Ultrastructure and origin of membrane vesicles associated with the severe acute respiratory syndrome coronavirus replication complex

Abstract

The RNA replication complexes of mammalian positive-stranded RNA viruses are generally associated with (modified) intracellular membranes, a feature thought to be important for creating an environment suitable for viral RNA synthesis, recruitment of host components, and possibly evasion of host defense mechanisms. Here, using a panel of replicase-specific antisera, we have analyzed the earlier stages of severe acute respiratory syndrome coronavirus (SARS-CoV) infection in Vero E6 cells, in particular focusing on the subcellular localization of the replicase and the ultrastructure of the associated membranes. Confocal immunofluorescence microscopy demonstrated the colocalization, throughout infection, of replicase cleavage products containing different key enzymes for SARS-CoV replication. Electron microscopy revealed the early formation and accumulation of typical double-membrane vesicles, which probably carry the viral replication complex. The vesicles appear to be fragile, and their preservation was significantly improved by using cryofixation protocols and freeze substitution methods. In immunoelectron microscopy, the virus-induced vesicles could be labeled with replicase-specific antibodies. Opposite to what was described for mouse hepatitis virus, we did not observe the late relocalization of specific replicase subunits to the presumed site of virus assembly, which was labeled using an antiserum against the viral membrane protein. This conclusion was further supported using organelle-specific marker proteins and electron microscopy. Similar morphological studies and labeling experiments argued against the previously proposed involvement of the autophagic pathway as the source for the vesicles with which the replicase is associated and instead suggested the endoplasmic reticulum to be the most likely donor of the membranes that carry the SARS-CoV replication complex.

FIG. 1.

SARS-CoV replicase polyprotein organization, depicted in the form of the 7,071-amino-acid pp1ab. The border of amino acids encoded in ORF1a and ORF1b is indicated as RFS (ribosomal frameshift), and arrowheads represent sites that are cleaved by the nsp3 PLpro (gray) or the nsp5 Mpro (black). The 16 proteolytic cleavage products (nonstructural proteins) are numbered, and within the cleavage products key replicase domains have been highlighted (see text also). These include putative transmembrane domains (TM) and the four ORF1b-encoded domains (RdRp, Z, Hel, and NendoU) that are conserved in all nidoviruses. Abbreviations, from the N terminus to the C terminus: aa, amino acids; ADRP, ADP-ribose-1′′-monophosphatase; RBD, RNA-binding domains; Z, (putative) zinc-binding domain; Hel, helicase domain; Exo, (putative) exonuclease; MT, (putative) ribose-2′-O-methyltransferase.

FIG. 2.

Time course IF labeling experiment showing the development of SARS-CoV replicase signal in infected Vero E6 cells, as exemplified by labeling for nsp3. The initially punctate cytoplasmic staining (6 h p.i.) develops into a number of densely labeled areas close to the nucleus later in infection (9 and 12 h p.i.). Bar, 10 μm.

FIG. 3.

Confocal IF microscopy analysis of the intracellular distribution of various SARS-CoV replicase subunits in infected Vero E6 cells. (A) Double-labeling experiments (9 h p.i.) using an AF488-coupled IgG fraction purified from an anti-nsp3 serum and antisera recognizing nsp5, nsp12, nsp13, and nsp15. Extensive colocalization of these five nonstructural proteins was observed throughout infection. (B) Double-labeling experiment (9 h p.i.) for SARS-CoV nsp3 and the ERGIC-53 cellular marker protein. (C) Double-labeling experiment (18 h p.i.) for the SARS-CoV nsp13 helicase and the ERGIC-53 cellular marker protein, illustrating the complete separation of the nsp13 and the ERGIC at late time points in infection. (D) Double-labeling experiment (6 h p.i.) for SARS-CoV nsp3 and the viral M protein, which localizes to the Golgi complex at this time point. (E) Labeling for the SARS-CoV M protein at 9 h p.i., showing the spread of the protein throughout the cytoplasm, presumably due to the traffic of progeny virions towards the plasma membrane. Insets illustrate the strong labeling of the region just beneath the plasma membrane. (F) Double-labeling experiment (18 h p.i.) for SARS-CoV nsp3 and M protein, confirming the almost-complete separation of the two proteins also at late time points in infection. (G) Double-labeling experiment (18 h p.i.) using an AF488-coupled IgG fraction purified from an anti-nsp3 serum and an antiserum recognizing nsp13, illustrating the colocalization of the two proteins also at late stages of infection. In general, late in infection, the nsp13 signal was found to decline more rapidly than that of nsp3, suggesting differences in turnover of these two proteins. Bar, 10 μm.

FIG. 4.

EM analysis of SARS-CoV-infected Vero E6 cells (panels A and B, 6 h p.i.; panel C, 9 h p.i.) fixed using conventional chemical fixation and embedded in epoxy LX-112 resin. (A) Low-magnification overview of a cluster of virus-induced vesicles in the perinuclear region of the cell (N, nucleus), which is also rich in mitochondria (M). Whereas other membranes, like those of mitochondria, were generally well preserved, the virus-induced vesicles were quite electron lucent and the surrounding membranes were poorly visible. (B) Virus-induced vesicles were often observed to occur in association with the ER or inside the lumen of the (dilated) ER (arrow). (C) Close-up of virus-induced vesicles, showing their electron-lucent interior with a spider web-like content. Only occasionally, a part of a surrounding double membrane was observed (arrow). The images presented in this figure illustrate the poor conservation of the virus-induced vesicles when standard procedures for fixation and embedment were used. Bar, 250 nm.

FIG. 5.

EM analysis of SARS-CoV-infected Vero E6 cells (panels A, B, D, and E, 9 h p.i.; panel C, 6 h p.i.) cryofixed by high-speed plunge freezing in liquid ethane, a step followed by freeze substitution with 1% osmium tetroxide and 0.5% uranyl acetate in acetone and embedment in epoxy LX-112 resin. (A) Low-magnification overview of a region rich in virus-induced DMVs (arrows) and mitochondria (M). The interior of the virus-induced vesicles was strikingly different from that in the images presented in Fig. 4, and clear double membranes were now found to surround the structures. (B) Close-up of virions outside of the cell, with the spikes on the virion surface illustrating the general high quality of samples prepared using cryofixation. (C) Close-up of virus-induced DMVs, showing the double membrane of the structure and the high electron density of the interior compared to those shown in Fig. 4C. (D) Example of apparent continuity (arrow) between the outer membrane of a DMV and a mitochondrion (M), as was occasionally observed. (E) Example of a possible intermediate (arrow) in DMV formation, reminiscent of the previously proposed “protrusion and detachment” model (40). Bars, 250 nm (A, C, D, and E) and 100 nm (B).

FIG. 6.

IEM analysis of SARS-CoV-infected Vero E6 cells (panels A, B, and D, 9 h p.i.; panel C, 6 h p.i.). Ultrathin cryosections of chemically fixed, SARS-CoV-infected Vero E6 cells were used for immunogold-labeling experiments. Although this protocol was not compatible with the preservation of the interior of DMV-infected cells, many virus-induced vesicles were observed. (A) Cluster of irregularly shaped vesicles in the perinuclear area, which again also contained many mitochondria (M). The boundary of the structures could be labeled specifically using the αnsp3 serum and protein A-gold (15 nm). (B) Higher magnification of structures as shown in panel A but now labeled with the antiserum directed against the viral helicase (αnsp13). (C) Example of ER stacks double positive for nsp13 (visualized using 15-nm gold; arrows) and the cellular protein PDI (visualized using 10-nm gold; arrowheads). (D) Double labeling using the αnsp13 serum (visualized using 10-nm gold; arrowheads) and the αM serum (visualized using 15-nm gold). The αM serum labeled the Golgi area on the infected cell and new virus particles but did not label the vesicles that were positive for nonstructural proteins (and vice versa for the αnsp13 serum). Bar, 250 nm.

FIG. 7.

IF microscopy analysis of the overlap between autophagosomes (visualized by means of the LC3 marker protein) and the SARS-CoV RC in infected Vero E6 cells (9 h p.i.). (A) Staining of GFP-LC3B-expressing, transfected Vero E6 cells with the αLC3B rabbit antiserum that was raised using an N-terminal synthetic peptide (see Materials and Methods). (B) IF double-labeling analysis showing a (relatively rare) example of a SARS-CoV-infected cell with a convincing LC3B labeling pattern which is clearly distinct from the staining for the viral replication complex (nsp3). (C to E) Staining of pGFP-LC3A, -LC3B, -LC3C-transfected and SARS-CoV-infected Vero E6 cells, showing complete separation of compartments positive for GFP-LC3A, GFP-LC3B, or GFP-LC3C and structures carrying the viral replication complex (stained with αnsp3). (F) Staining of pLAMP-GFP-transfected and SARS-CoV-infected Vero E6 cells, showing complete separation of compartments positive for LAMP1-GFP and structures carrying the viral replication complex (stained with αnsp3). Bar, 10 μm.