Self-Inhibition of Synthesis and Antigen Presentation by Epstein-Barr Virus-Encoded EBNA1: An Explanation

1. Self-Inhibition of Synthesis and Antigen Presentation by Epstein-Barr Virus-Encoded EBNA1: An Explanation Y. Yin, B. Manoury, R. Fahraeus

2. First: A Little About MHC-1 Major Histocompatibility Complex-1 presents viral peptides to CD8+ cytotoxic T cells (CTL in this paper). Interacts with CD8a, T cell Receptor (TCR) on T Cell

3. A Little About MHC-1: MHC-1 found on all cells in all tissues If it can be infected, it will produce MHC-1. Some tissues present more immune system cells Some present less hepatocytes Some almost none erythrocytes: no nucleus = no viral replication ? inefficient to have MHC-1

4. A Little About MHC-1:Structure Two polypeptide chains a chain: large, three domains Spans membrane a1 and a2 form walls of cleft on surface of molecule b2 Microglobulin: small, immunoglobulin-like domain MHC-1 binds short peptides of 8-10 a.a. in length Ends of peptide bind to groove Can also bind peptides at anchor residues polymorphisms

5. A Little About MHC-1:Peptide Processing In ER: MHC-1a stabilized by calnexin b2m then binds; a:b2m binds to chaperones, TAP via tapasin. In cytosol: Proteasome digests cytosolic proteins TAP channel delivers small peptides to MHC MHC exported to outer membrane

6. And Now the Paper: Question: how does GAr work to prevent EBNA1 antigen presentation by MHC-1?

7. Basics: What is known: GAr of EBNA1 inhibits antigen presentation on MHC-1 Original Hypothesis: GAr was first thought to inhibit proteasome degradation ? no peptide to be presented on MHC-1 New Hypothesis: Coupled with the above, GAr also inhibits mRNA translation of EBNA1 ? less protein to be digested by proteasome, presented by MHC-1

8. Old Hypothesis: Original Hypothesis: GAr was first thought to inhibit proteasome degradation ? no peptide to be presented on MHC-1 Observed: Cells fail to present antigen on MHC-1 when GAr is present on transcript Fusion of GAr to other protein prevents antigen presentation of fused protein GAr increases half-life of EBNA1 by inhibiting degradation by proteasome

9. Old Hypothesis: Original Hypothesis: GAr was first thought to inhibit proteasome degradation ? no peptide to be presented on MHC-1 Problems: No observed higher steady-state level of full EBNA1 vs. EBDGA ? Reverse has been found No way to prevent Defective Ribosomal Products (DRiPs) from proteasome digestion, MHC-1 presentation.

10. How Does GAr Inhibit Translation? Steady-state levels of EBNA1 increased in EBDGA vs. EBNA1wt Observed: GAr inhibits EBNA1 degradation, does not change EBNA1 mRNA levels In Vitro Translation Assays of above mRNAs: EBNA1wt transcript 11x less efficient than EBDGA transcript Stability of mRNAs were the same Cotranslated mRNAs: EBNA1wt mRNA did not attenuate EBDGA translation Conclusion: Presence of GAr inhibits transcription of EBNA1 mediated in cis both in vitro and in vivo (cis = only affects transcript GAr is coded on; does not affect other mRNA in solution)

11. Signal Transduction Cascade? No evidence of eIF2a phosphorylation by PKR with added GAr GAr on 3�-UTR of transcript does not have inhibitory effect in this same PKR model ? GAr depends on position in transcript Conclusion: GAr mRNA sequence does not hinder translation itself through signal transduction cascade or through interaction with ribosomes and other translation machinery

12. Do other parts of EBNA1 factor in? Placed only GAr on ovalbumin C- or N-terminus Truncated or full GAr Both N- and C-terminus GAr inhibit translation Only Truncated N-terminus GAr reduced translation, but not as much as in full GAr

13. Does GAr Interfere with Translation Machinery? Hypothesis: GAr peptide may interfere with activity of ribosomal factors needed for translation. GAr Ab added to IVT assays ? increased translation in EBNA1wt, not in EBDGA

14. DRiP! According to DRiP hypothesis, only proteasome inhibition is necessary to show results seen. Incubate Ova (no GAr action), Ova-GAC (degradation inhibition only) Ova-GAN (degradation and translation inhibition) with T cell line specific for SIINFEKL Ova-derived peptide Ova-GAC showed increased T cell activation over Ova-GAN GAr inhibition of proteasomal digestion not enough to prevent MHC-1 presentation of EBNA1 Slower translation of EBNA1 means less mistakes made ? less DRiP proteins created that are suitable for proteasome degradation

15. So What Does All This Mean? Removal of GAr will not only increase protease degradation of translation products, but will also increase EBNA1 protein created from set amount of mRNA Protease inhibition alone not sufficient to explain lack of MHC-1 presentation of EBNA1 Shows how efficient the GAr is in protecting EBV virus from �early� death from CD8+ T Cells

16. Sources: Yin et al: �Self-Inhibition of Synthesis and Antigen Presentation by Epstein-Barr Virus-Encoded EBNA1.� Science, vol 301. Sept. 5, 2003. 1371-4. Janeway et al: Immunobiology. New York: Garland Publishing, 2001.