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. 2007 May 10;361(2):304-15.
doi: 10.1016/j.virol.2006.11.027. Epub 2007 Jan 8.

The intracellular sites of early replication and budding of SARS-coronavirus

Silke Stertz  1 , Mike Reichelt, Martin Spiegel, Thomas Kuri, Luis Martínez-Sobrido, Adolfo García-Sastre, Friedemann Weber, Georg Kochs
Affiliations

The intracellular sites of early replication and budding of SARS-coronavirus

Silke Stertz et al. Virology. .

Abstract

In this study, we analyzed the replication and budding sites of severe acute respiratory syndrome coronavirus (SARS-CoV) at early time points of infection. We detected cytoplasmic accumulations containing the viral nucleocapsid protein, viral RNA and the non-structural protein nsp3. Using EM techniques, we found that these putative viral replication sites were associated with characteristic membrane tubules and double membrane vesicles that most probably originated from ER cisternae. In addition to its presence at the replication sites, N also accumulated in the Golgi region and colocalized with the viral spike protein. Immuno-EM revealed that budding occurred at membranes of the ERGIC (ER-Golgi intermediate compartment) and the Golgi region as early as 3 h post infection, demonstrating that SARS-CoV replicates surprisingly fast. Our data suggest that SARS-CoV establishes replication complexes at ER-derived membranes. Later on, viral nucleocapsids have to be transported to the budding sites in the Golgi region where the viral glycoproteins accumulate and particle formation occurs.

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Figures

Fig. 1
Fig. 1
Distribution of SARS-CoV N, SARS-CoV nsp3 and SARS-CoV S at early time points of infection. Vero cells were either mock-infected (a, d, g) or infected with SARS-CoV for 3 h (b, e, h) or 5 h (c, f, i) with an MOI of 10. Cells were fixed, permeabilized and analyzed by immunofluorescence using antibodies directed against SARS-CoV N (a–c), SARS-CoV nsp3 (d–f) or SARS-CoV S protein (g–i). Scale bars represent 10 μm.
Fig. 2
Fig. 2
Cytoplasmic accumulations of SARS-CoV N colocalize with nsp3 and viral genomic RNA; perinuclear N colocalizes with S and a Golgi marker. (a–c) Vero cells were infected with SARS-CoV (MOI of 10) for 3 h, fixed and stained with specific antibodies for SARS-CoV nsp3 (a, green) and N (b, red) in parallel. (d–f) Vero cells were infected with SARS-CoV (MOI of 10) for 5 h, fixed and in situ hybridization was performed with a FITC-labeled RNA probe of negative polarity directed against the nsp1-ORF (d, green). After in situ hybridization, immunofluorescence staining was performed with an antibody specific for SARS-CoV N (e, red). (g–l) Vero cells were infected with SARS-CoV (MOI of 10) for 5 h, fixed, permeabilized and stained with antibodies specific for SARS-CoV S or Giantin, a marker for cis-Golgi, (g, j, green) and SARS-CoV N (h, k, red). Areas of colocalization appear in yellow in the merged images (c, f, i, l). Scale bars represent 10 μm.
Fig. 3
Fig. 3
Budding of SARS-CoV occurs in the ERGIC and Golgi area already at 3 h p.i. (a–d) Vero cells were mock-infected (a) or infected with SARS-CoV (MOI of 10) for 3 h (b) or 5 h (c, d), fixed and analyzed by standard EM. The nuclei are marked with “Nu” and the Golgi with “G”. Viral particles are highlighted with arrows (b–d). Scale bars represent 200 or 500 nm as indicated. (e, f) Vero cells were infected with SARS-CoV (MOI of 10) for 3 h, fixed and analyzed by cryo-iEM with antibodies directed against SARS-CoV N (10 nm gold, filled arrowheads) and β′COP-I as a marker for the ERGIC and Golgi area (15 nm gold, open arrowheads). Budding profiles are marked with small arrows, large arrows point to viral particles. Scale bars represent 100 nm.
Fig. 4
Fig. 4
Ultrastructural analysis of the putative SARS-CoV replication sites. (a, b) Vero cells were mock-infected (a) or infected with SARS-CoV (MOI of 10) for 3 h (b), fixed and analyzed by standard EM. Double membrane vesicles characteristic for SARS-CoV replication sites are highlighted with arrows. (c–e) Vero cells were infected with SARS-CoV (MOI of 10) for 3 h, fixed and analyzed by cryo-iEM. Cells were labeled with antibodies directed against SARS-CoV N (15 nm gold, filled arrowheads) and SARS-CoV nsp3 (5 nm gold, open arrowheads). Areas of colocalization of N and nsp3 are shown in panels c and d. Membrane tubules specific for these sites are marked with arrows. Sites where viral particles (marked with stars) were present only contained N labeling but almost no nsp3 labeling (e). Scale bars represent 200 or 500 nm as indicated.
Fig. 5
Fig. 5
Membrane tubules within the SARS-CoV replication sites are derived from ER membranes. A) Vero cells were mock-infected (a) or infected with SARS-CoV (MOI of 10) for 3 h (b, c), fixed and analyzed by standard EM. ER cisternae are marked with large arrows, whereas the double membrane vesicles and the membrane tubules characteristic for the putative replication sites are highlighted with small arrows. Scale bars represent 200 nm or 500 nm as indicated. B) Vero cells were mock-infected (a) or infected with SARS-CoV (MOI of 10) for 3 h (b–e), fixed and analyzed by cryo-iEM. Cells were labeled with antibodies directed against SARS-CoV N (a–c, 10 nm gold, filled arrowheads) or SARS-CoV nsp3 (d–e, 15 nm gold, filled arrowheads) and PDI as a marker for ER (a–e, 5 nm gold, open arrowheads). Panel B c and e show putative replication complexes in a higher magnification. PDI labeling surrounded the N assembly in c but is not visible in this cutout. ER cisternae are marked with large arrows (a, b) and small arrows delineate membrane tubules characteristic for the putative replication sites (b, c). Scale bars represent 200 nm.
Fig. 5
Fig. 5
Membrane tubules within the SARS-CoV replication sites are derived from ER membranes. A) Vero cells were mock-infected (a) or infected with SARS-CoV (MOI of 10) for 3 h (b, c), fixed and analyzed by standard EM. ER cisternae are marked with large arrows, whereas the double membrane vesicles and the membrane tubules characteristic for the putative replication sites are highlighted with small arrows. Scale bars represent 200 nm or 500 nm as indicated. B) Vero cells were mock-infected (a) or infected with SARS-CoV (MOI of 10) for 3 h (b–e), fixed and analyzed by cryo-iEM. Cells were labeled with antibodies directed against SARS-CoV N (a–c, 10 nm gold, filled arrowheads) or SARS-CoV nsp3 (d–e, 15 nm gold, filled arrowheads) and PDI as a marker for ER (a–e, 5 nm gold, open arrowheads). Panel B c and e show putative replication complexes in a higher magnification. PDI labeling surrounded the N assembly in c but is not visible in this cutout. ER cisternae are marked with large arrows (a, b) and small arrows delineate membrane tubules characteristic for the putative replication sites (b, c). Scale bars represent 200 nm.
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