Article Figures & Data
Figures
- FIG 1
Engineering nsp4 mutants. (A) Proposed topology of nsp4: nsp4 has four transmembrane regions (TM1 to TM4) and three loops (loop 1 to 3). Mutations in nsp4 tested in this study are shown on the diagram. Red circles represent nonviable mutations; green circles represent viable viruses that were recovered in this study. The double-headed arrows represent native glycosylation sites within MHV nsp4. (B) nsp4 mutants were engineered with alternate NX(S/T) sites (gray double-headed arrows) in the DGM background. The locations of the native NX(S/T) sequons (black double-headed arrows) are shown. Viruses are identified with the introduced NX(S/T) site shown below the designation. *, previously recovered viruses (1).
- FIG 2
Electron microscopic analysis of alternate NX(S/T) sequon mutants. (A to F) DBT cells were mock infected (A) or infected with the WT (B), DGM (C), DGM-K85S (D), DGM-V129N/Q130I (E), DGM-N479S (F), or N479S (G) virus at an MOI of 5 PFU/cell for 8 h before being fixed in 2% glutaraldehyde and processed for TEM. Black arrowheads indicate normal DMVs; the white arrowheads indicate aberrant DMVs. CM, convoluted membranes; N, nucleus; M, mitochondrion; E, endosome; VV, virions in vesicles; ER, endoplasmic reticulum. Scale bar, 500 nm. (H) Normal and aberrant DMVs were quantified for each virus. Percentages are shown as indicated on the figure. The total number of DMVs analyzed is shown above each bar. (I) The numbers of DMVs per area of cytoplasm were calculated and are shown on the graph. Each circle represents an individual field, and the bars show the means ± standard deviations. Fields were chosen based on the presence of signs of virus infection that included DMVs, convoluted membranes, and virions. A Kruskal-Wallis test was used to analyze for significant differences in numbers of DMVs per area of cytoplasm. *, P = 0.008; **, P < 0.0001 (compared to the WT).
- FIG 3
Replication kinetics and glycosylation of alternate NX(S/T) sequon viruses. (A) DBT cells were infected with the indicated viruses at an MOI of 1 PFU/cell. Supernatants were sampled from 0 to 16 h p.i., and titers were determined by plaque assay. Error bars represent the standard errors of the means of three replicates plated in duplicate. (B) DBT cells were infected with the indicated viruses at an MOI of 10 PFU/cell. At 4 h p.i., cells were starved in DMEM lacking Met and Cys and treated with ActD for 1 h before being radiolabeled with [35S]Met-Cys. At 7 h p.i., lysates were harvested and immunoprecipitated with antibodies specific for nsp4 and treated in the presence or absence of endo-H. Proteins were resolved by SDS-PAGE (n ≥ 2). DGM-VQ/NI, DGM-V129N/Q130I.
- FIG 4
RNA synthesis of nsp4 NX(S/T) mutants. DBT cells were infected with the indicated viruses at an MOI of 1 PFU/cell for 10 h. Cells were then harvested in TRIzol, and genomic RNA was extracted. Genomic RNA levels were determined by qRT-PCR using primers specific to Orf1a. RNA levels were normalized using the 2−ΔCT (where CT is threshold cycle) method to endogenous GAPDH expression. Mean values ± standard errors of the means are shown (n ≥ 3). RNA synthesis levels were not significantly different from the WT level according to a Kruskal-Wallis test.
- FIG 5
nsp4 localization in NX(S/T) mutant-infected cells. DBT cells were infected with the indicated viruses at an MOI of 5 PFU/cell for 6.5 h. Cells were then fixed in 100% methanol and stained with antibodies specific for nsp4 (green) and nsp8 (red). Yellow pixels represent colocalization of red and green pixels. Scale bar, 20 μm.
- FIG 6
Replication and glycosylation of nsp4 mutants. (A) Schematic of nsp4 showing the location of the nsp4 mutations. (B) DBT cells were infected with the indicated viruses at an MOI of 1 PFU/cell for 24 h. At the indicated time points, supernatants were sampled, and titers were determined by plaque assay. Error bars represent the standard errors of the means of three replicates plated in duplicate. (C) DBT cells were infected with the indicated viruses at an MOI of 10 PFU/cell. Cells were starved with DMEM lacking Cys and Met in the presence of ActD for 1 h prior to being radiolabeled with [35S]Cys-Met for 2 h before lysates were harvested. Lysates were immunoprecipitated with antibodies specific to nsp4 in the presence or absence of endo-H, and proteins were resolved by SDS-PAGE (n = 2).
- FIG 7
EM of nsp4 mutant-infected cells. (A to F) DBT cells were mock infected (A) or infected with the WT (B), DGM (C), K44A/D47A (D), E226A/E227A (E), or N258T (F) virus for 8 h before fixation in 2% glutaraldehyde and processed for TEM. Black arrowheads indicate normal DMVs, and white arrowheads indicate aberrant DMVs. N, nucleus; M, mitochondrion; VV, virion in vesicles; E, endosome. (G) Normal and aberrant DMVs were quantified, and results are shown as percentages of total DMVs, as indicated on the figure. The total number of DMVs analyzed is shown above each bar. (H) The total number of DMVs per area of cytoplasm was calculated. Circles represent the number of DMVs per area of cytoplasm for a single field. Bars represent the means ± standard deviations. A Kruskal-Wallis test was used to analyze for significant differences in numbers of DMVs per area of cytoplasm. *, P = 0.008; **, P < 0.0001 (compared to the WT).
- FIG 8
Competition assay of nsp4 mutants. DBT cells were infected with the indicated pairs of viruses at a total MOI of 0.1 PFU/cell at a ratio of 1:1. When cells were at least 50% involved in cytopathic effect, supernatants were collected, and cell monolayers were harvested in TRIzol. Supernatants were used for subsequent passages, for a total of three passages. Total RNA was extracted, and nsp4 amplicons were generated by RT-PCR and sequenced. For residues of interest, the area under the peak was calculated using MacVector, version 13. Then, the percentage of nucleotides of virus A to virus B was calculated and plotted on the graph. Error bars represent standard errors of the means (n ≥ 2).
Tables
- TABLE 1
Primers used for alternate NXS/T mutagenesis
Primer name Sequencea Purpose K85S sense 5′-CCGCAACTCTTTCGCTTGTCCTG-3′ Mutagenesis for K85S K85S antisense 5′-CAGGACAAGCGGAAGAGTTGCGG-3′ Mutagenesis for K85S P106N sense 5′-CTTATTTAATGTTAACACCACAGTTTTAAG-3′ Mutagenesis for P106N P106N antisense 5′-CTTAAAACTGTGGTGTTAACATTAAATAAG-3′ Mutagenesis for P106N V129N sense 5′-CTACTGATAGCAACCAGTGTTACACGC-3′ Mutagenesis for V129N V129N antisense 5′-GCGTGTAACACTGGTTGCTATCAGTAG-3′ Mutagenesis for V129N Q130I sense 5′-CTACTGATAGCAACATATGTTACACGC-3′ Mutagenesis for Q130I Q130I antisense 5′-GCGTGTAACATATGTTGCTATCAGTAG-3′ Mutagenesis for Q130I C133N sense 5′-GATAGCGTGCAGAACTACACGCCAC-3′ Mutagenesis for C133N C133N antisense 5′-GTGGCGTGTAGTTCTGCACGCTAG-3′ Mutagenesis for C133N V214N sense 5′-GTGCGTGTTAACCGCACTCGC-3′ Mutagenesis for V214N V214N antisense 5′-GCGAGTGCGGTTAACACGCAC-3′ Mutagenesis for V214N E355N sense 5′-CAACACTTATATTCGAAGGG-3′ Mutagenesis for E355N E355N antisense 5′-CCCTTCGAATATAAGTGTTG-3′ Mutagenesis for E355N N479S sense 5′-CATAATAATGGTTCCGATGTTCTC-3′ Mutagenesis for N479S N479S antisense 5′-GAGAACATCGGAACCATTATTATG-3′ Mutagenesis for N479S ↵a Bold letters denote nucleotides used to introduce mutations.
- TABLE 2
Quantification of normal and aberrant DMVs
Virus No. of DMVs Total Normal Aberrant (% of total) WT 623 393 230 (37) DGM 198 65 133 (67) DGM-K85S 344 40 304 (88) DGM-V129I/Q130I 577 126 451 (78) DGM- N479S 438 202 236 (54) K44A/D47A 308 101 207 (67) E226A/E227A 326 96 230 (71) N258T 412 128 284 (69) N479S 587 310 277 (53)
