Coronavirus NSP6 restricts autophagosome expansion

Abstract

Autophagy is a cellular response to starvation that generates autophagosomes to carry long-lived proteins and cellular organelles to lysosomes for degradation. Activation of autophagy by viruses can provide an innate defense against infection, and for (+) strand RNA viruses autophagosomes can facilitate assembly of replicase proteins. We demonstrated that nonstructural protein (NSP) 6 of the avian coronavirus, infectious bronchitis virus (IBV), generates autophagosomes from the ER. A statistical analysis of MAP1LC3B puncta showed that NSP6 induced greater numbers of autophagosomes per cell compared with starvation, but the autophagosomes induced by NSP6 had smaller diameters compared with starvation controls. Small diameter autophagosomes were also induced by infection of cells with IBV, and by NSP6 proteins of MHV and SARS and NSP5, NSP6, and NSP7 of arterivirus PRRSV. Analysis of WIPI2 puncta induced by NSP6 suggests that NSP6 limits autophagosome diameter at the point of omegasome formation. IBV NSP6 also limited autophagosome and omegasome expansion in response to starvation and Torin1 and could therefore limit the size of autophagosomes induced following inhibition of MTOR signaling, as well as those induced independently by the NSP6 protein itself. MAP1LC3B-puncta induced by NSP6 contained SQSTM1, which suggests they can incorporate autophagy cargos. However, NSP6 inhibited the autophagosome/lysosome expansion normally seen following starvation. Taken together the results show that coronavirus NSP6 proteins limit autophagosome expansion, whether they are induced directly by the NSP6 protein, or indirectly by starvation or chemical inhibition of MTOR signaling. This may favor coronavirus infection by compromising the ability of autophagosomes to deliver viral components to lysosomes for degradation.

Keywords: MHV; SARS; autophagosome quantification; autophagy; coronavirus; nonstructural proteins; omegasome.

Figure 1. Analysis of autophagosomes induced by coronavirus NSP6 proteins or infection with infectious bronchitis virus. Autophagosomes were visualized in Vero cells by immunofluorescence staining for endogenous MAP1LC3B. Pixel densities in fluorescent images were analyzed in Imaris using spot function to generate rendered images. The diameters of MAP1LC3B-puncta were color coded using a heat map where blue represents puncta less than 3.0 µm, green puncta are 0.5 µm and red puncta 1.0 µm or greater. (A) cells starved in HBSS for 4 h. (B) cells expressing IBV NSP6-mCherry. (C) cells expressing MHV NSP6-mCherry. (D) cells imaged after overnight infection with IBV. Infected cells were detected by immunostaining for double-stranded RNA (dsRNA). Rendered images are shown below the fluorescence micrographs, the images are projections made of 9 z slices 300 nm apart. Cells expressing NSP6-mCherry, or infected with IBV are outlined with white hatched lines, mCherry signals and dsRNA are pseudocolored in black in rendered images. (E) Composite data calculated from from 3 replicate experiments. MHV NSP6: 867 puncta from 16 cells, IBV NSP6: 1137 puncta from 26 cells, IBV infection: 590 puncta from 24 cells, nutrient media control: 380 puncta from 21 cells, HBSS 1 h: 845 puncta from 23 cells, HBSS 4 h: 421 puncta from 25 cells. Significant differences in puncta per cell and puncta diameter were measured by a paired t test. ***A significant difference of P < 0.0001 ** A significant difference of P < 0.001* A significant difference of P < 0. 01.

Figure 2. Effect of HBSS on autophagosome maturation. (A) Analysis of autophagosome expansion during starvation. Vero cells were transferred from nutrient media to HBSS and fixed at the indicated times. Autophagosomes were visualized by immunofluorescence staining for endogenous MAP1LC3B. Pixel densities in fluorescent images were analyzed in Imaris using spot function to generate rendered images presented below the corresponding fluorescent images (the images are projections made of 9 z slices 300 nm apart). The diameters of MAP1LC3B-puncta were color coded using a heat map where blue represents puncta less than 3.0 µm, green puncta are 0.5 µm and red puncta 1.0 µm or greater. The images show examples from 3 replicate experiments. (B) Analysis of autophagosome numbers and diameters during starvation. The number of puncta per cell was calculated from ≥ 20 cells per time point taken from 3 starvation time courses and plotted with the error bars representing the standard deviation (left chart). The percentage of puncta identified with a diameter ≥ 0.75 µm was also calculated and plotted over time (right chart). (C–G) Time-lapse imaging of autophagosome lifetime. Vero cells were transduced with an adenovirus expressing mammalian GFP-MAP1LC3B. The following day the cells were transferred to HBSS and the time course of GFP-MAP1LC3B puncta formation and decay was determined by time-lapse imaging. GFP-MAP1LC3B puncta were tracked and analyzed using Imaris spot and surface functions. The left-hand image on each panel identifies the autophagosome of interest and shows its track as determined by Imaris. Color-coded rendered images are shown below corresponding fluorescence images from example time points indicated in min. (C) nutrient media. (D) 1 to 2 h after transfer to HBSS. (E) 4 h after transfer to HBSS. (F) cells in nutrient media expressing IBV NSP6 mCherry.

Figure 3. Effect of IBV NSP6 on maturation of autophagosomes induced by starvation. (A) Analysis of fixed cells. Vero cells expressing IBV NSP6-mCherry or infected with IBV were transferred from nutrient media to HBSS and fixed after 4 h. Autophagosomes were visualized by immunofluorescence staining for endogenous MAP1LC3B. Pixel densities in fluorescent images were analyzed in Imaris using spot function to generate rendered images (the images are projections made of 9 z slices 300 nm apart). The diameters of MAP1LC3B-puncta were color coded using the heat map, red puncta represent puncta 1.0 µm or greater. Rendered images are shown below the fluorescence micrographs. Cells expressing NSP6-mCherry, or infected with IBV are outlined with white lines, infected cells were detected by immunostaining for double-stranded RNA (dsRNA), mCherry signals and dsRNA are pseudocolored in black in rendered images. (B) Analysis of autophagosome numbers and diameters. The diameters (i) and numbers (ii) of GFP-MAP1LC3B puncta per cell were calculated from ≥ 20 cells and plotted with the error bars representing the standard deviation. Significant differences in puncta diameter and pucta per cell were measured by a paired t test. ***A significant difference of P < 0.0001; **a significant difference of P < 0.001; *a significant difference of P < 0. 01. (C) Analysis of autophagosome diameter and lifetime. Vero cells were transduced with an adenovirus expressing mammalian GFP-MAP1LC3B. The following day the cells were transferred to HBSS and autophagosome diameters and lifetimes were calculated from time-lapse imaging of GFP-MAP1LC3B puncta. Each data point represents an individual autophagosome. The lifetimes of GFP-MAP1LC3B puncta generated under the indicated conditions are plotted against maximum vesicle diameter. The vertical bar represents 0.75 μm which would provide orange spheres in rendered images. Charts (i–iv); control cells transferred to HBSS. Charts (v and vi); cells expressing NSP6 transferred to HBSS. Insets indicate numbers of puncta (n), mean diameters (D) and mean lifetimes (L).

Figure 4. Effect of IBV NSP6 on maturation of omegasomes. Vero cells were transferred from nutrient media to HBSS and fixed at the indicated times. Omegasomes were visualized by immunofluorescence staining for endogenous WIPI2. Pixel densities in fluorescent images were analyzed in Imaris using spot function to generate rendered images presented below the corresponding fluorescent images (the images are projections made of 9 z slices 300 nm apart). The diameters of WIPI2 puncta were color coded using a heat map red puncta are 1.0 µm or greater. The images show examples from 3 replicate experiments. (A) Immunofluorescence images. (i) Cells expressing IBV NSP6 maintained in nutrient media, (ii) cells expressing IBV NSP6 4 h after transfer to HBSS, (iii) control cells 4 h after transfer to HBSS. (B) Analysis of omegasome numbers and diameters. The numbers per cell (i) and % of WIPI2 puncta with a diameter greater than 0.75 μm (ii) were calculated from ≥ 17 cells per time point taken from 3 starvation time courses and plotted with the error bars representing the standard deviation. Composite data were calculated from 3 replicate experiments. Nutrient media (FM): 164 puncta from 21 cells, HBSS 4 h: 127 puncta from 17 cells, IBV NSP6-mCherry in nutrient media: 599 puncta from 26 cells, IBV NSP6-mCherry after 4 h HBSS: 641 puncta from 17 cells. Significant differences in puncta numbers (iii) and diameters (iv) were measured by a paired t test. ***Significant difference of P < 0.0001; **a significant difference of P < 0.001; *a significant difference of P < 0.01.

Figure 5. Effect of IBV NSP6 on incorporation of SQSTM1 into autophagosomes. (A–F) Vero cells, or Vero cells expressing IBV NSP6, were transferred from nutrient media to HBSS and fixed at the indicated times. Autophagosomes were visualized by immunofluorescence staining for endogenous MAP1LC3B (green) and endogenous SQSTM1 (red). Regions of interest are indicated by white boxes. SQSTM1 puncta (red) and MAP1LC3B puncta (green) were rendered and analyzed for degree of colocalization (yellow). (G) SQSTM1 and MAP1LC3B puncta were analyzed from 3 replicate experiments and extent of colocalization of SQSTM1 with MAP1LC3B determined from rendered images. (H) Vero cells, or Vero cells expressing IBV NSP6, were transferred from nutrient media to HBSS, or incubated with Torin1 as indicated. Cells were lysed and analyzed for SQSTM1 by western blot. Bar graphs show SQSTM1 levels relative to β-actin.

Figure 6. Effect of IBV NSP6 on lysosome and autolysosome expansion in response to starvation. Vero cells, or Vero cells expressing NSP6, were transferred from nutrient media to HBSS and fixed at the indicated times. Lysosomes were visualized by immunofluorescence staining for endogenous LAMP2 (green). IBV NSP6 expression was detected from mCherry tag (red). (A) Vero cells. Immunofluorescence images show cells maintained in nutrient media (i) or following 4 h in HBSS (iii). Rendered images (ii and iv) show LAMP2 staining pseudocolored blue using the surfaces function in Imaris. Regions of interest are indicated by white boxes with corresponding high magnification images presented at the bottom of each panel. High magnification images show side-by-side comparisons of regions of interest taken from cells in nutrient media (v), or following 4 h in HBSS (vi). (B) Vero cells expressing IBV NSP6-mCherry. Immunofluorescence images show cells maintained in nutrient media (i) or following 4 h in HBSS (iii). Rendered images (ii and iv) show LAMP2 staining pseudocolored blue using the surfaces function in Imaris. Cells expressing NSP6 are outlined in red, and the region of interest is identified by the red box. Cells negative for NSP6 are outlined in white and region of interest is identified by the white box. High magnification images show side-by-side comparisons of the region of interest taken from cells in nutrient media (v), or following 4 h in HBSS (vi).

Figure 7. Effect of IBV NSP6 on MTOR association with lysosomes in response to starvation. Vero cells, or Vero cells expressing NSP6, were transferred from nutrient media to HBSS and fixed at 1 and 4 h. Lysosomes were visualized by expression of GFP-LAMP1 (green). IBV NSP6 expression was detected from mCherry tag (red). MTOR was detected by immunostaining (far-red). Regions of interest are indicated by white boxes with corresponding high magnification merged images presented at the top left of each panel. (A) Nutrient media; (B) 1 h in HBSS; (C) 4 h in HBSS.