Phosphatidylinositol 4-kinases (PI4Ks) catalyze the synthesis of phosphatidylinositol 4-phosphate (PI4P), an important intermediate for the synthesis of membrane polyphosphoinositides, regulators of multiple cellular functions. PI4P defines the membranes of Golgi and trans-Golgi network (TGN) and regulates trafficking to and from the Golgi. Based on enzymatic differences, two classes of PI4K have been distinguished termed Types II (PI4KII) and III (PI4KIII), and each of which contains α and β isoforms. PI4KII alpha and beta have similar biochemical properties. PI4KIIIs (α- and β-forms) are soluble enzymes structurally related to PI3-kinases, and sensitive to PI3-kinase inhibitors, such as Wortmannin. PI4KIIs produce PtdIns 4-phosphate, an early key signaling molecule in phosphatidylinositol cycle, which is indispensable for T cell activation. PI4KIIIs plays a key role in the production of replication complexes (viral factories) of a number of positive-sense RNA viruses and represents a potential target for novel pan-viral therapeutics.