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. 2002 Nov;76(21):11113-22.
doi: 10.1128/jvi.76.21.11113-11122.2002.

Differential requirements for COPI coats in formation of replication complexes among three genera of Picornaviridae

Elena V Gazina  1 , Jason M Mackenzie, Rebecca J Gorrell, David A Anderson
Affiliations

Differential requirements for COPI coats in formation of replication complexes among three genera of Picornaviridae

Elena V Gazina et al. J Virol. 2002 Nov.

Abstract

Picornavirus RNA replication requires the formation of replication complexes (RCs) consisting of virus-induced vesicles associated with viral nonstructural proteins and RNA. Brefeldin A (BFA) has been shown to strongly inhibit RNA replication of poliovirus but not of encephalomyocarditis virus (EMCV). Here, we demonstrate that the replication of parechovirus 1 (ParV1) is partly resistant to BFA, whereas echovirus 11 (EV11) replication is strongly inhibited. Since BFA inhibits COPI-dependent steps in endoplasmic reticulum (ER)-Golgi transport, we tested a hypothesis that different picornaviruses may have differential requirements for COPI in the formation of their RCs. Using immunofluorescence and cryo-immunoelectron microscopy we examined the association of a COPI component, beta-COP, with the RCs of EMCV, ParV1, and EV11. EMCV RCs did not contain beta-COP. In contrast, beta-COP appeared to be specifically distributed to the RCs of EV11. In ParV1-infected cells beta-COP was largely dispersed throughout the cytoplasm, with some being present in the RCs. These results suggest that there are differences in the involvement of COPI in the formation of the RCs of various picornaviruses, corresponding to their differential sensitivity to BFA. EMCV RCs are likely to be formed immediately after vesicle budding from the ER, prior to COPI association with membranes. ParV1 RCs are formed from COPI-containing membranes but COPI is unlikely to be directly involved in their formation, whereas formation of EV11 RCs appears to be dependent on COPI association with membranes.

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Figures

FIG. 1.
FIG. 1.
Time course of EV11, ParV1, and EMCV production. BS-C-1 cells were infected with EV11 (A), ParV1 (B), or EMCV (C) at an MOI of 3 and treated with 10-μg/ml BFA starting from 1 h p.i. (▴) or left untreated (○). Cells were harvested into culture medium at indicated times, and virus yield was measured by plaque assay.
FIG. 2.
FIG. 2.
Time course of EV11, ParV1, and EMCV RNA synthesis. BS-C-1 cells were infected with each virus at MOI of 3 and labeled with [5,6-3H]uridine starting from 1 h p.i. Cytoplasmic RNA was extracted from the cells at indicated times (hours) after infection and analyzed by formaldehyde-agarose gel electrophoresis and autoradiography. The positions of viral (V) and cellular ribosomal (28S and 18S) RNAs are marked on the right. M, RNA extracted from mock-infected cells labeled with [5,6-3H]uridine for 10 h.
FIG. 3.
FIG. 3.
Distribution of dsRNA in BS-C-1 cells infected with EMCV, ParV1, or EV11 in the absence or presence of BFA. Cells were infected with EMCV (A and B), ParV1 (C and D), or EV11 (E and F) at an MOI of 3 and treated with 10-μg/ml BFA starting from 1 h p.i. (B, D, and F) or left untreated (A, C, and E). The cells were fixed at 7.5, 6, and 5.5 h p.i. for EMCV, ParV1, and EV11, respectively, and immunostained with anti-dsRNA antibody and Alexa Fluor 488 conjugate.
FIG. 4.
FIG. 4.
Morphology of the replication complexes of EMCV, ParV1 and EV11. BS-C-1 cells were infected with EMCV (A and D), ParV1 (B and E), or EV11 (C and F) at an MOI of 3; incubated for 7.5, 6, or 5.5 h, respectively; and then harvested and fixed for electron microscopy. (A to C) Cells were embedded in Epon resin, and ultrathin sections were cut and stained with uranyl acetate and lead citrate. All viruses induced the clustering of vesicles in the perinuclear region indicative of picornavirus RCs. Nu, nucleus. Bars, 1 μm. (D to F) Ultrathin cryosections of infected cells were immunolabeled with anti-dsRNA antibody and visualized with either 5-nm anti-IgG gold (D) or 10-nm protein A-gold (E and F). (D) In EMCV-infected cells, labeling of dsRNA was localized to electron-dense structures (indicated by arrows) within the clusters of heterogeneous vesicles. (E) ParV1-infected cells were labeled on clusters of homogeneous vesicles ranging in size between 70 and 100 nm. The anti-dsRNA antibody appeared to specifically label the membrane of the vesicles. (F) EV11-infected cells displayed labeling of dsRNA within the clusters of heterogeneous vesicles, associated with electron-dense structures (indicated by arrows), as for EMCV, and also with the membrane of the vesicles (indicated by arrowheads). Bars, 200 nm.
FIG. 5.
FIG. 5.
The distribution of β-COP and dsRNA in cells infected with EMCV, ParV1, and EV11 visualized by confocal IF microscopy. Cells were infected as in Fig. 4, then fixed and double-labeled with anti-dsRNA and anti-β-COP antibodies. (A, D, and G) Staining with anti-β-COP antibody and Alexa Fluor 568 conjugate (red). (B, E, and H) Staining with anti-dsRNA antibody and Alexa Fluor 488 conjugate (green). (C, F, and I) Merge of first two columns; the sites of colocalization of the two antibodies are highlighted in yellow. (A to C) EMCV-infected cells; no colocalization. (D to F) ParV1-infected cells; strong reduction in anti-β-COP staining is obvious in comparison with an uninfected cell present in this sample. (G to I) EV11-infected cells; partial colocalization of the two antibodies.
FIG. 6.
FIG. 6.
β-COP is present within the RCs of ParV1 and EV11 but is absent from the RCs of EMCV. Cells were infected as in Fig. 4, then harvested, processed for cryosectioning, and double-immunolabeled with anti-dsRNA (5-nm gold conjugate) and anti-β-COP (10-nm gold conjugate) antibodies. In EMCV-infected cells (A and B) the two markers (large arrowheads indicating β-COP and small arrowheads indicating dsRNA) appeared distinct from each other. ParV1 (C and D)- and EV11 (E)-infected cells showed coincidental labeling as indicated by the arrows. In panel E, anti-β-COP and anti-dsRNA antibodies are bound to a vesicular structure budding from a membrane in close proximity to the ER. (F) Both anti-β-COP (large arrowhead) and anti-dsRNA (small arrowhead) antibodies within an EV11 RC, although not very close to each other. Bars, 200 nm.
FIG. 7.
FIG. 7.
Distribution of β-COP and dsRNA in the cells at early times in EMCV, ParV1, and EV11 infections. Cells were infected with EMCV (A to C), ParV1 (D to F), or EV11 (G to I) at an MOI of 3. The infected cells were fixed at 5 h p.i. (EMCV) or 4 h p.i. (ParV1 and EV11), double-labeled with anti-dsRNA and anti-β-COP antibodies, and visualized by confocal IF microscopy. (A, D, and G) Staining with anti-β-COP antibody and Alexa Fluor 568 conjugate (red). (B, E, and H) Staining with anti-dsRNA antibody and Alexa Fluor 488 conjugate (green). (C, F, and I) Merge of first two columns; the sites of colocalization of the two antibodies are highlighted in yellow.
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