Visualising the autophagy pathway in avian cells and its application to studying Infectious Bronchitis Virus

Autophagy, 2013 Apr; 9(4):496-509.

Helena J. Maier, Eleanor M. Cottam, Phoebe Stevenson-Leggett, Jessica A. Wilkinson, Christopher J. Harte, Tom Wileman and Paul Britton.

The Pirbright Institute, Compton Laboratory, Compton, Newbury, Berkshire, UK.

 

Abstract

Autophagy is a highly conserved cellular response to starvation that leads to the degradation of organelles and long-lived proteins in lysosomes and is important for cellular homeostasis, tissue development and as a defence against aggregated proteins, damaged organelles and infectious agents. Although autophagy has been studied in many animal species, reagents to study autophagy in avian systems are lacking. Microtubule-associated protein 1 light chain 3 (LC3) is an important marker for autophagy and is used to follow autophagosome formation. Here we report the cloning of avian LC3 paralogues A, B and C from the domestic chicken, Gallus gallus domesticus, and the production of replication deficient, recombinant adenovirus vectors expressing these avian LC3s tagged with EGFP and FLAG-mCherry. An additional recombinant adenovirus expressing EGFP-tagged LC3B containing a G120A mutation was also generated. These vectors can be used as tools to visualise autophagosome formation and fusion with endosomes/lysosomes in avian cells and provide a valuable resource for studying autophagy in avian cells. We have used them to study autophagy during replication of Infectious Bronchitis Virus (IBV). IBV induced autophagic signalling in mammalian Vero cells but not primary avian chick kidney cells or the avian DF1 cell line. Furthermore, induction or inhibition of autophagy did not affect IBV replication, suggesting that classical autophagy may not be important for virus replication. However, expression of IBV non-structural protein 6 alone did induce autophagic signalling in avian cells, as seen previously in mammalian cells. This may suggest that IBV can inhibit or control autophagy in avian cells, although IBV did not appear to inhibit autophagy induced by starvation or rapamycin treatment.

 

Summary

Infectious Bronchitis is an economically important respiratory disease of chickens, caused by the gammacoronavirus Infectious Bronchitis Virus (IBV). In addition to respiratory symptoms, IBV causes damage to the egg producing organs of hens and results in reduced egg production, as well as reduced meat quality. This costs the UK poultry industry an estimated £23 million per year. Vaccines are available against IBV, but due to high virus variation and poor cross protection between strains, better vaccines and new vaccine design strategies are required. A detailed understanding of the cellular machinery required for virus replication is critical to enable design of novel vaccines through targeted attenuation.

In this work we studied the role of autophagy in the replication cycle of IBV in avian cells. Reagents were generated to allow the study of autophagy in avian cells with all 3 paralogues of autophagy marker protein microtubule-associated protein 1 light chain 3 (LC3) being cloned from Gallus gallus domestius, the domestic chicken. The proteins were tagged with EGFP or FLAG-mCherry to allow direct visualisation and were validated for use in both primary avian cells and avian cell lines. The reagents were then used to study autophagy during IBV infection. We found that although IBV replication in mammalian cells induced autophagic signalling, no induction was detected during IBV replication in avian cells. This highlights a potential difference between the autophagy pathways in mammalian and avian systems. However, viral non-structural protein 6, previously shown to induce autophagy in mammalian cells when expressed individually, was also found to induce autophagic signalling in avian cells. This may indicate that IBV has developed mechanisms to inhibit autophagy.

 

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