Structural and functional characterization of the poliovirus replication complex

Abstract

Two populations of membrane-bound replication complexes were isolated from poliovirus-infected HEp-2 cells by sucrose gradient centrifugation. The two fractions show similar ultrastructural features: the replication complex is enclosed in a rosettelike shell of virus-induced vesicles and contains a very tightly packed second membrane system (compact membranes). The vesicular fraction, which bands in 30% sucrose, contains replicative intermediate (RI) and 36S RNA. The fraction banding in 45% sucrose contains only minute amounts of RI and contains mainly 36S RNA, two-thirds of which is encapsidated. In vitro, the two fractions show similar RNA synthesizing capacities and produce 36S plus-strand RNA. Dissolving the membranes within and around synthetically active replication complexes with sodium deoxycholate abolishes the completion of 36S RNA but still allows elongation in the RI. Our findings suggest an architecture of the replication complex that has the nascent plus strands on the RI enclosed in the compact membranes and the replication forks wrapped additionally in protein. Plus-strand RNA can be localized by in situ hybridization with a biotinylated riboprobe between the replication complex and the rosette of the virus-induced vesicles. It was found that the progeny RNA strands are set free soon after completion from the replication complex at the sites where the compact membranes within the replication complex are in close contact with the surrounding virus-induced vesicles. The replication of poliovirus RNA is carried out by the primer-dependent viral polymerase 3DPo' and proceeds * Corresponding author.

Viral rewiring of cellular lipid metabolism to create membranous replication compartments

“…poliovirus, coxsackievirus, and rhinovirus) ( Table 1). Protrusion-type VRCs often form a network of tightly packed membranes, which has been proposed to protect and hide the viral RNA [3][4][5][6][7][8]. Biochemical support for this idea comes from work with poliovirus.…”

Interactions Between Virus Proteins and Host Cell Membranes During the Viral Life Cycle

“…Soon after translation, newly synthesized nonstructural viral proteins become associated with the viral RNA into an RNA replication complex, which is assembled on intracellular membranes (Bienz et al, 1987(Bienz et al, , 1990. Poliovirus replication complexes consist of groups of vesicles of 70-400 nm in diameter, and after isolation become associated as large rosette-like shells of many vesicles interconnected with tubular extensions (Bienz et al, 1992). In vitro, the rosettes can dissociate reversibly into tubular vesicles, and are able to resume RNA synthesis (Bienz et al, 1992).…”

“…Poliovirus replication complexes consist of groups of vesicles of 70-400 nm in diameter, and after isolation become associated as large rosette-like shells of many vesicles interconnected with tubular extensions (Bienz et al, 1992). In vitro, the rosettes can dissociate reversibly into tubular vesicles, and are able to resume RNA synthesis (Bienz et al, 1992). Immunoisolated poliovirus-specific vesicles contain viral nonstructural proteins, and cellular markers of the ER, lysosomes, and trans-Golgi network, suggesting a complex biogenesis of the RNA replication complexes (Schlegel et al, 1996).…”

(+)RNA viruses rewire cellular pathways to build replication organelles

“…In the invaginations, the replication machinery is located on the inner membrane surface and the replication compartment is connected to the cytoplasm with an opening wide enough to provide a supply of nucleotides and to export synthesized RNA [2 ,3 ]. In tubulo-vesicular replication structures, the viral replication proteins are localized on the external membranous surface facing the cytoplasm [24]. This division may reflect an evolutionary divergence between (+)RNA virus groups and suggest that the mechanisms of membrane remodeling may be significantly different between picorna-like and other (+)RNA viruses ( Figure 1).…”