Transcript BDV

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
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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)
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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
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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.
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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)
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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).
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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.
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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.
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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
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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%
…
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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
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Significantly differentiated transcription factors mapped onto the
KEGG database
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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
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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.
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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.
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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.
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RESULTS
Bioinformatic analysis
the amyloid precursor protein mitochondrial translocase
APPTOMM40 and IFP35-NMI
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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.
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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
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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.
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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.
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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
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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.
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