This study has investigated the ability of individual replicase nonstructural proteins encoded by coronaviruses to activate autophagy. The first experiment showed that IBV infection induced redistribution of LC3 to vesicular structures indicative of autophagosomes. A series of 10 IBV-derived replicase nonstructural proteins derived from the IBV replicase polyprotein were tagged with m-Cherry and expressed in a GFP-LC3 reporter cell line to determine whether they could induce autophagosome formation. Eight proteins showed diffuse fluorescence and were unable to change the distribution of GFP-LC3. However, two of the proteins that contained membrane-spanning motifs, nsp4 and nsp6, localized to the ER, and nsp6 caused redistribution of LC3 to punctate structures indicative of autophagosomes. Recent studies showed that DMVs formed from the ER during coronavirus MHV infection can recruit LC3.29
These DMVs recruited the non-lipidated LC3 (LC3I) by a pathway that is independent of autophagy and linked to the export of ER chaperones from the ER to endosomes.36
The MHV-derived DMVs therefore differ from autophagosomes where recruitment of LC3II to membranes is dependent on autophagy, and requires the Beclin 1/vps34 class III PI3 kinase complex, Atg12-Atg5 and Atg16 and the addition of PE to the C-terminus of LC3. Our results showed that these three stages of autophagy were required for the redistribution of LC3 by IBV nsp6. The LC3 puncta were positive for Atg5, and were not formed in cells lacking Atg5, or when class III PI3 kinase activity was inhibited by wortmannin. In addition, LC3 puncta induced by IBV nsp6 were unable to incorporate a modified G120A LC3 that lacked the glycine required for addition of PE. These observations show that LC3 puncta generated by IBV nsp6 are autophagosomes rather than DMVs related to EDEMosomes. Further evidence for formation of bona fide autophagosomes by IBV nsp6 comes from the observation that the LC3 puncta induced by nsp6 complete the autophagosome-lysosome pathway and deliver LC3 to the interior of lysosomes for degradation. This demonstrates that the autophagosomes generated by IBV nsp6 therefore recruit and recycle the proteins needed for autophagosome nucleation, expansion, cellular trafficking and delivery of cargo to the lysosomes.
The origins of the membranes that generate autophagosomes in response to starvation remain the subject of intense research, and organelles as diverse as the Golgi, ER and mitochondria have been implicated.17,19
Our observation that IBV nsp6 located to the ER raised the possibility that the IBV nsp6 could drive autophagosome formation from the ER. Live cell imaging of autophagosomes formed from the ER in response to starvation has shown that a ring of membrane enriched for PtdIns(3) P, called an omegasome, expands from the ER and recruits LC3 to its interior.19
The LC3 signal subsequently moves from the omegasome and the Ptdlns(3)P signal decays rapidly. The formation of autophagosomes by IBV nsp6 also involved an omegasome intermediate. Nsp6 generated wortmannin-sensitive vesicular structures associated with the ER that recruited Atg5 and LC3. Importantly, the structures induced by nsp6 recruited the DFCP1-GFP probe specific for Ptdlns(3)P showing they contained high levels of the Ptdlns(3)P necessary to nucleate autophagosome formation. The half-life of DFCP-1-GFP recruitment was short and decayed at a similar rate to omegasomes in the presence of wortmannin. The results suggest that IBV nsp6 can increase Ptdlns(3)P concentration at the ER, independently of starvation, and induce omegasomes which then seed formation of autophagosomes. It is important to note that while we showed that exogenously expressed nsp6 activates autophagy, we do not have direct evidence that nsp6 activates autophagy during virus infection. It is possible that during infection autophagy is activated by other viral components, for example dsRNA, or complexes of viral proteins that we have not reconstituted in our experiments. It is also possible that nsp6 is part of a protein complex during viral infection and this may modulate the ability of nsp6 to activate autophagy.
The seven transmembrane domain topology of the IBV nsp6 protein is shared with the nsp6 protein of the mammalian coronaviruses SARS-CoV and MHV, and the nsp5–7 protein of the arterivirus, PRRSV. The ability to generate autophagosomes directly from the ER was retained by these nsp6 orthologs throughout the Nidovirales
order. All four proteins located to the ER where they induced the formation of autophagosomes. Notably the coronavirus nsp6 protein described in this study is the first identified ER resident protein that induces omegasome/autophagosome progression. This provides additional support for the idea that at least some autophagosomes are formed from ER-connected omegasomes and that this pathway can be activated by viruses. Electron micrographs of cells expressing a defective form of the Atg4 protease (Atg4C74A
) required for addition of PE to LC3 reveal crescent-shaped phagophores (IM) sandwiched between ER cisternae.37
These ER-IM complexes contain high concentrations of DFCP1 and are interconnected by narrow membrane bridges. It has been suggested that these specialized domains of the ER facilitate the formation of phagophores.38
It will be interesting to see if similar ER-IM domains are generated by IBV nsp6 to seed autophagosome formation.
The omegasomes and autophagosomes induced by IBV nsp6 formed independently of starvation. A potential explanation was that nsp6 protein could mimic starvation by inhibiting mTOR kinase signaling directly or, as seen for the inhibition of Akt by VSV G protein,39
inhibit nutrient-sensing pathways upstream of mTOR. mTOR signaling, however, appeared to be unaffected since phosphorylation of 4E-BP1 did not change in cells expressing nsp6. Given that all the coronavirus nsp6 ortholog proteins analyzed located to the ER, it was possible that they activated autophagy by inducing ER stress. This appeared unlikely since it was not possible to detect increased levels of CHOP in cells expressing nsp6. Splicing of XBP1 mRNA is an early upstream ER stress response. The nsp6 proteins activated splicing of XBP1 mRNA, however the degree of splicing was less than seen in response to thapsigargin and the same as splicing induced by nsp4, which was unable to induce autophagy. Autophagy can be induced independently of mTOR signaling by activation of sirtuin 1 but inhibition of the sirtuin 1 deacetylase failed to inhibit autophagy induced by nasp6 and the precise mechanism of activation remains unknown. IBV nsp6 did however recruit DFCP-GFP to the ER indicating that induction of autophagosome formation by nsp6 may involve increased production of Ptdlns(3)P. Nsp6 may mimic Atg14 and recruit the Vps34/p150/Beclin 1 complex directly to the ER. Alternatively, nsp6 may inhibit the phosphatase Jumpy that can inhibit autophagosome nucleation by removing phosphate from Ptdlns(3) P.40
Deletion analysis showed that the cytoplasmic domain of nsp6 is not required for activation of autophagy and does not therefore play a role in recruiting proteins to the cytoplasmic face ER.
Large numbers of DMVs are generated in the cytoplasm of cells infected with mammalian coronaviruses and arteriviruses. Electron tomography shows that DMVs generated by SARS-CoV are connected to the ER, with the interior of the DMVs labeling for dsRNA.24
The observation that MHV is unable to generate DMVs in mouse embryonic stem cells lacking Atg5, and that replication is reduced 1,000-fold in the absence of Atg5, suggested a functional link between DMVs, autophagy and virus replication.41
Other studies suggest that MHV does not require Atg5,30
for replication in primary fibroblasts or macrophages and question a role for autophagosomes in virus replication. Our study of the avian coronavirus IBV also failed to provide a link between autophagosomes and virus replication. Double-stranded RNA did not colocalize with LC3 and virus replication was unaffected by inhibitors of autophagy or silencing of Atg5. The DMVs induced by mammalian coronavirus and arteriviruses are approximately one-third the diameter of cellular autophagosomes, suggesting that they are modified autophagosomes, or independent structures induced by assembly of viral proteins. Recent work suggests that the DMVs are related to EDEMosomes, rather than autophagosomes, and recruit LC3I by a pathway independent of autophagy. Autophagy may not, therefore, be required to generate DMVs for replication but, in common with other virus infections, the activation of autophagy seen during coronavirus and arterivirus replication may represent an innate defense against infection to clear virus particles, or a modification of the adaptive immune response by increasing MHC class II antigen presentation. In addition, nsp6 may alter adaptive immune responses by directing immunomodulatory proteins synthesized by the ER into autophagosomes for degradation.