Formation of the poliovirus replication complex requires coupled viral translation, vesicle production, and viral RNA synthesis - PubMed (original) (raw)
Formation of the poliovirus replication complex requires coupled viral translation, vesicle production, and viral RNA synthesis
D Egger et al. J Virol. 2000 Jul.
Abstract
Poliovirus (PV) infection induces the rearrangement of intracellular membranes into characteristic vesicles which assemble into an RNA replication complex. To investigate this transformation, endoplasmic reticulum (ER) membranes 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-binding proteins were superinfected with PV and then analyzed for virus replication, location of membranes, viral protein, and RNA by immunofluorescence and fluorescent in situ hybridization. Cultures expressing cellular or viral membrane-binding proteins, but not those expressing soluble proteins, showed a markedly reduced ability to support PV replication as a consequence of the modification of ER membranes. The altered membranes, regardless of their morphology, were not used for the formation of viral replication complexes during a subsequent PV infection. Specifically, membrane sheets were not substrates for PV-induced vesicle formation, and, surprisingly, vesicles induced by and carrying one or all of the PV replication proteins did not contribute to replication complexes formed by the superinfecting PV. The formation of replication complexes required active viral RNA replication. The extensive alterations induced by membrane-binding proteins in the ER resulted in reduced viral protein synthesis, thus affecting the number of cells supporting 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.
Figures
FIG. 1
Schematic representation of the constructs used in this study. PV and pPVΔP1 have authentic PV 5′ NTRs and are replication competent. pE5PVΔP1 and the remainder of the constructs contain the EMCV IRES instead and are not replication competent. The construction of the plasmids is described in Materials and Methods. ∗, mutations; Fg, Flag sequence; An, poly(A)tail.
FIG. 2
Electron micrographs of membrane alterations in HeLa cells. Cell cultures were transfected with plasmids expressing the following membrane-binding proteins: Cyt _b_5 (a) and PV proteins 3AB (b), 2C (c), 2BC (d), 2C(K135S) (e), and 2C(1–274) (f). Arrowheads indicate the characteristic alterations for each protein: concentric, myelin-like membranes (a and b); vesicles surrounding lipid droplets (b); rigid, extended membrane sheets (c); and vesicles similar to those found during a PV infection (d to f). The micrographs were obtained 9 to 14 h posttransfection. Bars, 500 nm.
FIG. 3
Expression of membrane-binding proteins reduces the susceptibility of cells to a PV infection. (A) Cells were transfected with one of the constructs indicated and superinfected with PV 6 to 7 h later. At 5 h p.i., the cells were subjected to IF with MAb against the Flag epitope of the transfected protein and with an Ab detecting PV (see Table 1). PV-infected cells are indicated as the percentage of the subpopulation of cells which express the indicated protein. (B) The upper and lower panels show the identical area of micrographs of cells transfected with pTM-Fg-2BC and superinfected with PV. (Top) IF performed with MAb against Flag to detect Fg-2BC. (Bottom) IF performed with anti-VPg Ab to detect PV. Most cells productively infected with PV were not expressing 2BC. Solid arrowheads indicate cells infected with PV and not expressing 2BC. Open arrowheads indicate cells expressing 2BC and not infected with PV. The asterisk indicates a cell expressing 2BC and infected with PV. Magnification, ×320.
FIG. 4
Localization of membrane-binding proteins and viral products in transfected, infected cells. HeLa cells expressing membrane-binding proteins Fg-Cyt _b_5 (a to c), Fg-PV 2C(1–274) (d to f), and Fg-PV 2BC (g to m) were superinfected with PV at 6 to 7 h posttransfection. Cells that were both transfected and infected were selected for analysis by CLSM. (a, d, g, and k) IF with anti-Flag MAb and Texas red-labeled secondary Ab to detect the expressed membrane-binding proteins. (b, e, and h) PV replication complex visualized at 5 h p.i. with anti-VPg Ab and FITC-coupled secondary Ab. (l) PV plus-strand RNA detected by ISH with FITC-labeled riboprobe. (c, f, i, and m) Overlay of the corresponding individual reactions: PV replication complex-associated proteins and RNA stay separate from membrane-binding proteins. Each image area is 28 by 35 μm.
FIG. 5
Localization of viral proteins and RNA in transfected HeLa cells. (a to f) Cells transfected with pE5PVΔP1 (a to c) or the replicon pPVΔP1 (d to f) were treated with actinomycin D at 2.5 h posttransfection to stop T7-mediated transcription. 2B and 2BC were visualized by IF and CLSM with anti-2B MAb and Texas red-labeled secondary Ab (a and d). RNA of the transfected construct was localized by ISH with FITC-labeled riboprobe (b and e) at 7 h posttransfection. (c and f) Overlay. Replication complexes, similar to those found in a PV-infected cell, can be formed only with pPVΔP1-derived replicating RNA (f). (g to i) Cells dually transfected with pTM-Fg-2BC for 7 h and supertransfected with pE5PVΔP1 for another 7 h. 2BC-induced vesicles were visualized by IF with anti-Flag MAb and Texas red-labeled secondary Ab (g), and pE5PVΔP1-induced vesicles were visualized with anti-VPg Ab and FITC-labeled secondary Ab (h). (i) Overlay. 2BC- and pE5PVΔP1-induced vesicles, not engaged in RNA-dependent RNA synthesis, mix and associate with each other. Each image area is 26 by 33 μm.
FIG. 6
HeLa cells were transfected with pE5PVΔP1 and superinfected with PV at 6.3 h posttransfection. (A) The number of PV-susceptible cells was found to be reduced compared to a mock-transfected culture. Since pE5PVΔP1 proteins cannot be discriminated from PV proteins, the conclusion that pE5PVΔP1 inhibits PV replication was drawn from counting infected or transfected cells in parallel cultures which were mock transfected and PV infected, transfected with pE5PVΔP1 only, or transfected with pE5PVΔP1 and PV superinfected. n.a., not applicable. (B) The upper picture shows cells transfected with pE5PVΔP1. IF performed with anti-2B MAb showed that 95% of the cells were efficiently transfected. The lower picture shows a parallel culture transfected with pE5PVΔP1 and superinfected with PV. The number of PV-infected cells was determined by IF with anti-VP1 MAb at 5 h p.i. Magnification, ×100.
FIG. 7
Schematic diagram illustrating that the PV replication complex is formed in cis. (A) ER membranes are altered by the expression of membrane-binding proteins, e.g., Cyt _b_5 or PV protein 2C, or all of the PV nonstructural proteins encoded in the plasmid pE5PVΔP1. The altered membranes are not utilized (crossed arrows) in trans for PV replication complex formation during a subsequent PV infection. (B) Replication complex formation as it occurs during PV replication. Membrane-bound translation of viral RNA into protein (step 1) triggers the formation of vesicles on the ER (step 2). Vesicles carrying PV nonstructural proteins and previously translated RNA with initiated minus strand (step 3) form a viral replication complex (vesicular rosette) with replicating RNA in the replicative intermediate (RI) configuration (step 4). Hatched lines, RNA; stippled symbols, viral proteins.
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