Extensive coronavirus-induced membrane rearrangements are not a determinant of pathogenicity

Abstract

Positive-strand RNA (+RNA) viruses rearrange cellular membranes during replication, possibly in order to concentrate and arrange viral replication machinery for efficient viral RNA synthesis. Our previous work showed that in addition to the conserved coronavirus double membrane vesicles (DMVs), Beau-R, an apathogenic strain of avian Gammacoronavirus infectious bronchitis virus (IBV), induces regions of ER that are zippered together and tethered open-necked double membrane spherules that resemble replication organelles induced by other +RNA viruses. Here we compared structures induced by Beau-R with the pathogenic lab strain M41 to determine whether membrane rearrangements are strain dependent. Interestingly, M41 was found to have a low spherule phenotype. We then compared a panel of pathogenic, mild and attenuated IBV strains in ex vivo tracheal organ culture (TOC). Although the low spherule phenotype of M41 was conserved in TOCs, each of the other tested IBV strains produced DMVs, zippered ER and spherules. Furthermore, there was a significant correlation for the presence of DMVs with spherules, suggesting that these structures are spatially and temporally linked. Our data indicate that virus induced membrane rearrangements are fundamentally linked to the viral replicative machinery. However, coronavirus replicative apparatus clearly has the plasticity to function in different structural contexts.

Figure 1. Replication of Beau-R and M41 are comparable in CK cells.

(a) CK cells were infected with Beau-R (open circles) or M41 (closed circles) at an MOI of 0.005. Release of progeny virus was determined by plaque assay. Mean and standard error of three independent experiments are shown. (b) Genomic RNA levels were measured by two-step RT-qPCR at 8 and 24 hours after inoculation of CK cells with 20 pfu per cell of Beau-R or M41. Results from three independent experiments are shown. Non-significant differences by t-test are indicated (ns). CK cells were inoculated with (c) Beau-R for 4 hours or (d) M41 for 3 hours. Cells were fixed with 4% paraformaldehyde and labelled with anti-dsRNA (green) and nuclei were labelled with TO-PRO-3 (red).

Figure 2. M41 has a low spherule phenotype in CK cells.

Primary CK cells were infected with Beau-R or M41. After 24 hours, cells were chemically fixed and visualised by TEM. DMVs are indicated with a star, zippered ER is indicated with a black arrowhead and spherules with black brackets.

Figure 3. Pathogenic and apathogenic viruses replicate in tracheal organ culture resulting in ciliostasis.

(a) TOCs were mock infected or infected with H120, Beau-R, M41, D1466, 4/91 or Italy02. Ciliary activity was assessed and scored at 24 hour intervals. The mean and SEM of three independent experiments are shown. (b) TOCs were mock infected or infected with H120, Beau-R, M41, D1466, 4/91 or Italy02. After 24 hours, cells were fixed with 4% paraformaldehyde and labelled with anti-dsRNA (red) and anti-tubulin (green, c). Nuclei are stained with DAPI (blue).

Figure 4. Pathogenic and apathogenic strains of IBV induce zippered ER, spherules and DMVs.

TOCs were mock infected or infected with (a) H120, D1466, 4/91 or Italy02, (b), Beau-R or (c) M41. After 24 hours, cells were chemically fixed. DMVs are indicated by a star, zippered ER with black arrowheads and spherules with black brackets.

Figure 5. Spherule and DMV diameters are not altered by virus strain.

Spherule (a) and DMV (b) diameters were measured and divided into 2 nm (spherules) or 10 nm (DMV) size classes. The number of spherules or DMVs in each size class is shown for each of the viral strains.

Figure 6. Spatial and temporal correlation between DMVs and other paired membrane structures but not intracellular virions across diverse coronaviruses.

Transmission electron micrographs of (a) tracheal organ cultures infected with Beau-R and chemically fixed after 24 hours and (b) DBT cells infected with MHV-A59 and chemically fixed after 5.5 hours. DMVs (blue), spherules (purple), zippered ER (red in a) and convoluted membranes (red in b) are highlighted. Enlarged images of DMVs and paired membrane structures from (c) IBV and (d) MHV infected cells. (e) Pearson linear correlation coefficients and p values (ns = not significant) for comparisons between the number of DMVs, spherules, convoluted membrane regions and intracellular virions in randomly oriented ultrathin sections through infected cells.