Direct interactions between picornavirus 3CD protein and phosphoinositide lipids in virus replication organelles.

Abstract

Positive-strand RNA viruses disrupt host membrane systems and reconstitute these materials into so-called replication organelles, which serve as sites for virus replication and genome packaging. In picornaviruses, these new membrane systems are enriched in phosphoinositide lipids, including phosphoinositide-4-phosphate (PI4P). The 3CD polyprotein is central to membrane biogenesis, as it was previously shown that ectopic expression of 3CD leads to PI4P induction and redistribution, a phenotype similar to that of poliovirus-infected cells. Here, we have shown direct interaction between poliovirus 3CD and PI4P using nuclear magnetic resonance (NMR) spectroscopy, especially providing atomic-level insights into PI4P interaction with both the 3C protease and 3D polymerase derived domains of 3CD. Our studies using paramagnetically-labeled lipid nanodiscs demonstrated that 3C interacts peripherally with the membrane through its positively charged N-terminal helix, which also interacts with genome RNA sequences important for coordinating replication and translation processes. Extension of the C-terminus of 3C increases its affinity to PI4P-containing membranes, suggesting a means by which interactions may change between 3CD and its processed counterparts. Adjustments to virus protein-membrane interactions may be an important component of the switch from replication to packaging modes on these membranes.