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.

Ultrastructural and biochemical analyses of hepatitis C virus-associated host cell membranes

“…We demonstrate here that the replication of a subgenomic replicon of the HCV JFH-1 strain induces numerous 200-500 nm wide DMVs (Fig. 1a-c), similar to those described for poliovirus (Schlegel et al, 1996), severe acute respiratory syndrome (SARS) coronavirus (Snijder et al, 2006) and coxsackievirus B3 (Wong et al, 2008). We also identified some DMVs on EM sections of cells harbouring a subgenomic HCV 1b replicon but in much smaller numbers.…”

“…In the SARS virus model these virus-induced, ER-derived vesicles seem to have a thick, dense membrane. However, careful EM examination has revealed the presence of double membrane (Snijder et al, 2006), a feature also found in the HCV-induced vesicles. These DMVs were initially thought to carry the SARS virus replication complexes (Snijder et al, 2006) but more recent studies have clearly demonstrated that DMVs contain some non-structural proteins, mostly dsRNA, and that their contents do not appear to be connected to the cytoplasm (Knoops et al, 2008).…”

“…However, careful EM examination has revealed the presence of double membrane (Snijder et al, 2006), a feature also found in the HCV-induced vesicles. These DMVs were initially thought to carry the SARS virus replication complexes (Snijder et al, 2006) but more recent studies have clearly demonstrated that DMVs contain some non-structural proteins, mostly dsRNA, and that their contents do not appear to be connected to the cytoplasm (Knoops et al, 2008). These findings led to suggestions that DMVs may help to conceal the viral RNA by enabling the virus to evade the dsRNAtriggered antiviral responses of the host such as those mediated by the dsRNA-dependent protein kinase (Knoops et al, 2008).…”

“…However, careful examination at high magnification revealed the presence of a double membrane in some areas suggesting a tight association of the two membranes in these vesicles (inset in Fig. 1a, b), as reported for similar structures in cells infected with coronavirus (Snijder et al, 2006). We estimated the frequency of these DMVs by determining the number of these vesicles in 60 consecutive cell sections for Huh7.5-C5 and Huh7.5 cells.…”

The SARS-Unique Domain (SUD) of SARS Coronavirus Contains Two Macrodomains That Bind G-Quadruplexes

“…Coronavirus replication in the perinuclear region of the cell is localized to double-membrane vesicles that have been hijacked from the endoplasmic reticulum or late endosomes [37][38][39][40]. These vesicles are around 200-350 nm in diameter and present alone or as clusters in the cytosol [38].…”