Figure 2.

Influenza polymerase. (a) A self-processing recombinant polyprotein was used to express influenza polymerase complex in high quality and quantity in insect cells by using the MultiBac system [37] as illustrated (left). The polymerase was expressed from a single open reading frame encoding for tobacco etch virus NIa protease (TEV), the polymerase subunits PA, PB1 and PB2 and a fluorescent protein (CFP). A second, yellow fluorescent protein (YFP) was inserted elsewhere into the MultiBac baculoviral genome as an expression performance marker. The resulting polyprotein is processed into the individual subunits by highly specific proteolysis mediated by TEV. The polyprotein construct is shown schematically (right) illustrating tag placement and design of spacers in between the subunits (adapted from [6^••]). The C-terminal part of PB1 and the N-terminal part of PB2 co-fold into a helical bundle necessitating careful linker design (boxed). (b) Crystal structure of influenza polymerase bound to cognate viral RNA (vRNA) is shown in a ribbon representation. PA is colored in blue, PB1 in green and PB2 in red. RNA substrate is colored in orange. Cap-binding domain and endonuclease domain are indicated. The structure motif formed by PB1 C-terminal domain and PB2 N-terminal domain is shown in a magnification (right). PB2 N-terminus and PB1 C-terminus are marked.