Visualization and quantification of autophagy in primary gastrointestinal epithelia cell cultures.

Autophagy is an intracellular bulk degradation pathway which can be used during cellular maintenance to encapsulate and target intracellular content such as organelles and proteins to be degraded by the lysosome. The autophagosome is a double membrane vesicle which is used to encapsulate this cellular content. During the cellular response to starvation, autophagy can degrade cytoplasmic organelles and long-lived proteins in lysosomes, to recover amino acids within the cell and form new proteins. Stimulation of autophagy during infection can enhance immune responses by degrading pathogens and increasing presentation of microbial components to the immune system. Genome wide association studies (GWAS) have identified mutations in the autophagy related (Atg) protein Atg16L1, and NOD2 as risk alleles for Crohn’s disease. Atg16L1 is essential for the formation of autophagosomes and autophagosome formation is stimulated following recognition of bacterial cell wall peptide muramyl dipeptide (MDP) by NOD2. Crohn’s disease results from an autoimmune reaction to gut epithelial cells, and this raises the possibility that the risk alleles alter autophagy and/or NOD2-mediated microbial sensing in gut epithelial cells. The aim of this thesis has been to develop methods to quantifying autophagy in primary epithelial cells cultured from human intestinal biopsy material. Atg8/LC3 is the major structural protein of the autophagosome. Images of GFP-Atg8/LC3 puncta generated in response to starvation, or NOD2 signalling, were taken by fluorescence microscopy, and pixel densities were rendered to generate digital datasets that were amenable to statistical analysis. Rendered puncta were displayed graphically to indicate autophagosome diameters, spatial location within the cell, lifetime, expansion and movement. The methods were validated in tissue culture cell lines and then extended to study human colon crypts cultures transduced by adenoviral vectors expressing GFP-LC3. In comparison to cell lines after starvation, there were less GFP LC3 puncta in the smaller epithelial cells from colon crypt tissue (20-30 compared to 5.8+/-2.2). MDP generated significantly more LC3 puncta (9.5+/-4) in colon crypt epithelial cells compared to starvation (5.8+/-2.2). This work provides a method to quantify autophagy in colon crypt cultures, and if applied to biopsies taken from patients will be able to determine if autophagy and NOD2 sensing are altered in patients carrying with Crohn’s disease risk alleles. Keywords: Autophagy, in vitro culture, adenoviral transduction, Imaris quantification

The pestivirus N terminal protease N(pro) redistributes to mitochondria and peroxisomes suggesting new sites for regulation of IRF3 by N(pro.)

The N-terminal protease of pestiviruses, N(pro) is a unique viral protein, both because it is a distinct autoprotease that cleaves itself from the following polyprotein chain, and also because it binds and inactivates IRF3, a central regulator of interferon production. An important question remains the role of N(pro) in the inhibition of apoptosis. In this study, apoptotic signals induced by staurosporine, interferon, double stranded RNA, sodium arsenate and hydrogen peroxide were inhibited by expression of wild type N(pro), but not by mutant protein N(pro) C112R, which we show is less efficient at promoting degradation of IRF3, and led to the conclusion that N(pro) inhibits the stress-induced intrinsic mitochondrial pathway through inhibition of IRF3-dependent Bax activation. Both expression of N(pro) and infection with Bovine Viral Diarrhea Virus (BVDV) prevented Bax redistribution and mitochondrial fragmentation. Given the role played by signaling platforms during IRF3 activation, we have studied the subcellular distribution of N(pro) and we show that, in common with many other viral proteins, N(pro) targets mitochondria to inhibit apoptosis in response to cell stress. N(pro) itself not only relocated to mitochondria but in addition, both N(pro) and IRF3 associated with peroxisomes, with over 85% of N(pro) puncta co-distributing with PMP70, a marker for peroxisomes. In addition, peroxisomes containing N(pro) and IRF3 associated with ubiquitin. IRF3 was degraded, whereas N(pro) accumulated in response to cell stress. These results implicate mitochondria and peroxisomes as new sites for IRF3 regulation by N(pro), and highlight the role of these organelles in the anti-viral pathway

Coronavirus NSP6 restricts autophagosome expansion

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 SA RS 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

N^pro co-distributes with peroxisomes marker PMP70.

<p>(A). Peroxisomes were localized by immunostaining with anti-peroxisome marker PMP70 in MEF cells expressing N^pro cherry i) control cells (CON) ii) treatment with sodium arsenate (+NaA) for 4 hours iii) Digital rendering using Imaris software is shown for the images in ii) (scale bars are 5 um). (B). Statistical analysis of average number of N^pro puncta per cell and average number co-distributing with PMP70 after sodium arsenate treatment for 4 hours. (C). Boxplot: distribution of the fraction of N^pro distributing with PMP70 compare to total number of N^pro granules. A Welch two sample t-test was applied, two-sided, not paired, (assuming that the variances are unequal) and showing t = 0.5867, df = 25.712, p-value = 0.5625 with the lower end of the box = 25% of the data, bold line in the middle = median, Upper end of the box = 75%, lower whisker = 5%.</p

N^pro and IRF3 co-distribute to cytoplasmic peroxisomes containing ubiquitin.

<p>(A). Cells expressing IRF3 GFP and N^pro cherry i) Control cells (CON) were stained for ubiquitin with monoclonal antibody FK2 visualized with anti-mouse Cy5. ii) Cell treated with sodium arsenate (+NaA) for 60 mins and ubiquitin was stained with monoclonal antibody FK2 and visualized with anti-mouse Cy5. iii) Cells treated with sodium arsenate (+NaA) for 60 mins and peroxisomes were stained with anti-rabbit PMP70 and visualized with anti- rabbit Cy5. (B). N^pro is stabilized and IRF3 degraded following sodium arsenate treatment. Control cells, cells expressing N^pro and cells expressing N^pro C112R mutant were untreated (−) or treated with sodium arsenate for 4 hours (+NaA). Lysates were probed by Western blot for N^pro, IRF3 and actin. Actin is shown for equal lane loading and bands were scanned and normalize with actin for N^pro levels in treated and untreated cells (bottom graph).</p

Inhibition of apoptosis by N^pro correlates with degradation of IRF3.

<p>(A). Western blotting of lysates from MEF cell expressing N^pro cherry with antibodies against N^pro, IRF3 and actin shows loss of IRF3 from cells expressing N^pro. Lane 1: Control MEF cells not expressing plasmid. Lane 2: MEF cells stably expressing N^pro mcherry. Lane 3 : MEF cells expressing N^pro C112R mcherry mutant. Lane 4: MEF cells expressing N^pro D136N cherry mutant. Graph shows relative intensities of IRF3 compared to actin for the image scanned with Scion image software <a href="http://scion-image.software.informer.com" target="_blank">http://scion-image.software.informer.com</a> (representative of Western blot repeated three times for each lane). (B). N^pro but not N^pro C112R inhibits caspase 3/7 activity. MEF cells stably expressing wild type N^pro or mutant N^pro C112R mcherry were treated with staurosporine, interferon, dsRNA (poly I:C), sodium arsenate (NaA) or hydrogen peroxide (H₂O₂) for 4 hrs. Fold increase in caspase activity was normalized to negative (untreated cells) for each cell line and analyzed with a two-sided t-test with unequal variance relative to the untransfected cells for each cell line, and significance values are: bars marginally significant (0.05–0.1), * significant (0.01–0.05), ** highly significant (0–0.01) (n = 5 to 8). The table shows significance of drug treatment relative to control treated cells for cells expressing N^pro WT or N^proC112R and analysis was conducted using similar tests as described above.</p

N^pro is recruited to mitochondria after cell stress and protects from apoptosis.

<p>(A). MEF cells were transfected with Bax-GFP alone (a–f) or tBid-GFP alone (g–j) or N^pro cherry alone (k–n) and either untreated (No NaA, left panel) or treated with sodium arsenate for 4 hours (+NaA, right panel). Cells were incubated with Mitotracker (MT) CM Ros (b,e,i) or Green FM (m) for 30 mins before fixation. (B). Protection from apoptosis induced by sodium arsenate requires IRF3. MEF cells co-expresssing Bax-GFP and either wild type N^pro-cherry (a–b) or mutant N^pro C112R-cherry (c–d) were treated with sodium arsenate (+NaA). MDBK primary cells, transfected with Bax GFP (e–f) alone or infected with BVDV overnight and then transfected with Bax GFP (g–h), were treated with sodium arsenate (+NaA) for 4 hours. BVDV was detected with bovine hyperimmune serum V182 and detected with Cy3 rabbit anti bovine antibody (red) and DNA was stained with DAPI.</p

N^pro rapidly redistributes with IRF3 to mitochondria and cytoplasmic puncta following induction of stress.

<p>(A). MEF cells co-expressing N^pro -cherry (red) and IRF3-GFP (green) were treated with sodium arsenate (+NaA) for 30 minutes and examined by confocal microscopy. (B). Treatment of MEF cells co-expressing N^pro-cherry and IRF3-GFP for 60 minutes.</p