Audrey Lullo, Brett Seaholm , Socheata Vong , Jamie islas , Cody Carini ,

1. Two Novel Human Cytomegalovirus nk cell evasion functions target mica for lysosomal degradation Audrey Lullo, Brett Seaholm, SocheataVong, Jamie islas, Cody Carini,

2. Immune Evasion • “Methods used by pathogenic organisms to evade a host’s immune system.”

3. Viral Immune Evasion Strategies • Reducing Class 1 expression to evade the cytotoxic T lymphocytes (CTL). • Suppression of Class 1 proteins from ever reaching the surface of the cell. • Producing “decoy” Class 1 homologs to evade Natural Killer cells (NK). • Preventing apoptosis (programmed cell death).

4. Two Novel Human Cytomegalovirus Nk cell evasion functions target mica for lysosomal Degradation • By: Ceri A. Fielding,Rebecca Aicheler, Richard J. Stanton, Eddie C. Y. Wang, Song Han, Sepehr Seirafian,James Davies, Brian P. McSharry, Michael P. Weekes, P. Robin Antrobus, Virginie Prod'homme, Fabien P. Blanchet, Daniel Sugrue, Simone Cuff, Dawn Roberts, Andrew J. Davison,Paul J. Lehner, Gavin W. G. Wilkinson, and Peter Tomasec • Funding: Medical Research Council and the Wellcome Trust • Published: online, May 1, 2014

5. Human Cytomegalovirus (HCMV) • Herpes virus (life long infection). • High morbidity and mortality in those with weakened immune systems.

6. Human Cytomegalovirus • Can cause graft rejection, pneumonia, enteritis, hepatitis, retinitis in immune compromised individuals. • Major cause of birth defects (mental retardation and hearing loss). • Correlation between infection and two brain tumors (medulloblastomaand glioblastomamultiforme). • Can cause heart disease and arthritis. • However, most infections do not get this severe.

7. HCMV and Immune Evasion • HCMV evades natural killer (NK) cells by suppressing the stress proteins from ever reaching the infected cells surface. These stress proteins would normally signal the NK cells of a cell’s abnormality. However, they never reach the surface, allowing HCMV to “hide” in the host cell. • A vast majority of HCMV’s large (263 kb) genome is dedicated to evading the immune system.

8. NKG2D and NKG2dL’s • NKG2D is an activating receptor on NK cells that recognize 8 ligands (NKG2DL’s) present in HCMV cells: • Ligands that are recognized by NKG2D

9. NKG2DL’s • Become present on the cell surface when the cell undergoes a form of stress such as: • Genotoxic damage • Growth stimulation • Viral infection

10. Overview Genes UL122 & UL123 • Ligands • MICA/B • ULBP(1-6) Immediate Early proteins IE1/2 NK Cell Evasion Genes US18 & US20

11. Hypothesis • “We hypothesized that HCMV infected cells may be vulnerable to NK cell surveillance during the early phase if there were a temporal window between activation of NKG2DL’s by IE1 and IE2 and the expression of effective HCMV counter measure.” • The results and findings of this study will hopefully help to fight HCMV lifelong infections.

12. METHODS • Cell lines • MICA typing • Viruses • Infect cells with mutated virus • Analysis: • Flow cytometry- cell surface marker expression • Triton X-114 membrane protein extraction • Immunoblotting of target cells • Immunofluorescence staining • Intracellular flow cytometry • NK cytotoxicity assay • NK degradation assay

13. Cell lines used- • immortalized human fetal foreskin fibroblasts with human telomerase- HF-TERTS • transfected with adenovirus receptor and cells expressing U373 MICA-YFP • MICA typing • DNA extracted • Viruses • mutated HCMV with deleted genes, Rad-1E1 and 1E2 from HCV strain • Infected cells with mutated virus

14. Flow cytometry • Analyze cell surface marker expression • of viable and infected cells Picture source: http://www.abcam.com/index.html?pageconfig=resource&rid=11446

15. Immunoblotting • Identify target protein • Using antibodies http://www.virology.ws/2010/07/07/virology-toolbox-the-western-blot/

16. Immunofluorescence staining http://www.wieslab.se/index.php?langId=1&headId=72&subId=92&pageId=123 http://fanosmcb.blogspot.com/2010/06/immunofluorescence.html

17. NK (natural killer cells) analysis • Standard 51Cr chromium release assay • Target: labelledRAd-infected • fibroblast target cells • Effector: NK cells • Analyze cytotoxicity and • Degranulation of NK cells http://www.perkinelmer.com/resources/technicalresources/applicationsupportknowledgebase/radiometric/chromium51.xhtml

18. Figure 8a-1 “Before designating a virus gene as being an NK cell evasion function, it is important to monitor its biological activity during infection. The effects of US18 and US20 on NK cell recognition were therefore analyzed using the HCMV deletion mutants.”

19. Figure 8A-2 “Relative to uninfected cells, infection with strain Merlin elicited robust protection against NK cells in all donors tested. A significant increase in NK cell degranulation was associated with loss of US18 or US20 or the US18–22 ‘block’ deletion, and an additive effect was observed when both genes were absent.”

20. Figure 8B “The use of a MICA blocking antibody led to a small decrease in NK degranulation in response to targets infected with the US18 and US20 double deletion virus, whilst the same antibody had no effect on NK activation in response to Mock or HCMV-infected targets.”

21. What Does it mean? • ‘It was concluded that US18 and US20 were effective in suppressing NK cell activation in the context of a productive HCMV infection.’ • ‘These data suggest that either the blockade of MICA was incomplete, or that US18 & US20 target other cellular molecules capable of regulating NK cell activation.’

22. Why Is It Important? • In layman’s terms, the two genes – US18 and US20 – successfully prevent Natural Killer cells from being activated. • It also points out the possibility that the Major Histocompatibility Complex A was altered or incompletely formed, otherwise genes US18 and US20 could be targeting other molecular components that activate Natural Killer cells.

23. Results • When expressed using ad vectors, IE1 induced relatively modest increase in MICA and MICB, but provided for a major upregulation in ULBP2 both as the level of total protein expression and specifically on the cell surface. • In contrast, IE2 induced strong activation of MICA and MICB, yet only a small increase of ULBP2 levels. • IE1 and IE2 were thus found to differentially activate individual ligands recognized by the NKG2D activator receptor.

24. Results • Expression of US18 or US20 led to reductions in MICA/B levels • Deletion of US18 alone had no visible effect on cell surface • Deletion of US20 induced a modest but significant increase in surface levels of MICA compared to parental virus • Deletion of both US18 and US20 caused a much more dramatic increase in MICA levels

25. In conclusion: • The infected cell responded to differential sensing of the: • Expression of IE1 to promote the upregulationof ULBP2 • Expression of IE2 to promote the upregulationof MICA/B • Expression of the US18–US22 genes individually was each associated with a minor reduction in MICA cell surface expression • Deletion of both US18 and US20 caused a much more dramatic increase in MICA levels

26. REferences • Fielding, C. A., & et al. (2014). Two Novel Human Cytomegalovirus NK Cell Evasion Functions Target MICA for Lysosomal Degradation. PLoS Pathogens,10(5). Retrieved May 26, 2014, from http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4006889/ • Immune Evasion (definition). (n.d.).Immune Evasion (definition). Retrieved May 28, 2014, from http://www.reference.md/files/D057/mD057131.html • Ploegh, H. L. (1998). Viral Strategies of Immune Evasion. Science Magazine,280(5361), 248-253. Retrieved May 28, 2014, from http://www.sciencemag.org/content/280/5 • Trasnfection Protocols & Applications (definition). Sample & Assay Technologies. Retrieved May 28, 2014. from http://www.qiagen.com/resources/molecular-biology-methods/transfection/ • Copeland, N., Jenkins, N. & Court, D. Et al. (2001). Recombineering: A Power New Tool for Mouse Functional Genomics. Nature Reviews Genetics 2 (769-779). Retrieved May 28, 2014. from http://www.nature.com/nrg/journal/v2/n10/full/nrg1001-769a.html • Magi, B., Liberatori, S. et al. (2005). Immunoblotting Techniques. Methods in Molecular Biology. Retreived May 28, 2014, from http://www.ncbi.nlm.nih.gov/pubmed/15596900

27. Team analysis • The researchers started out exploring if there was a temporal window of activation pre-expression of suppression genes. • The methods were appropriate for their alternate exploration, however not for their initial hypothesis. • The conclusions were not overstated—if anything they were understated. • This research could potentially have an effect on any individual who is infected with a herpes virus. • It is super complicated but it HCMV is a perfect virus to study immunize evasion because of how much of its viral genome is dedicated to immunize evasion • The experimental step would be to take the procedure into animal testing