Formation of the poliovirus replication complex requires coupled viral translation, vesicle production, and viral RNA synthesis

Poliovirus (PV) infection induces the rearrangement of intracellular membra nes into characteristic vesicles which assemble into an RNA replication com plex. To investigate this transformation, endoplasmic reticulum (ER) membra nes in HeLa cells were modified by the expression of different cellular or viral membrane binding proteins. The membrane-binding proteins induced two types of membrane alterations, i.e., extended membrane sheets and vesicles similar to those found during a PV infection. Cells expressing membrane-bin ding proteins were superinfected with PV and then analyzed for virus replic ation, location of membranes, viral protein, and RNA by immunofluorescence and fluorescent in situ hybridization. Cultures expressing cellular or vira l membrane-binding proteins, but not those expressing soluble proteins, sho wed a markedly reduced ability to support PV replication as a consequence o f the modification of ER membranes. The altered membranes, regardless of th eir morphology, were not used for the formation of viral replication comple xes during a subsequent PV infection. Specifically, membrane sheets were no t substrates for PV-induced vesicle formation, and, surprisingly, vesicles induced by and carrying one or all of the PV replication proteins did not c ontribute to replication complexes formed by the superinfecting PV. The for mation of replication complexes required active viral RNA replication. The extensive alterations induced by membrane-binding proteins in the ER result ed in reduced viral protein synthesis, thus affecting the number of cells s upporting PV multiplication. Our data suggest that a functional replication complex is formed in cis, in a coupled process involving viral translation , membrane modification and vesicle budding, and viral RNA synthesis.