Fig 1.
MNV infection leads to apoptosis and loss of cell viability in iBMDMs.
(A) iBMDMs were infected with MNV (MOI 5) and supernatant was collected at 3-hour intervals between 9- and 24- hours post infection. Infectious virus was quantified for each time point by plaque assay and plotted on the right y-axis (Blue line; n = 2; SEM). LDH release assay to calculate % cell viability relative to total cell lysis control was performed on each time point and plotted on the left y-axis (Red line; n = 2; SEM). (B) iBMDMs infected with MNV (MOI 5) or left uninfected were observed under 5x magnification at 24 hours post infection. (C) Lysates from iBMDMs infected with MNV (MOI 5) were collected at the indicated times and immuno-blotted for markers of apoptosis, cleaved (CL)-caspase- 3 and poly(ADP-ribose) polymerase (Full-length [FL] and cleaved [CL]-PARP, along with pro-survival BCL-2 family proteins, MCL-1, BCL-XL, BCL-2. (D) The relative levels of pro-survival proteins compared with 0 h.p.i were estimated from immuno-blots using image-based quantification (n = 2; SEM).
Fig 2.
MNV NS3 protein induces translational shut-off and apoptosis.
(A) A puromycin incorporation assay was assessed on lysates harvested from 293T cells transfected with expression plasmids encoding MNV nonstructural proteins fused with His tag or left untransfected. MNV nonstructural proteins were observed by staining with Anti-6X His tag antibody. The NS4 and NS6 proteins could not be observed by western blot on this occasion but can be observed in Panel (B). NaAs is sodium azide and was used to invoke a stress response in cells. Host cell translation was measured by treating with puromycin for 30 mins and measuring incorporation with anti-puromycin antibody. Mock represents cells not transfected but pulsed with puromycin, -puro represents mock-transfected cells without addition of puromycin and -ve control are cells transfected with the control plasmid and pulsed with puromycin. (B) Immuno-blot of lysates harvested from 293T cells transfected with expression plasmids encoding MNV nonstructural proteins fused with His tag. The membrane was probed with anti-6X His tag, PARP, MCL-1 and actin antibodies. Proteins were visualised with species-specific IgG conjugated to HRP and ECL. (C) Immuno-blot of lysates collected from cells transfected with expression plasmid encoding NS3 fused with mCherry (NS3-mC), treated with caspase inhibitor QVD throughout transfection or left untreated and pulsed with puromycin as above. (D) Immuno-blot of lysates from 293T cells transfected with expression plasmid encoding NS3 fused with His tag (NS3-His), mCherry (NS3-mC) or left untransfected. Cell treated were either untreated (i), treated with caspase inhibitor QVD (ii), or treated with proteosome inhibitor MG-132 (iii) throughout transfection. The membrane was incubated with antibodies specific for PARP, MCL-1 and actin.
Fig 3.
MNV NS3 localises predominantly to the viral replication complex during infection.
RAW264.7 cells were infected with MNV (MOI 5) and at 6 (A), 12 (B) or 24 (C) hrs p.i. the cells were collected and processed for cryosectioning and immunogold labelling with anti-NS3 antibodies and 10nm Protein-A gold. (D-F) Quantitation of the anti-NS3 labelling on defined cellular organelles at each time point as specified. Cytoplasmic localisation was defined as any gold particle observed not associated with a membrane or organelle. The MNV replication complex (RC) and Mitochondria (Mito) are highlighted. Magnification bars in all panels are 500nm.
Fig 4.
Cellular localisation of MNV truncation mutants.
(A) Schematic of NS3 truncation mutants generated in pcDNA3.1 expression constructs with key domains illustrated. Arrows indicate the amino acid positions of the three NS3 domains. (B) Immunofluorescent staining of HeLa cells transfected with mCherry-tagged NS3 expression constructs (white) and fixed and permeabilised before counterstaining with DAPI (blue). Images were captured using a Zeiss LSM 710 confocal microscope and analysed with ZEN software.
Fig 5.
N-terminal region of MNV NS3 induces translation repression and apoptosis.
293T cells were transfected with expression plasmids encoding NS3 and truncation mutants for 24 hours. (A and B) Host cell translation was measured by treating with puromycin for 30 mins before (A) harvesting lysates and staining with anti-puromycin antibody by immuno-blot or (B) fixing cells and performing flow cytometry with anti-puromycin antibody. Mean fluorescent intensity (MFI) was normalised to untransfected cells. (C-E) immuno-blots were performed on lysates harvested from transfected cells following (C) no treatment (D) treatment with caspase inhibitor QVD or (E) treatment with proteosome inhibitor MG-132 for the duration of transfection.
Fig 6.
Identification of key NS3 residues for translational shut-off and apoptosis.
(A) Schematic of mCherry-tagged NS3 tripartite alanine mutants generated in pcDNA3.1 expression constructs. Amino acid residues in region between 70 to 100 is illustrated in the expanded box along with changes in red. * denotes where generation of mutant was unsuccessful. (B) Table outlining positions of NS3 tripartite mutations, the original amino acids (A.A) and the specific change (Δ A.A). (C) Immuno-blot of lysates harvested 24 h.p.t. from 293T cells transfected with NS3 expression constructs encoding full length, truncation, or tripartite alanine mutant proteins. (D) 293T cells transfected with full length NS3 or tripartite alanine mutant constructs for 24 hours were treated with puromycin for 30 mins before fixation and flow cytometry was performed. Mean fluorescent intensity (MFI) of puromycin staining was normalised to control cells transfected with mCherry only.
Fig 7.
HuNoV NS3 induces translation repression and apoptosis.
(A) Amino acid homology between MNV NS3 and HuNoV NS3. Alignment generated with EMBOSS Needle tool using Needleman-Wunsch algorithm. MNV NS3 amino acids 68 to 100 have been highlighted in red (B) Immuno-blot of lysates transfected with expression plasmids encoding MNV NS3 or HuNoV NS3 fused with either His Tag or mCherry for 24 hours. Cells were treated with puromycin 30 mins before harvest and translational assessed by measuring incorporation with anti-puromycin antibody. (C) Immunofluorescence was performed on Hela cells transfected with either HuNoV NS3, MNV NS3 or left untreated and treated with puromycin 30 mins before fixation. Cells were stained with DAPI (blue) anti-puromycin (green) and mCherry-tagged HuNoV or MNV NS3 visible (red). (D) Fluorescence intensity was quantified in mCherry positive cells relative to untransfected bystanders. Images were captured using a Zeiss LSM 710 confocal microscope and analysed with ZEN software.
Fig 8.
Predictive modelling of the WT and mutant MNV NS3 protein.
The sequence of the WT CW1 NS3 protein (orange) and NS3(70,71,72) (green) protein were imported into AlphaFold2 to derive a predicted model of the proteins. The models were overlaid to indicate the changes in protein structure upon mutation and the amino acids mutated are indicated in yellow.