma/leukosis virus (ASLV, including the A-J ten subgroups) has been studied intensively and many milestone discoveries have been achieved based on work with ASLV. Here, we used a DF1 cell line expressed viral receptor-binding protein to efficiently identify chicken Annexin A2 (chANXA2) as a novel receptor for retrovirus ALV-J (avian leukosis virus subgroup J). Our data demonstrate that antibodies or siRNA to chANXA2 significantly inhibited ALV-J infection and replication, and over-expression of chANXA2 permitted the entry of ALV-J into its non-permissible cells. Our findings have not only identified chANXA2 as a novel biomarker for anti-ALV-J, but also demonstrated that cell lines with the expression of viral receptor-binding protein could be as efficient tools for isolating functional receptors to identify novel anti-viral targets.

Analysis of protein expression profiles in the thymus of chickens infected with Marek's disease virus

BackgroundMarek’s disease virus (MDV) is a highly cell-associated oncogenic α-herpesvirus that causes a disease characterised by T-cell lymphomas. The pathogenesis, or the nature of the interaction of the virus and the host, in the thymus are still unclear.ResultsIn this study, we identified 119 differentially expressed proteins using two-dimensional electrophoresis and mass spectrometry from the thymuses of chickens infected with the RB1B strain of MDV. These differentially expressed proteins were found mainly at 21, 28 and 35 days post-infection. More than 20 of the differentially expressed proteins were directly associated with immunity, apoptosis, tumour development and viral infection and replication. Five of these proteins, ANXA1, MIF, NPM1, OP18 and VIM, were further confirmed using real-time PCR. The functional associations and roles in oncogenesis of these proteins are discussed.ConclusionsThis work provides a proteomic profiling of host responses to MDV in the thymus of chickens and further characterises proteins related to the mechanisms of MDV oncogenesis and pathogenesis.

Activation of Toll-like receptor 3 inhibits Marek's disease virus infection in chicken embryo fibroblast cells.

Toll-like receptor 3 (TLR3) is a critical component of the innate immune system against viral infection and controls the activation of adaptive immunity. The role of TLR3 in Marek’s disease virus (MDV) infection is not clear. In this study, we found that the abundance of TLR3 mRNA was significantly higher in chicken embryo fibroblast cells (CEF) infected with MDV than in a control group. Activated TLR3 signaling via TLR3 ligand stimulation inhibited replication of the RB1B strain of MDV in CEF cells. In contrast, CEF cells transfected with TLR3 siRNA promoted RB1B infection and replication. However, treatment with other TLR ligands, whether stimulatory (LPS, imiquimod and CpG) or inhibitory (TLR2/4 inhibitor and/or MyD88 inhibitor), had little effect on RB1B infection and replication. In addition, we found that the expression trend of TLR3 mRNA in RB1B-infected CEF cells was similar to that of mdv1-mir-M4-5p (a functional ortholog of oncogenic miR-155 encoded by MDV). Inconsistent with this, the TLR3 protein level was sharply reduced in RB1B-infected CEF cells at 96 hpi, while there was an at least 200-fold increase in miR-M4-5p at the same time point. Additionally, CEF cells transfected with an mdv1-mir-M4-5p mimic promoted RB1B infection and replication, while an mdv1-mir-M4-5p inhibitor inhibited RB1B infection and replication. Similar results were observed in CEF cells transfected with a gga-miR-155 mimic or inhibitor. These findings suggest that TLR3 and MDV-encoded miRNAs might be involved in MDV infection.

Proteomics of DF-1 cells infected with avian leukosis virus subgroup J

Avian leukosis virus subgroup J (ALV-J) is an avian oncogenic retrovirus that has led to severe economic losses in the poultry industry in China in recent years. The pathogenesis of virus infection and virus-host interactions are still not well elucidated. In this paper, we investigated the expression changes for cellular proteins in DF-1 cells infected with ALV-J. Comparative analyses revealed that the majority of the altered proteins in DF-1 cells appeared at 6-12h after ALV-J infection. Mass spectrometry identified 74 altered cellular proteins, including 30 up-regulated proteins and 44 down-regulated proteins. Some of these proteins are involved in cell cytoskeleton, metabolic processes, response to stimulus and immune responses. Other proteins, such as DJ-1, UCHL1, VDAC1 and HMGB1, have some relationship to apoptosis or oncogenesis. The changes in the transcriptional profile of DJ-1, UCHL1, VDAC1 and HMGB1 in infected as compared to uninfected DF-1 cells were confirmed by real-time RT-PCR. Our work gives some information about differential protein expression in cells infected with ALV-J, which will help us to understand viral pathogenicity.

Transcriptional analysis of host responses to Marek's disease virus infection in chicken thymus.

Mareks disease virus (MDV) is a cell-associated alpha-herpesvirus that causes T-cell lymphomas and nervous disorders in chickens. Different from other lymphoid organs, the thymus is the site of T-cell maturation and differentiation. However, the transcriptional response to MDV infection in the chicken thymus is still not known. In this study, we performed genome-wide expression analysis in thymus tissues of RB1B-infected chickens at different time points to investigate the molecular mechanisms of MDV pathogenesis. The number of differentially expressed genes with 2-fold or higher changes (>2) are as follows: 1,250 genes (7 dpi), 834 genes (14 dpi), 1,958 genes (21 dpi), and 2,306 genes (28 dpi). Gene ontology enrichment analysis revealed that the upregulated genes were involved in immune and inflammatory response at 7 dpi; angiogenesis, cytoskeleton organization, cell adhesion, and signal transduction showed different expressions at 21 and 28 dpi. The expression pattern of 18 randomly selected genes was confirmed by real-time RT-PCR. Several differently expressed host genes associated with tumor development are discussed. We identified the global host-gene expression pattern in the thymus of chickens that responded to MDV infection. The present data may provide groundwork for future investigation in the biology and pathogenesis of MDV.

Expression patterns of endogenous avian retrovirus ALVE1 and its response to infection with exogenous avian tumour viruses

Endogenous retroviruses (ERVs) are genomic elements that are present in a wide range of vertebrates and have been implicated in a variety of human diseases, including cancer. However, the characteristic expression patterns of ERVs, particularly in virus-induced tumours, is not fully clear. DNA methylation was analysed by bisulfite pyrosequencing, and gene expression was analysed by RT-qPCR. In this study, we first found that the endogenous avian retrovirus ALVE1 was highly expressed in some chicken tissues (including the heart, bursa, thymus, and spleen) at 2 days of age, but its expression was markedly decreased at 35 days of age. In contrast, the CpG methylation level of ALVE1 was significantly lower in heart and bursa at 2 days than at 35 days of age. Moreover, we found that the expression of ALVE1 was significantly inhibited in chicken embryo fibroblast cells (CEFs) and MSB1 cells infected with avian leukosis virus subgroup J (ALVJ) and reticuloendotheliosis virus (REV) at the early stages of infection. In contrast, the expression of the ALVE1 env gene was significantly induced in CEFs and MSB1 cells infected with Marek’s disease virus (MDV). However, the methylation and expression levels of the ALVE1 long terminal repeat (LTR) did not show obvious alterations in response to viral infection. The present study revealed the expression patterns of ALVE1 in a variety of chicken organs and tissues and in chicken cells in response to avian tumour virus infection. These findings may be of significance for understanding the role and function of ERVs that are present in the host genome.

Generation of Cas9 transgenic zebrafish and their application in establishing an ERV-deficient animal model

ObjectivesTo investigate the effect of endogenous Cas9 on genome editing efficiency in transgenic zebrafish.ResultsHere we have constructed a transgenic zebrafish strain that can be screened by pigment deficiency. Compared with the traditional CRISPR injection method, the transgenic zebrafish can improve the efficiency of genome editing significantly. At the same time, we first observed that the phenotype of vertebral malformation in early embryonic development of zebrafish after ZFERV knockout.ConclusionsThe transgenic zebrafish with expressed Cas9, is more efficient in genome editing. And the results of ZFERV knockout indicated that ERV may affect the vertebral development by Notch1/Delta D signal pathway.

Reactivation of development-related genes by the DNA methylation inhibitor 5-Aza-2′-deoxycytidine in chicken embryo fibroblasts

&NA; It has been clearly demonstrated that DNA methyltransferase inhibition can induce embryonic stem cells to differentiate into cells of specific tissues by altering the genes expression. Chicken embryonic fibroblasts (CEFs) are mesenchymal stem cell (MSC)‐like multipotent progenitor cells and can both self‐renew and undergo differentiation. In this study, we hypothesize that DNA demethylation may lead to cell differentiation and the re‐expression of development‐related genes in chicken embryo fibroblasts. Thus, we investigated which development‐related genes are affected when CEFs are treated with DNA methyltransferase inhibitor 5‐Aza‐2′‐deoxycytidine (5‐Aza‐dC) and how those genes are affected. In this study, we analyzed the expression profiles of genes by RNA sequencing of CEFs treated with 5‐Aza‐dC. A total of 40 differentially expressed genes (DEGs) (>2‐fold change) related to chicken development were identified. Co‐expression cluster analysis revealed that these genes are mainly related to development of bone, feather follicle, epidermis, and blood vessels. Moreover, the molecular interaction network analysis showed that canonical Wnt signaling tended to play a central role in development associated with these genes. These findings will help us to understand the molecular regulatory mechanisms of development.