Membrane alterations induced by nonstructural proteins of human norovirus

Abstract

Human noroviruses (huNoV) are the most frequent cause of non-bacterial acute gastroenteritis worldwide, particularly genogroup II genotype 4 (GII.4) variants. The viral nonstructural (NS) proteins encoded by the ORF1 polyprotein induce vesical clusters harboring the viral replication sites. Little is known so far about the ultrastructure of these replication organelles or the contribution of individual NS proteins to their biogenesis. We compared the ultrastructural changes induced by expression of norovirus ORF1 polyproteins with those induced upon infection with murine norovirus (MNV). Characteristic membrane alterations induced by ORF1 expression resembled those found in MNV infected cells, consisting of vesicle accumulations likely built from the endoplasmic reticulum (ER) which included single membrane vesicles (SMVs), double membrane vesicles (DMVs) and multi membrane vesicles (MMVs). In-depth analysis using electron tomography suggested that MMVs originate through the enwrapping of SMVs with tubular structures similar to mechanisms reported for picornaviruses. Expression of GII.4 NS1-2, NS3 and NS4 fused to GFP revealed distinct membrane alterations when analyzed by correlative light and electron microscopy. Expression of NS1-2 induced proliferation of smooth ER membranes forming long tubular structures that were affected by mutations in the active center of the putative NS1-2 hydrolase domain. NS3 was associated with ER membranes around lipid droplets (LDs) and induced the formation of convoluted membranes, which were even more pronounced in case of NS4. Interestingly, NS4 was the only GII.4 protein capable of inducing SMV and DMV formation when expressed individually. Our work provides the first ultrastructural analysis of norovirus GII.4 induced vesicle clusters and suggests that their morphology and biogenesis is most similar to picornaviruses. We further identified NS4 as a key factor in the formation of membrane alterations of huNoV and provide models of the putative membrane topologies of NS1-2, NS3 and NS4 to guide future studies.

Fig 1. Ultrastructure of MNV infected Huh7-CD300lf cells.

Huh7-CD300lf cells were infected (A, B) or mock infected (C) with MNV for 16 h (MOI = 1) before being chemically fixed and subjected to EM. Boxed and numbered areas are shown in higher magnification in subsequent panels. SMV, single membrane vesicle; DMV, double membrane vesicle; MMV, multi membrane vesicle; LD, lipid droplet. Note that SMVs are separated from the surrounding cytosol by a unique lipid bilayer. DMVs are delimited from the cytosol via two lipid bilayers and have a diameter below 300 nm. DMVs have normally a more electron-dense content than SMVs, likely due to engulfment of cytosolic content. MMVs contain more than 2 membranes. Yellow arrowheads indicate virions (Vi). White scale bars, 5 μm. Red scale bars, 200 nm.

Fig 2. Expression model used in this study and ultrastructural analysis of membrane alterations induced by MNV ORF1 expression.

(A) Schematic representation of the ORF1 polyprotein of MNV (top) and huNoV GII.4, indicating alternative nomenclature for the non-structural proteins (NS). The terminology in the boxes is used throughout this study. (B) Schematic representation of the protein expression model used in this study. Huh7 cells stably expressing T7-RNA polymerase are transfected with plasmids encoding ORF1 under transcriptional control of a T7 promoter and translational control of an EMCV IRES element, indicated by a secondary structure. Cytoplasmic expression of ORF1 proteins induces membrane alterations similar to infected cells in absence of RNA replication, indicated by a crossed box. (C) Huh7 T7 cells transfected with a construct expressing MNV ORF1 were processed for analysis by transmission EM (TEM) 20 h p.t. upon chemical fixation (CF) or high pressure freezing (HPF). Samples designated CF were grown on glass coverslips, fixed chemically with aldehydes, post-fixed with heavy metals, dehydrated with ethanol and resin embedded for further TEM analysis. Samples designated HPF were grown on sapphire discs, cryo-fixed by high pressure freezing, freeze substituted with heavy metals dissolved in acetone, dehydrated with acetone and resin embedded (reviewed in [97]). Representative images of both fixation techniques are shown as specified on the left. Magnified views of the boxed areas are shown to the right and on top. Single membrane vesicles (SMVs), double membrane vesicles (DMVs), multi-membrane vesicles (MMVs), lipid droplets (LDs) and mitochondria (m) are indicated in the high magnification views.

Fig 3. Ultrastructural analysis of pTM ORF1 expression.

(A) Huh7 T7 cells transfected with a construct expressing NO ORF1 were processed for analysis by transmission EM 20 h p.t., using HPF and CF, as indicated (for further detail see the legend to Fig 2C). Double membrane vesicles (DMVs), multi membrane vesicles (MMVs) and lipid droplets (LDs) are indicated. (B) Comparison of DMV sizes induced by ORF1 expression of the New Orleans (NO), Den Haag (DH) and Sydney (Syd) strains, using both fixation protocols. The diameter of 60 DMVs for each condition was determined. Mean values and SEM are shown in violet. (C) Relative abundance of SMVs, DMVs and MMVs in cells expressing ORF1 of the different GII.4 isolates or MNV and in MNV infected cells after chemical fixation. ORF1 expressing cells and MNV infected cells were identified by the appearance of typical virus induced membrane structures and all vesicles in the respective cell sections were classified and counted. Mean values and SD from at least six cells per condition. For clarity, error bars are shown below the column for SMVs and above the column for DMVs and MMVs.

Fig 4. Electron tomography of vesicle clusters induced by ORF1 of the NO strain.

(A) 3D rendering of a dual-axis tomogram of S1 Movie showing ER cisternae (in dark brown), a cluster of single (in white) and multi membrane (in blue) vesicles, and a double membrane vesicle (in yellow). A tomographic xy slice of the yellow dashed area containing the cluster of vesicles is shown in (1). Several virtual slices extracted from the tomogram of the green dashed area are shown in (2). Note the continuity of the DMV membranes with the ER membrane (pink arrowheads). (B) Serial single xy slices through the tomogram shown in S2 Movie revealing the different membrane numbers of the vesicles within the cluster: from one lipid bilayer (SMVs) to two (DMVs) or more than two (MMVs). (1) Left: 3D rendering of the same tomogram showing a cluster of single (in white) and multi-membrane (in blue) vesicles in close proximity to late endosomes (in red) and a microtubule (in green). Right: Higher magnification picture of the cluster of vesicles. (2) MMVs are composed of several single membrane tubules that close up around a single membrane vesicle. rER, rough endoplasmic reticulum; SMV, single membrane vesicle; DMV, double membrane vesicle; MMV, multi membrane vesicle; MVB, multivesicular body. Note that we defined endosomes or MVBs as rounded organelles delimited from the cytosol by one lipid bilayer, containing a highly heterogeneous lumen composed of multiple vesicles with different sizes and electron densities. ALSs were defined as rounded organelles having two lipid bilayers that separate them from the cytosol and a diameter larger than 300 nm. Their lumen, in contrast to MVBs, was only composed of cytosolic content and/or one engulfed vesicle. We cannot rule out, however, that ALSs are larger DMVs.

Fig 5. Subcellular localization of NS1-2 analyzed by IF and CLEM.

A plasmid encoding eGFP-NS1-2 (green) was transfected into Huh7-T7 cells. Twenty hours post transfection cells were fixed and analyzed by confocal microscopy (A, B) or CLEM (D). Cellular markers (red) were stained using monoclonal or polyclonal antibodies, LDs were stained with LipidTox and Mitochondria were stained with Mitotracker. A white and a yellow asterisk mark a cell with a focal and an intermediate focal/filamentous distribution of NS1-2, respectively. Mito, mitochondria. (B) Pearson correlation of the eGFP-NS1-2 signal with different cellular markers shown in (A). Each dot represents a single cell. (C) HA-NS1-2 was expressed in Huh7-T7 cells and detected by immunofluorescence using a HA-specific antibody (red). Nuclei were counterstained with DAPI (right panel, blue). (D) For CLEM, cells were seeded onto gridded coverslips, fixed and subjected to optical sectioning using a confocal microscope. Maximum-intensity Z-projection of a selected cell is shown on the top. eGFP-NS1-2 signal is depicted in green, LDs in red and the nucleus (DAPI) in blue. Samples were subsequently processed for electron microscopy by using the coordinates etched onto the surface of the gridded coverslips to record the position of the selected cells. The correlated panel was obtained by re-orientation and superimposition of light and electron micrographs as described in M&M. Boxed and numbered areas are shown in higher magnification in subsequent panels. White scale bars, 5 μm. Yellow scale bar, 1 μm. Red scale bars, 200 nm.

Fig 6. Subcellular localization of eGFP-NS3 analyzed by IF and CLEM.

A plasmid encoding eGFP-NS3 (green) was transfected into Huh7-T7 cells. Twenty hours post transfection, cells were fixed and analyzed by confocal microscopy (A) or CLEM (B). (A) Cellular markers are depicted in red and eGFP-NS3 in green in the merge panels. Mito, mitochondria; Autophag., autophagosome. (B) Huh7-T7 cells expressing eGFP-NS3 were processed for CLEM. For further details see the legend to Fig 5D and M&M. Maximum-intensity Z-projection of selected cell is shown on the top. The eGFP-NS3 signal is depicted in green, lipid droplets (LD) in red and the nucleus (DAPI) in blue. Boxed and numbered areas are shown in higher magnification in subsequent panels. CM, Convoluted Membranes. White scale bars, 5 μm. Yellow scale bar, 1 μm. Red scale bars, 200 nm. (C) Pearson correlation of the eGFP-NS3 signal with different cellular markers shown in (A). Each dot represents a single cell.

Fig 7. Subcellular localization of eGFP-NS4 analyzed by IF and CLEM.

A plasmid encoding eGFP-NS4 (green) was transfected into Huh7-T7 cells. Twenty hours post transfection cells were fixed and analyzed by confocal microscopy (A) or CLEM (B-C). (A) Cellular markers are shown in red and eGFP-NS4 in green in the merge panels. Pearson correlation of the eGFP-NS4 signal with different cellular markers is given below, each dot representing a single cell. Mito, mitochondria. (B-C) Huh7-T7 cells expressing eGFP-NS4 were processed for CLEM. For further details see the legend to Fig 5D and M&M. eGFP-NS4 signal is depicted in green, lipid droplets (LD) in red and the nucleus (DAPI) in blue. Boxed and numbered areas are shown in higher magnification in subsequent panels. CM, convoluted membranes; SMV, single membrane vesicle; DMV, double membrane vesicle. White scale bars, 5 μm. yellow scale bar, 1 μm. red scale bars, 200 nm. (D) Higher magnification images showing eGFP-NS4 induced DMVs (left panel). Quantification of the DMVs diameters calculated from 19 DMVs from three different cells. Mean and SD are shown. (E) Diameter of SMVs generated by eGFP-NS4 expression. SMVs were grouped into three different size classes as indicated and their relative proportion is given. Data are based on 350 individual, randomly chosen SMVs in three different cells.

Fig 8. Modeling of GII.4 NS1-2, NS3 and NS4 and analysis of mutants of the putative hydrolase domain.

(A) Modeling. Parts for which modeling is most reliable are displayed in secondary structure representation: First the two domains that were homology modeled, namely the central domain of NS1-2 (the catalytic C205 and H139 residues are displayed as spheres) and the NTPase domain of NS3; Second the membrane association helices of the three proteins. For NS1-2, the alternate possibilities for its C-terminal membrane association, as one membrane-peripheral and one transmembrane helix, or as two transmembrane helices, are indicated by a question mark. Similarly for NS3, the alternate possibilities for its N-terminal helix as transmembrane or as membrane-peripheral are indicated with a question mark. The natively unfolded N-terminus of NS1-2 and C-terminus of NS4 are displayed as random coils. The N-terminus of NS4, folded according to secondary structure predictions but with no homolog of known structure, is displayed as a blob with a putative amphipathic helix leading to the unstructured C-terminus in secondary structure representation. (B-E) pTM plasmids encoding NS1-2 variants as indicated N-terminally fused to eGFP were transfected into Huh7-T7 cells and harvested 20 hours post transfection. Cells were fixed and permeabilized with Triton-X100 (0.5%), and ER (red) was stained using a RTN-3 monoclonal antibody. (B) Cells expressing eGFP NS1-2 C205A, with representative examples of the filamentous, intermediate and focal phenotype, encircled in red, green and violet, respectively. (C) Percentage of cells showing a filamentous, focal or intermediate phenotype for cells expressing eGFP-NS1-2 wt or the indicated mutant. Mean values and SD from at least a total of 100 cells from two independent experiments are shown for each condition. (D) A representative picture showing the filamentous phenotype for wt, the focal phenotype for mutant H139A and the intermediate phenotype for mutant C205A. (E) Pearson correlation of the eGFP signal with ER marker shown in (D); each dot representing one cell. (F) TCID50/ml values obtained from wt and mutant MNV variants in HEK293-T cells. Plasmids encoding a wildtype or mutant MNV full-length cDNA clone were transfected into HEK293T cells. Virions were harvested 72 hours post transfection and titrated on RAW 264.7 cells. Titers of infectious virus was determined by TCID50 assay and calculated using the Spearman-Karber method. Values represent the mean of three independent experiments with error bars representing the standard deviation. n.d.: not detectable. Note that positions H139 and C205 in GII.4 NS1-2 correspond to H150 and C216 in the MNV genome.