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.

New insights on the role of paired membrane structures in coronavirus replication

“…The authors conclude that the presence of exogenous, individually expressed IBV nsp6, which localizes to the ER, induces the formation of autophagosomes in contrast to other IBV replicase proteins. Additionally, although autophagosomes induced by IBV nsp6 or IBV infection appeared smaller than conventional autophagosomes observed after starvation of cells, they were similar in size to DMVs (Cottam et al, 2014). However, the data reported here do not appear to support the assumption that there is a functional link between IBV nsp6 and autophagosomes, and a role of the autophagy in the formation of IBV replicative structures can hereby not be demonstrated.…”

“…TM3 largely consists of transmembrane regions, without the hallmarks of amino acid conservation or predicted structural conservation that would be expected for an enzyme. Overexpression of TM3 alone disturbs intracellular membrane trafficking (Cottam et al, 2011(Cottam et al, , 2014, resulting in an accumulation of singlemembrane vesicles around the microtubule organization complex (Angelini et al, 2013). However, quantitative electron microscopy revealed that expression of TM2 with TM3 prevents the membrane disruption seen with TM3 expression alone (Angelini et al, 2013).…”

Biogenesis and architecture of arterivirus replication organelles

“…This effect was also described for nsp6 of MHV and SARS-CoV, as well as the arteriviral PRRSV nsp5-7 (Cottam et al, 2011). The autophagosomes induced by these proteins appeared to be smaller in size than normal autophagosomes, as judged from light-microscopy images, and therefore closer to viral DMVs (Cottam et al, 2014). However, in contrast with individually expressed nsp6, IBV infection did not induce LC3 relocalization and autophagic signalling in avian cells (Maier et al, 2013a).…”

Manipulation of autophagy by (+) RNA viruses

“…However, when compared to the properties of starvation-induced autophagosomes, those generated by Coronavirus infection or NSP6 proteins presented significant differences. NSP6-induced autophagosomes displayed limited ability to undergo expansion, preventing formation of large autolysosomes, and hence circumvented degradation of viral particles through the lysosomal pathway [43]. Results obtained with IBV, SARS and MHV nsp6 was recapitulated with Middle eastern respiratory syndrome coronavirus (MERS-CoV), where a similar phenomenon was observed for nsp6 [44].…”

“…Infectious bronchitis virus (IBV) -an avian coronavirus responsible for major losses to the poultry industry, is one such example where a significant portion of the genome encodes nonstructural proteins (NSP) dedicated to virus replication. Expression of NSP6 proteins resulted in increased levels of Ptdlns3P on ER membranes, recruitment of Ptdlns3P effector protein WIPI2 and the generation of autophagosomes directly from the ER [42,43]. However, when compared to the properties of starvation-induced autophagosomes, those generated by Coronavirus infection or NSP6 proteins presented significant differences.…”