Zhen F. Fu Department of Pathology University of Georgia

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

% survival

0102030405060708090

100

1 2 3 4 5 6 7 8 9 10 11 12

Days

% s

urv

ival

B2C 10(3)

B2C 10(4)

B2C 10(5)

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

2002004004006006008008001000100012001200m/zm/z

tandem mass spectrumtandem mass spectrum

4. automated database searching

theoretical observed

protein protein identificationidentification

2002004004006006008008001000100012001200m/zm/z

2002004004006006008008001000100012001200m/zm/z

peptide peptide identificationidentification

fragment peptide

Ar

Ar

ArAr

2. select specific peptide2. select specific peptide

ESIESI

1. MS “survey” scan1. MS “survey” scan

600600 800800 10001000 12001200 1400140000

100100

5050

Relat

ive A

bund

ance

600600 800800 10001000 12001200 1400140000

100100

5050

Relat

ive A

bund

ance

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

100100

6060

5050

4040

3535

3030

2525

2020

Molec

ular W

eight

Molec

ular W

eight

ppII

peptides

trypsin

gel

µLCµLC

Gygi et al.

+TOF MS: 36 MCA scans from MWmems50_18.wiff Max. 185.0 counts.

1200 1300 1400 1500 1600 1700 1800 1900 2000 2100 2200 2300 2400 2500 2600 2700 2800 2900 3000m/z, amu

0

10

20

30

40

50

60

70

80

90

100

110

120

130

140

150

160

170

180

In

te

ns

ity

, c

ou

nts

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!!