Poliovirus 3Dpol polymerase region is essential for cleavage of poliovirus 3AB in vivo

Poliovirus (PV) genome encodes a large single polyprotein that is processed by viral proteases to form an active replication complex through either cis or trans interactions between the viral proteins (i.e., interactions between viral proteins encoded on the same polyprotein molecule or between those encoded on different polyprotein molecules, respectively). In the processing of polyprotein, the cleavage of viral 3AB into 3A and 3B is unique, as it requires host factors (PI4KB/OSBP) and viral protease (3Cpro/3CDpro) in cultured cells (i.e., in vivo). Here, we show viral/host requirements for the cleavage of 3AB in vivo. In a polyprotein encoding 2BC3ABCD of PV, cleavage of 3AB requires the activity of PI4KB as well as the entire 3Dpol region; even a partial deletion of the 3Dpol region severely affects the cleavage in the polyprotein. The activity of OSBP and the binding activity of 3CDpro to negatively charged molecules are not required for the cleavage in the polyprotein. PV mutants with premature termination codons or in-frame deletions in the 3Dpol-coding region are generally quasi-infectious in trans-rescued replication with 3CDpro, causing extensive in-frame genome duplication or deletion. Surprisingly, some PV mutants lacking the C-terminal peptides of 3Dpol showed stable replication without any reversion in the presence of 3CDpro provided in trans; 3Dpol provided in trans could rescue the defect in 3AB cleavage via amino acid residues involved in 3Dpol-3AB and 3Dpol-3Dpol interactions, indicating a remarkable overlap with those required for the uridylylation of 3B. This work reveals novel roles of the 3Dpol region, offering insights into the polyprotein processing and recombination.