Pathogenic and attenuated rabies viruses induces differential host protein
expression in the central nervous system: Implication of neuronal
dysfunction
Zhen F. Fu
Department of Pathology
University of Georgia
Robert Hurt-USC
Rabies PathogenesisRabies Pathogenesis
Patients die of circulatory insufficiency, Patients die of circulatory insufficiency, cardiac arrest and respiratory failure.cardiac arrest and respiratory failure.
Despite extensive research in the past 100 years, we still know very little about the pathogenic mechanism by which rabies virus infection of neurons causes rabies.
There are scarce neuropathology with mild inflammation and little neuronal loss, which cannot explain the lethality of the disease.
It has been hypothesized that rabies results from neuronal dysfunction rather than structural damage. However, it is not known how RV infection leads to neuronal dysfunction.
To better understand rabies pathogenesis, we initiate a project to determine how the host responds to rabies virus infections using one street and one fixed virus. This is accomplished by using proteomics technologies.
Two viruses were used in this study:
SHBRV: Wt virus, normally circulating in silver-haired bats and responsible for most of the human rabies in the US.
CVS-B2C: Lab-adapted attenuated virus derived from CVS-24 by passaging in BHK cells.
Survival curve
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B2C 10(6)
SHBRV 10(3)
neg control
Detection of Differential Protein Levels in the Proteome
pH
Outline of 2-D Proteomics Strategy
MW
pH
In-gel digestion with protease
Identify the protein by Mass Spectrometry
mutant/Infected Wild type
Identification of gel-separated proteins by mass spectrometry
3. detect fragments3. detect fragments
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tandem mass spectrumtandem mass spectrum
4. automated database searching
theoretical observed
protein protein identificationidentification
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peptide peptide identificationidentification
fragment peptide
Ar
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2. select specific peptide2. select specific peptide
ESIESI
1. MS “survey” scan1. MS “survey” scan
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10.010.0 7.57.5 6.36.3 6.06.0 5.85.8 5.55.5 5.25.2 5.05.0 4.04.0 3.03.09.09.0 8.08.0
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µLCµLC
Gygi et al.
+TOF MS: 36 MCA scans from MWmems50_18.wiff Max. 185.0 counts.
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Software is essential !
The situation to avoid…
Proteins differentially expressed in response to SHBRV infection
Category Protein PI Molecular weight (kDa)
Up/down-regulation
Fold change
Ion Homeostasis
Na+/K+ ATPase 5.3 110 ↑ +3.0
H+ ATPase subunit a isoform 1
6.0 96 ↑ +2.6
Synaptic Physiology
TRIM 9 6.6 90 ↓ -3.0
-SNAP 5.3 33 ↓ -3.0
Pallidin (Syntaxin 13)
5.8 20 ↓ -2.2
Synexin 5.9 50 ↓ -2.0
Syntaxin 18 5.2 35 ↓ -1.8
Proteins differentially expressed in response to B2C infection in mice
Category Protein PI Molecular weight (kDa)
Up/down regulation
Fold change
Innate immunity
G protein coupled receptor 44
4.8 88 ↑ 3
Hsp 60 5.9 60 ↑ 3.2
Ion Homeostasis
Villin 1 5.1 89 ↓ 2
Calretinin 4.9 31 ↓ 2.2
H+ ATPase synthase subunit b
5.5 58 ↓ 2.8
Apoptosis APAF 1 6.0 143 ↑ 2.4
SH3 domain binding protein 2
7.6 62 ↑ 2.6
Neuro-Physiology
Neurofilament protein NF 66
5.1 60 ↓ 3
CRMP-2 6.6 62 ↑ 3.6
Western blotting of proteins involved in ion homeostasis and synaptic physiology
No Docking
No release of Neurotransmitters
Accumulation of vesicles
SH
BR
V
B2C
C
ontr
ol
Proteomics data indicate that wt RV infection resulted in up-regulation of proteins involved in ion homeostasis and down-regulation of synaptic proteins.
The altered protein expression as detected by 2D-gel analysis is confirmed by Western blotting in animals infected either ic or im as well as in primary neuron.
Up-regulation of Na/K-ATPase leads to decrease in Na+ concentrations in infected cells. Likewise, down-regulation of Ca-ATPase resulted in decrease of Ca++ concentration in infected cells.
Changes in Na/Ca concentration affects membrane potential and thus leading to alteration of neuronal transmission.
Synaptic proteins such as syntaxin, -SANP, and TRIM9 play important roles in synaptic-vesicle fusion and docking of synaptic vesicles. Down-regulation of these proteins prevented the docking and fusion of synaptic vesicles with presynaptic membrane, thus resulting in accumulation of synaptic vesicles in the presynapses.
Thus our data may provide structural and metabolic basis by which RV infection causes neuronal dysfunction.
Conclusions
Acknowledgements
Vikas Dhingra
Xia-qing Li
Luciana Sarmento
UGA Proteomics Facility
Tracy Andachtc
Thank you!!