Proteomics And Transcripteomics Of Marek’s Disease Virus Infection And Innate Immune Machnism

Marek’s disease virus (MDV) is a highly oncogenic alpha-herpesvirus that causes immune suppression and T-cell lymphoma in chickens. Despite of that Marek’s disease (MD) latency and transformation are key stages of the disease, significant information on critical aspects of virus latency in T-lymphoid cells and the virus-host interaction in MDV-induced T cell lymphoma remain unknown. The thymus is the specific organ in which the maturation and differentiation of avian T lymphocytes take place, and thus can be good organ model for study of host immune and the mechanism of pathogenesis induced by MDV infection.In addition, MDV latent infection mainly related to CD4+T cells and eventually the lymphoma are T cells. Therefore, T cells play a key role in immunity and pathogenesis of MDV infection. Recent papers show that toll like receptor3(TLR3) plays a dominant role in antiviral T cell immune and tumor immunity and it is required for T cell immune response to herpes simplex virus infection. TLR3gene expression was significantly changed after MDV infection, which may be indicated that TLR3involve in T cells immune response induced by MDV. A recent study found that microRNA-155involved in regulation of TLR3pathway. MDV encoded the functional homologues of miR-155, mdvl-mir-M4share the same seed sequence with gga-mir-155, suggesting a role for this microRNA in the biology and pathogenesis of MDV. It is unclear that mdvl-mir-M4whether control host TLR3signals during MDV infection.In this study, we explored the change of proteome and transcriptome in RB1B infected chicken thymus and the role of microRNA-155regulated TLR3signals in RB1B infection and replication by proteomics, transcriptomics, bioinformatics and microRNA research methods. The present study provides important data and clues for understanding host immunity and pathogenic mechanism of RB1B infection.1. Proteomics analysis of host response to RB1B infection in the thymus of chickenIn this study, we successfully established the two dimensional gel electrophoresis (2-DE) method for proteomic of thymus by optimizing the conditions, including sample preparation, rehydration, isoelectric focusing, equilibration, and other steps. This method provides a basis for further elucidation of the development and immune function of thymus. Next, we observed that chicken thymus showed severe atrophy after RB1B infection at21,28and35days post-infection (dpi), and gradually returned to normal at42dpi. Compared with the control group, RB1B-infected groups showed significant difference at7,21,28and35dpi analyzed by the thymus/body weight parameters. Using2-DE method and mass spectrometry, we identified119differentially expressed proteins from the thymus of chicken infected with the RB1B strain of MDV. These differentially expressed proteins were found mainly at21,28and35days post-infection. More than20of the differentially expressed proteins were directly associated with immunity, apoptosis, tumour development and viral infection and replication. Five of these proteins, ANXA1, MIF, NPM1, OP18and VIM, were further confirmed using real-time PCR. The functional associations and roles in oncogenesis of these proteins are discussed. This work provides a proteomic profiling of host responses to MDV in the thymus of chickens and further characterized proteins related to the mechanisms of MDV oncogenesis and pathogenesis.2. Transcriptome analysis of host response to RB1B infection in the thymus of chickenIn this study, genome-wide microarray analysis using Affymetrix GeneChip Chicken Genome Arrays, containing32,773chicken and684viral transcripts, was performed in chicken thymus. In this study,86Marek’s disease virus (MDV) transcripts were detected in chicken thymus infected with RB1B strain.47transcripts of them mainly involved in viral replication and immune escape were detected at7day post infection (dpi). Most of genes expression were increased at21and28dpi but reduced or shut down at14dpi. Unlike others tissues, we found that a latent infection was established at14dpi in infected thymus. We provided the kinetics of expression of MDV transcripts and their relative expression in infected thymus.A total of594genes associated with immune response, cell cycle regulation, apoptosis, tumor development and metastasis showed differential expression. These genes are involved in22signaling pathways including the Toll-like receptor, MHC, p53, JAK-STAT, Wnt, MAPK and VEGF signaling pathways. These results suggest that the virus uses various strategies to activate or inactivate these pathways to evade host immune surveillance and induce T-cell lymphoma. In the present study, we revealed that MDV infection has an extensive effect on gene expression in chicken thymus tissue. The observed gene expression profile provides new insights into the mechanisms of MDV pathogenesis and contributes to a better understanding of the crosstalk between host and pathogen.3. The influence of TLR3on RB1B infection and replicationMarek’s disease virus (MDV) is a highly oncogenic alpha-herpesvirus that causes T cell immune suppression and malignant lymphomas in chickens. Toll-like receptor (TLR) plays a dominant role in antiviral T cell immunity. However, it is unclear whether MDV induced T cell immunity is associated with TLR-mediated immunity. In this study, the expression of28host genes that are involved in TLR-mediated immunity and MHC-medicated T cell immunity was evaluated in chicken thymus at7,14,21and28days post-infection (dpi). Our results demonstrated that24host immune-related genes were upregulated during MDV infection at7dpi; however, the expression of most of these genes decreased at21and28dpi. Notably, a positive correlation was detected between the downregulation of CD4, CD8and TLR3signals but not the MyD88-dependent TLR pathway. The present study expanded our knowledge of host immune responses against MDV infection and our results might be provide a clue about that MDV may interfere with T cell immune response through TLR3signals.Next, we found that the abundance of TLR3mRNA was significantly increased in infected chick embryo fibroblast (CEF) compared to control group. The biggest change in TLR3mRNA expression was observed at96hpi (at least40-fold change). Infection of RB1B did not affect the expression of TLR2,4,7,15and21mRNA, which indicated that MDV had a specific effect on the expression of TLR3mRNA. We activated TLR3signals by TLR3ligand stimulation and found this treatment consistently suppressed MDV infection and replication. In contrast, we transfected CEF cells with TLR3siRNA or control siRNA, and monitored the effect on the expression of viral genes (gB and Meq) at24h after MDV infection. We found that infection and replication of MDV was promoted in CEF cells transfected with TLR3siRNA. However, other TLR ligands (LPS, Imiquimod and CpG) stimulation or inhibitor (TLR2/4inhibitor and/or MyD88inhibitor) treatment had little effect on MDV infection and replication.4. TLR3mRNA coding region targeted by mdvl-miR-M4and gga-miR-155We found the most likely target site in the coding region sequences of TLR3mRNA, focusing on base pairing in the seed sequence of the miRNA, because the seed sequence is considered a critical determinant in the recognition of target mRNA by miRNA. We mutated five nucleotides in the predicted seed binding sites of the coding region sequences of TLR3mRNA, then did a luciferase reporter assay in HEK293T cells. Notably, we observed that mdvl-mir-M4and gga-miR-155significantly downregulated the expression of firefly luciferase fused to the TLR3wild-type ORF, whereas mir-NC did not. In the reciprocal experiment in which we transfected cells with the firefly luciferase vector fused to the mutated TLR3ORF, neither gga-miR-155and mdvl-mir-M4nor mir-NC had an effect on the expression of firefly luciferase. Western blotting showed that TLR3protein expression was decreased in cells transfected by gga-miR-155mimic or mdvl-miR-M4-5p mimic. This finding showed the specificity of mdvl-mir-M4and gga-miR-155target to the coding region of TLR3mRNA and could be revealed an important mechanism of TLR3expression.5. Response of TLR3signals regulated by gga-miR-155on RB1B infection and replicationWe observed that the expression kinetics of mdvl-miR-M4-5p and TLR3during MDV infection. Infection of MDV resulted in production of IFN-β and the increased expression of gga-miR-155while did not affect the expression of gga-miR-21from8hpi to120hpi; however, mdvl-miR-M4-5p showed a different expression trend in MDV-infected CEF cells, which sharply shut down its expression from96hpi to120hpi while the virus levels still continuelly increased. Notably, TLR3mRNA expression level was also reduced from96hpi to120hpi, similar to mdvl-mir-M4-5p, which indicated that mdvl-miR-M4-5p had a specific effect on TLR3expression. To confirm that the effect of mdvl-mir-M4-5p on TLR3expression, we transfected CEF cells with increasing amounts of mdvl-mir-M4-5p mimic, the abundance of TLR3protein was significantly decreased. Again we examined the effect of mdvl-mir-M4-5p mimic on production of IFN-β and found that it significantly inhibited IFN-P production.Next, we transfected CEF cells with mdvl-mir-M4-5p mimic or mimic Ncontrol and then infected with MDV at48h after transfection, we observed this treatment promoted the expression levels of gB and Meq at24h and48h after virus infection. In contrast, this effect was impaired in CEF cells transfected with mdvl-mir-M4-5p inhibitor, which significantly suppressed expression levels of gB and Meq. Same results were observed in CEF cells transfected with gga-miR-155mimic or inhibitor.