Shannon Kenney (McArdle Laboratory for Cancer Research, University of Wisconsin-Madison, USA) “Differentiation-Dependent Lytic EBV Reactivation in Epithelial Cells”
- Two models of herpesvirus infection (B cell and epithelials)
- Latent —> Lytic
- Latent/B cells
- Few viral genes expressed
- Replicated by host cell DNA polymerase
- Episomal viral DNA helps viruses evade immune response
- EBV genome is methylated in the B cells
- Lytic / oropharyngeal
- Oral hairy leukoplakia: completely lytic replication
- EBV infection of normal epithelial cells is restricted to differentiated layers and is lytic. (Young et al, J Virol 1991)
- How does EBV achieve the transforming (latent) form of infection in NPC?
- A) epigenetic modifications f EBV genome (5-MC and 5-HMC) that regulate viral reactivation are altered in NPC EBV infection normal (telomerase-immortalized) cells depends on BRLF1 (preferentially activate unmethylated promoters)
- DNA methylation inhibits cellular gene depression
- The unique serine residue 186 is required fro Z binding to methylated ZREs
- DNA methylation f lytic EBV promoters increases Z activation
- – Wille et al. J Virol 2013
- – R transactivates unmethylated BALF2, BMRF1 promoters
- EBV genome methylation differentials affects Z versus R mediated viral reactivation
- Establishment of EBV-positive normal oral keratinocytes (NOKs-Akata) line 2013. J Virol Wille CL, Nawandar DM
- R but not Z expression induces lytic reactivation in EBV-infected telomerase-immortalized normal oral epithelial (NOKs)
- Lytic EBV promoters are hypo-methylated in EBV-infected NOKs cells by hypermethylated in NPC cells
- Differences between EBV infection in normal oral epithelium versus NPC
- EBV genome become methylated in NPC cells but is hypomethylated in NOK cells
- 5mC is converted into 5hmC by the TET family of hydroxyl ages (Mph et al, Exp. Hematol., 2010)
- Undifferentiated nasopharyngeal carcinoma tumor cells have very little 5hmC modified DNA
- 5hmC accumulates furring differentiation of normal tonsil epithelial cells. Wille et al PNAS 2016
- 5hmC inhibits Z binding but does not affect R binding
- TET2 inhibits Z-mediated promoter activation (on Rp)
- TET2 augments ability of R to activate methylated promoters (BALF2 SSBP)
- IDH1(R132H) expression in NOKs-EBV cells inhibits TET activity and reduces global 5hmC. —> restore the ability of Z to activate lytic cycle
- Differences in TET/DNMT activity in normal NP cells versus NPC Nawandar et alm PLoS pathogens 2016
- KLF4 and BLIMP1 high
- KLF4 cooperates with BLIMP1 to activate Rp and Zp, irrespective of the promoter methylation site
- KLF4 synergies with BLIMP1 to us dice lytic EBV reactivation in latently infected
- LMP1 Expression is activated by differentiation in aged NOKs-Akata cells even in the absence of Z/R. Nawandar et al J Virol 2017
- LMP1 is expressed before Z and ER during methyl cellulose-mediated differentiation
- Knocking down LMP1 expression inhibits methylation cellular…
- LMP1 enhances R-mediated lytic reactivation in EBV-infected NOKs cells (!)
- Unanswered issues