for phosphorylation of this residue. However, in the context of the non-culture-adapted infectious clones (e.g. H77, J4, Con1), the phosphorylation of serine 249 might be important in some aspect of NS5A function. Indeed, the acquisition of culture-adaptive mutations in NS5A has been shown to correlate with a loss of hyperpho- sphorylation, even when the mutations were not at serine residues (Blight et al., 2000). It will be of interest to investigate the potential role of serine 249 phosphoryla- tion in the context of the infectious genotype 1b virus described recently (Pietschmann et al., 2009). However, as acknowledged in that publication, the very low replicative capacity of this isolate in Huh7 cells will render such an investigation technically challenging. One attractive hypothesis that may be more tractable to investigation is the potential structural role of serine 249. As it is followed by a proline residue, phosphorylation of serine 249 might influence the recognition of this proline by peptidyl–prolyl isomerases such as cyclophilin A, a known NS5A-interacting partner that is important for viral RNA replication. The location of serine 249, precisely at the N terminus of domain II of NS5A, might allow phosphorylation to influence the spatial orientation of that domain with respect to domain I by mediating the cis–trans isomerization of the peptidyl– prolyl bond. Structural studies (e.g. NMR) on purified wild-type and mutant NS5A, phosphorylated in vitro, might shed light on this issue, but represent significant technical challenges. Acknowledgements This work was supported by the Wellcome Trust (078358). We thank Volker Lohmann and Ralf Bartenschlager (University of Heidelberg, Germany) for pFK5.1, and Takaji Wakita (National Institute for Infectious Diseases, Tokyo, Japan) for pJFH-1. 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