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Shanghai Fog city Chongqing Human borna disease virus infection impacts host proteome and histone lysine acetylation in human oligodendroglia cells Xia Liu July 12, 2016 1 INTRODUCTION BDV genome and proteins Non-segmented, single-stranded RNA-genome with negative polarity (8910 bp) N P X M G L-Pol • M N p40 P p24 G L-Pol Major Antigens Linked to disease BORNA DISEASE VIRUS – Borna=Town in Saxony – Conserved RNA genome – Nuclear replication – own unique virus family – Six proteins – Virus in brain and blood N and P disease-associated Virus model (Ludwig and Bode 1997, Intervirology 40: 185-197) 2 INTRODUCTION Animals naturally infected or experimentally infectable by BDV BDV replicates in the cell nucleus. Host spectrum •Human • Horse • Sheep • Cat • Dog • Rabbit • Rat • Birds • Monkeys etc.. BDV persistently infects a wide variety of mammal species including humans. BDV strains are genetically closely related and occur globally Occurrence of human infection around the globe 3 INTRODUCTION Most publications implicating BDV in human disease have focused on neuropsychiatric disorders including unipolar depression, bipolar disorder and schizophrenia. Patients’ suffering from depression Reviews: Bode and Ludwig, 2003; Ikuta et al 2002 Infection Meanwhile, BDV has also been found in patients with brain tumours (glioblastoma multiforme), even though a link, whatsoever, remained largely elusive. 4 INTRODUCTION BDV Hu-H1- a human strain Strain Hu-H1 was isolated from freshly PBMC of a female bipolar disorder patient in Germany who was admitted to hospital during a severe depressive episode(Bode et al. 1996). Sequencing displayed single but meaningful mutations vs. Str. V (Torre et al 1996) Recently, members of our group could demonstrate that strain Hu-H1 inhibited proliferation and supported apoptosis, whereas lab. Strain V did quite the opposite (Li et al, 2013) Both strains displayed different metabolic phenotypes (Liu et al 2015) 5 INTRODUCTION Oligodendroglia (OL) cells a cell line derived from human fetal oligodendrocytes. a major component of the brain white matter that play a pivotal role in maintaining neurological function. support both natural and experimental infection with several neurotropic NNS RNA viruses including BDV, canine distemper virus, and measles virus (Ibrahim et al., 2002; Koster-Patzlaff et al., 2007). 6 INTRODUCTION Why do we explore the histone lysine acetylation (Kac) profile of BDV-infected OL cells BDV affects the expression of several host mRNA transcripts (Carbone et al., 2001) , but the mechanisms remain unclear. The recent discovery of histone Kac as a modulator of gene expression in response to virus infection has brought fresh insight into viral epigenetic regulation. BDV infection has been shown to affect site-specific histone Kac in cortical neurons in vitro. However, the histone Kac profile of BDV-infected OL cells remains unknown. We hypothesized that BDV infection epigenetically impacts the OL cell proteome through histone Kac. 7 Tan MJ, et al. Cell, 2011 METHODS scheme of the experimental workflow . We applied SILAC labeling-based proteomics to comparatively quantify the host proteome of OL/BDV cells and control cells. 8 RESULTS BDV infection of OL/BDV cells Human BDV Strain Hu-H1 and human OL cells were kindly provided by Professor Hanns Ludwig (Free University of Berlin, Berlin, Germany). BDV p24 and p40 Detection. (A) Detection of BDV p24 and p40 RNA by reverse transcription polymerase chain reaction (RT-PCR). Detection of BDV p24 and p40 proteins by (B) Western blotting and (C) immunofluorescence assay. 100% of OL/BDV cells were 9 infected, and 100% of control cells were non-infected. RESULTS Proteomic profiling Non-redundant proteins 4436 Proteins with quantifiable differential expression levels in response to BDV infection 4383 Proteins displayed a greater than or equal to 1.5-fold increased expression 1572 Proteins displayed a lesser than or equal to 1.5-fold decreased expression 165 Four quantiles(Q1-Q4) based on the cumulative distribution of SILAC L/H ratios Q1 less than 15% Q2 15–50% Q3 50–85% Q4 greater than 85% … 10 RESULTS BDV infection affects transcription factors Transcription factors Transcription factors in the whole OL cell proteome 201 Transcription factors showed significantly increased expression 84 Transcription factors showed significantly decreased expression 11 Significantly differentiated transcription factors mapped onto the KEGG database 30 11 RESULTS and DISCUSSION Bioinformatic analysis ---Gene ontology (GO) categories Bioinformatic analysis --- Protein domain --- Kyoto Encyclopedia of Genes --- and Genomes (KEGG) --- Protein complexes Cellular compartment Protein complexes Biological process Molecular function Protein domain 12 RESULTS and DISCUSSION Gene ontology (GO) categories ---Membrane proteins and immune response BDV enters cells by plasma membrane fusion. Remarkably, it replicates in the nucleus of the infected cell, requiring nucleocytoplasmic trafficking of BDV macromolecules. As these processes intimately involve the plasma and nuclear membranes, changes in membrane protein expression should be associated with BDV infection. This study confirms that BDV infection mainly upregulated membrane-associated proteins in Q4. Proteins in Q4 were also enriched for immune response, considering the pivotal function of membrane proteins in the immune response. As successful viral infection and replication requires evasion from the host immune response, it is reasonable to surmise that BDV would affect host cellular proteins with an antiviral immune response function. 13 RESULTS and DISCUSSION Gene ontology (GO) categories ---Nuclear proteins and DNA replication As the host cellular chromatin inhibits viral gene expression and replication by uppressing DNA accessibility, viruses that enter and persist in the nucleus have evolved chromatin associated mechanisms to efficiently propagate the viral genome(Lieberman, 2006). For example, BDV‘s viral ribonucleoprotein(RNP) directly interacts with the mitotic host chromosome using core histones as a docking platform (Matsumoto et al., 2012). Here, BDV infection was found to differentially impact on nuclear and chromosomal proteins enriched in DNA replication and repair, transcription regulation, and chromosomal shape regulation in Q1, most of which were downregulated in response to BDV infection. 14 RESULTS and DISCUSSION Kyoto Encyclopedia of Genes and Genomes (KEGG) --- Metabolic pathways Through KEGG analysis, metabolic pathways were identified as the most significantly altered set of host biological pathways. Alterations were found in phospatidylinositol signaling, GABAergic synapse, CAMs, glycerophospholipid metabolism, immune response pathways, nucleic acid processes, cell cycle, ribosomal processes, purine metabolism, and pyrimidine metabolism Consistent with the current KEGG findings, our previous metabonomic profiling study in BDV-infected OL cells revealed significant perturbations in myo-inositol, α-glucose, acetate, pyruvate, and nicotinamide adenine dinucleotide (NAD) (Huang et al., 2012). Thus, these proteomic data and the previous metabonomic data, also based on human BDV Hu-H1 strain, mutually support each other. 15 RESULTS Bioinformatic analysis the amyloid precursor protein mitochondrial translocase APPTOMM40 and IFP35-NMI 16 RESULTS and DISCUSSION BDV infection affects histone Kac As BDV infection impacts transcription factor expression and histone Kac regulates gene transcription, we investigated the affect of BDV infection on site-specific histone Kac. A total of 30 Kac sites in core histones were identified. The illustration of identified histone Kac sites in OL cells in response to BDV infection. The identified sites in core histones were numbered and highlighted. 17 RESULTS and DISCUSSION BDV infection affects histone Kac Kac sites in core histones Total 30 Significantly decreased 15 significantly increased 1(H2BK15) no significant changes 14 Histone modification at the N-terminal plays a central role in chromatin remodeling and transcriptional regulation. Here, most of the quantifiable Kac sites were at N-terminals of core histones, suggesting pronounced epigenetic modulation of BDV-infected OL cells. 18 RESULTS Western blot analysis of some histone Kac To further validate the different histone acetylation profiles Western blot analysis was performed with histone Kac sequence specific antibodies. The expression of histone specific site Kac in the figure were consistent with the quantitative results. 19 RESULTS Histone acetyltransferase(HATs) and deacetylase(HDACs) HAT/ HDAC Protein Changed HAT GCN5 Down↓ HAT PCAF Down↓ HDAC SIRT1 Up↑ HDAC SIRT2 Up↑ HDAC HDAC1 - HDAC HDAC2 - HDAC HDAC3 - HDAC HDAC4 Up↑ HDAC HDAC5 - HDAC HDAC7 Up↑ Histone acetylation is dynamic and regulated by HATs and HDACs. The expression of several HATs and HDACs was significantly altered by BDV infection. Two HATs were found to be significantly downregulated, and four HDACs significantly 20 upregulated in response to BDV infection. HIGHLIGHTS •A human strain of BDV (BDV Hu-H1) was used to infect human oligodendroglial cells (OL cells). •BDV infection appears to preferentially dysregulate membrane, nuclear, and chromosomal host protein expression while affecting metabolic pathways, immune response, DNA replication, DNA repair, and transcription regulation. •BDV infection was found to affect histone acetylation of specific lysine residues. • BDV infection affected the expression of many transcription factors and several HATs and HDACs. •The results of the study will allow a better understanding of infection-driven pathophysiology in vitro. 21 INSTITUTE OF FORENSIC SCIENCE SHANGHAI Thank you ! Welcome to Shanghai China Yu Garden China Pavilion City God temple 22