Zhen F. Fu Department of Pathology University of Georgia

1. 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

2. Rabies Pathogenesis Robert Hurt-USC Patients die of circulatory insufficiency, cardiac arrest and respiratory failure.

3. 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.

4. 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.

5. Survival curve

6. Detection of Differential Protein Levels in the Proteome

7. Outline of 2-D Proteomics Strategy MW pH pH mutant/Infected Wild type In-gel digestion with protease Identify the protein by Mass Spectrometry

8. 2. select specific peptide 3. detect fragments ESI Ar Ar µLC fragment peptide Ar 1200 1000 200 400 600 800 Ar m/z tandem mass spectrum 1. MS “survey” scan peptides trypsin 4. automated database searching gel peptide identification 1200 1200 1000 1000 200 400 600 800 200 400 600 800 theoretical observed m/z m/z protein identification Identification of gel-separated proteins by mass spectrometry Gygi et al.

9. The situation to avoid… Software is essential !

11. Proteins differentially expressed in response to SHBRV infection

12. Proteins differentially expressed in response to B2C infection in mice

13. Western blotting of proteins involved in ion homeostasis and synaptic physiology

17. Accumulation of vesicles No Docking No release of Neurotransmitters

18. SHBRV B2C Control

19. Conclusions 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, a-SANP, and TRIM9 play important rolesin 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.

20. Acknowledgements Vikas Dhingra Xia-qing Li Luciana Sarmento UGA Proteomics Facility Tracy Andachtc

21. Thank you!!