NS5A phosphorylation can be studied in two ways: in living cells and in vitro. The former has several advantages: NS5A phosphorylation takes place in a cellular background and therefore might mimic more closely the real in vivo situation. Viral proteins and cellular kinases are in the correct cellular compartments, and dynamic processes like viral polyprotein processing and cellular signaling are in place. The disadvantage of this system is its great complexity, which makes limiting an observed effect to a single, well-defined agent, for example, a kinase, very difficult. NS5A phosphorylation in cells can easily be followed by metabolic labeling with either (35)S-methionine or (32)P-orthophosphate. The effect of a single, well-defined kinase on NS5A phosphorylation can be investigated in cells either by overexpression of this kinase in the presence of NS5A or by RNA interference of this kinase. If available, specific kinase inhibitors can be used to reveal the effect of this inhibition on NS5A phosphorylation. The problem with this approach is that only very few really specific kinase inhibitors are available. Biochemical in vitro experiments use purified components. This type of experiment allows direct investigation of the activity of a single kinase on NS5A as a substrate. In addition, the precise phosphorylation sites of a kinase can be mapped when NS5A-derived peptides are used instead of a full-length recombinant protein. Kinase inhibitors, which show a particular effect on NS5A phosphorylation in cells, can be retested in vitro on a particular kinase candidate. The problem with this approach is that purified components, like the purified NS5A substrate and the kinase of interest, are not always available.
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