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. 2014 Jun:458-459:125-35.
doi: 10.1016/j.virol.2014.04.027. Epub 2014 May 13.

Membrane rearrangements mediated by coronavirus nonstructural proteins 3 and 4

Affiliations

Membrane rearrangements mediated by coronavirus nonstructural proteins 3 and 4

Marne C Hagemeijer et al. Virology. 2014 Jun.

Abstract

Coronaviruses replicate their genomes in association with rearranged cellular membranes. The coronavirus nonstructural integral membrane proteins (nsps) 3, 4 and 6, are key players in the formation of the rearranged membranes. Previously, we demonstrated that nsp3 and nsp4 interact and that their co-expression results in the relocalization of these proteins from the endoplasmic reticulum (ER) into discrete perinuclear foci. We now show that these foci correspond to areas of rearranged ER-derived membranes, which display increased membrane curvature. These structures, which were able to recruit other nsps, were only detected when nsp3 and nsp4 were derived from the same coronavirus species. We propose, based on the analysis of a large number of nsp3 and nsp4 mutants, that interaction between the large luminal loops of these proteins drives the formation of membrane rearrangements, onto which the coronavirus replication-transcription complexes assemble in infected cells.

Keywords: Coronavirus; Membrane rearrangements; Nsps; Replication–transcription complex; Transmembrane proteins.

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Figures

Fig. 1
Fig. 1
Overview of CoV nsp3C and nsp4 constructs used in this study. (A) A schematic representation of the membrane topology of the C-terminal part of nsp3 (nsp3C), and of nsp4. The approximate location of the cysteine residues (*) and N-glycosylation sites (-«) in the large luminal loops are indicated, the latter either in gray (SARS-CoV) or in black (MHV-A59). (B) Schematic representation of nsp4 hybrid proteins. Nsp4 sequences derived from MHV or SARS-CoV are indicated by black (MHV) or red (SARS-CoV) lines or rectangles. Rectangles indicate the approximate location of transmembrane domains. Nsp4SM contains the amino-terminal half of SARS-CoV, while its carboxy terminus is derived from MHV. Nsp4MS refers to the reciprocal hybrid protein. (C) Schematic representation of the MHV nsp3 and nsp4 deletion mutants used in this study. Rectangles indicate transmembrane domains and the blue lines indicate either the luminal loop of nsp3 or the large luminal loop of nsp4.
Fig. 2
Fig. 2
Analysis of colocalization of cellular markers with nsp3C- and nsp4-positive foci. OST7-1 cells (co)-expressing nsp3C-GFP (green) and/or nsp4-mCherry (red) were stained with antibodies directed either against PDI, GM130, EDEM1, or LC3 (blue). Representative confocal microscopy images are shown.
Fig. 3
Fig. 3
Immunogold labeling of nsp3C and nsp4 at the induced membrane rearrangements. Immuno-gold labeling of cryo-sections from OST7-1 cells expressing nsp3C-GFP (A and A′), nsp4-mCherry (B and B′) or co-expressing nsp3C-GFP and nsp4-mCherry (C, C′,D and D′). Binding of antibodies directed against the GFP (A, A′, C and C′) or the mCherry (B, B′, D and D′) was visualized by protein A-gold conjugates. ER: endoplasmic reticulum; M: mitochondria; L: lysosome. Scale bar, 200 nm.
Fig. 4
Fig. 4
Nsp3C and nsp4 interact and induce membrane rearrangements in a virus-specific manner. (A) Fluorescence-lifetime imaging microscopy (FLIM) of OST7-1 cells expressing GFP fusion proteins of nsp3C (derived either from SARS-CoV [nsp3S] or MHV A59 [nsp3M]) either alone or in combination with mCherry fusion proteins of nsp4 (derived either from SARS-CoV [nsp4S] or MHV A59 [nsp4M]). Fluorescent lifetimes of GFP normalized against the lifetime of GFP when expressed alone are displayed. (B) Nsp3C-GFP and nsp4-mCherry fusion proteins derived from MHV and SARS-CoV were co-expressed in OST7-1 cells in different combinations as indicated. Representative confocal microscopy images are shown.
Fig. 5
Fig. 5
Induction of membrane rearrangements by nsp3C and nsp4 hybrid proteins. Representative confocal images of OST7-1 cells expressing nsp4-mCherry hybrid proteins (see Fig. 1B) together with nsp3C-GFP derived from either MHV or SARS-CoV. The presence or absence of membrane rearrangements is indicated by the ‘+’ and ‘−’ signs, respectively.
Fig. 6
Fig. 6
Induction of membrane rearrangements by nsp4 mutant proteins. (A) Full-length and truncated forms of MHV nsp4-mCherry (see Fig. 1C for details) were co-expressed in OST7-1 cells together with MHV nsp3C–GFP. The presence or absence of membrane rearrangements is indicated by the ‘+’ and ‘−’ signs, respectively. ‘+/−’ refers to an intermediate phenotype. (B) Mutant forms of MHV nsp4-mCherry, each carrying a cysteine to serine substitution in the large luminal loop, were analyzed for their ability to induce membrane rearrangements when co-expressed with MHV nsp3C-GFP (Fig. S1). Co-expression of nsp4-mCherry C233S with nsp3C-GFP is shown as representative example.
Fig. 7
Fig. 7
Recruitment of MHV nsps to the nsp3C- and nsp4-induced membrane rearrangements. GFP-tagged MHV nsp proteins (green) were expressed singly (single) or together (co-expression) with MHV nsp3C-HA (blue) and MHV nsp4-mCherry (red) in OST7-1 cells. Expression of nsp3-HA was visualized using antibodies against the HA tag. Representative confocal images are shown.
Fig. 8
Fig. 8
Model for the interplay between nsp3 and nsp4 in the formation of the CoV replicative structures. The rearrangement of cellular membranes into CoV replicative structures requires the concerted action of both viral and host proteins. We propose that among others the virus contributes to these rearrangements via the expression of nsp3 and nsp4, which interact via their large luminal loops. This interaction “zippers” the ER membranes and induces membrane curvature, two likely requirements for the formation of DMVs that are observed in CoV-infected cells.

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