When poliovirus (PV) starts to multiply, membranes are produced that contain phosphatidylinositol-4-phosphate (PI4P) and appear as vesicular clusters in cross section. The authors were interested in the induction and remodelling of PI4P and these membranes in relation to genome replication. They used two PV mutants, EG and GG, that experience aberrant proteolytic processing of the P3 precursor, substantially delaying the onset of genome replication and/or virus assembly, to examine the formation of these PV-induced membranous structures. For WT PV, changes to the PI4P pool were seen as early as 30 minutes post-infection. PI4P remodelling was found to occur in the presence of guanidine hydrochloride, a replication inhibitor, and was accompanied by formation of membrane tubules throughout the cytoplasm. Vesicular clusters appeared in the perinuclear region of the cell 3 hours following infection, which would be too slow for these structures to be responsible for genome replication. Delays seen in the onset of genome replication for EG and GG PVs were similar to the delays seen in virus-induced remodelling of the PI4P pools, which is consistent with PI4P being a marker of the genome-replication organelle. GG PV was then unable to convert virus-induced tubules into vesicular clusters, leading to a 5-log reduction in infectious virus. Therefore the authors findings suggest that PV induces temporally distinct membranous structures (organelles) for genome replication (tubules) and virus assembly (vesicular clusters). The pace of this formation, spatiotemporal dynamics and the efficiency of the replication-to-assembly-organelle conversion can be set by the rate of P3 polyprotein processing and the capacity for P3 processing to yield 3AB and/or 3CD proteins.
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