S T R U C T U R A L B I O L O G Y

S C I E N T I F I C H I G H L I G H T S

4 0 H I G H L I G H T S 2 0 2 2 I

to be determined in complex with a CpG-methylated viral DNA site (Figure 30a). Comparison with the AP-1-bound structure identified specific interactions as potentially important for high-affinity methyl-CpG recognition (Figure 30b). This hypothesis was confirmed by structure-based point mutations that decreased ZEBRA s binding affinity for methylated DNA. Mutants with reduced binding activity were defective for transactivating a CpG-methylated promoter and were unable to induce the EBV lytic cycle and viral production.

The study revealed a key role for one of ZEBRA s serine residues, Ser186, which is unique to ZEBRA among bZIP proteins and directly contacts the methyl-CpG motif (Figure 31a). Replacing Ser186 by the corresponding

alanine residue present in cellular bZIP proteins compromised ZEBRA s ability to bind CpG-methylated sites preferentially over AP-1 sites (Figure 31b) and completely abolished the ability of the virus to replicate (Figure 31c). These findings reveal that ZEBRA s DNA- binding interface is fine-tuned to discriminate between its two classes of DNA target sites rather than simply to differentiate methylated from unmethylated DNA.

In summary, the study revealed how ZEBRA recognises methylated DNA and how it switches between AP-1 and CpG-methylated sites, providing insights into ZEBRA s role as a master regulator that alternatively promotes the establishment of viral latency or triggers activation of lytic replication.

Fig. 31: A unique serine residue plays a key role in DNA site selectivity. a) Details of methyl-CpG recognition. H-bond and van der Waals interactions involving ZEBRA residue Ser186 are indicated by black and grey dashed lines,

respectively. b) DNA binding assays showing that the replacement of Ser186 by an alanine inverts ZEBRA s selectivity between an AP-1 site and a CpG-methylated site. c) The S186A mutation compromises ZEBRA s ability to transactivate

a CpG-methylated promoter (top) and to induce viral lytic replication (bottom).

PRINCIPAL PUBLICATION AND AUTHORS

Structural basis of DNA methylation-dependent site selectivity of the Epstein-Barr virus lytic switch protein ZEBRA/Zta/BZLF1, F. Bernaudat (a,b), M. Gustems (c), J. Günther (d,e), M.F. Oliva (a,f), A. Buschle (c), C. Göbel (c), P. Pagniez (a), J. Lupo (a,g), L. Signor (a), C.W. Müller (h), P. Morand (a,g), M. Sattler (d,e), W. Hammerschmidt (c), C. Petosa (a), Nucleic Acids Res. 50, 490-511 (2022); https:/doi.org/10.1093/nar/gkab1183 (a) Institut de Biologie Structurale, Grenoble (France) (b) ESRF (c) Helmholtz Zentrum München and German Centre for Infection Research (DZIF), Munich (Germany) (d) Institute of Structural Biology, Helmholtz Zentrum München, Neuherberg (Germany) (e) Technical University of Munich, Garching (Germany) (f) Institut Laue-Langevin, Grenoble (France) (g) Centre Hospitalier Universitaire Grenoble-Alpes, Grenoble (France) (h) European Molecular Biology Laboratory, Heidelberg (Germany)

REFERENCES

[1] C. Petosa et al., Mol. Cell 21, 565-572 (2006).