Coronavirus nsp-6 and the inhibition of autophagy

The Coronaviridae, which include viruses capable of infecting  animals as well as humans, belong to order of the Nidovirales and as such are enveloped positive strand ssRNA viruses. 
As described before, they induce the formation of Replication Transcription Complexes (RTCs), essentially double membrane vesicles (DMVs) derived from the ER containing enzymes and viral RNA. The biogenesis of these DMVs has been connected with the early secretory pathway and involves components of the autophagic pathway which in tun is triggered by he accumulation of viral nonstructural proteins (nsps) in the ER, although CoV nsps involved in the formation of DMVs lack conventional signal sequences found in ER or Golgi resident proteins. As described in the earlier post, in the case of CoV the viral nsps-3,-4, and -6 allow the formation of RTCs by not only forming the DMV but also by recruiting the viral replication complex, although DMV, although DMV-like structures can be formed by nsp-3 and nsp-4 in the absence of nsp-6 as well as vice versa. DMVs as well as RTCs have been shown to co-localise with components of the autophagy machinery such as microtubule-associated protein light-chain 3 (LC-3) in both its non-lipidated (LC3-I) and lipidated form (LC3-II) as outlined and discussed before in both CoV infected cells and cells expressing nsp-3,-4, and -6. 

                       nsp-6 and the formation of autophagosomes

Autophagy is normally induced in cells as a response to starvation or to the accumulation of damaged organelles or misfolded proteins that are delivered to lysosomes for degradation. The process begins with the formation of the phagosome (also termed phagophore), which is extended to the double membrane autophagosome that sequesters proteins or organelles marked for degradation by p62/SQSTM-1, NBR, or NIX. 

The formation of the phagophore is initiated by inactivation of mTOR/mTORC-1

As outlined before and shown in the figure the formation of autophagosomes is initiated by interactions of mTOR with the ULK complex and mediated by a complex involving Beclin, ATG14, and Vps34 (Class 3 phosphatidylinositol 3-kinase) thus forming the omegasome enriched in phosphatidylinositol- 3-phosphate (Ptdlns3P). Ptdlns3P in turn recruit Double FYVE-domain containing protein 1 (DFCP-1) and subsequently WD repeat domain phosphoinositide-interacting protein 2 (WIPI2/ATG18), thus allowing the formation of vesicle-like structures. The conversion of LC3-I to LC3-II is facilitated by the Beclin-1/ATG14/Vp34 complex via an ATG12– ATG5-ATG16L conjugate.

The expression of CoV nsp-6 derived from the avian IBV, the murine MHV, and the human SARS-CoV has been shown not only to induce autophagy-like vesicles but also lead to increased levels of Ptdlns3P on ER membranes, leading to the recruitment of effector proteins DFCP-1 and WIPI2 as well as conversion of LC3-I to LC3-II indicating that nsp-6 is sufficient to induce the formation of autophagosomes or autophagosome-like structures. 

CoV nsp-6 initiates the formation of autophagosome-like vesicles via sequestering PtdIns

If these structures however resemble autophagosomes, then the RTCs would subsequently targeted to the lysosomes where viral components would either be degraded or transferred either to multivesicular bodies and/or to endosomes where an antiviral response could be initiated. In order to prevent the degradation of viral components, Polio Virus inhibits the formation of autolysosomes via the viral protein 3A, Influenza Virus via M2, and HIV via the viral Nef protein (to name a few).  in the case of CoV, the expression of mCherry tagged IBV derived nsp-6 induces the formation of LC3-II positive punctae which are negative for LAMP-2, a lysosomal marker, suggesting that nsp-6 inhibits the maturation of autophagosomes into autolysosomes both in starved and non-starved cells. 

CoV nsp-6 inhibits mTORC-1 thus facilliating the formation of LC3-I and LC3-II positive vesicles and preventing the formation of autolysosomes 

Further analysis showed that nsp-6 does so by inhibiting the activity of the mTOR complex-1 (mTORC1) which under conditions of starvation localises to the surface of lysosomes via the Ragulator, the GDP/GFP exchange factor for the RagA/C complex. In the opinion of the author of this post, under normal conditions this may be achieved by nsp-6 localizing to Rab5-GTPase containing early endosomes thus not only inhibiting the RAGA/C complex but also leading to the formation of hybrid endosomes, positive for both Rab5-GTPase and Rab7-GTPase. 

Potential hybrid endosome formation by inhibiting the exchange of Rab5 for Rab7

These then might or might not fuse with LC-3II positive and/or LC3-I motive RTCs. Indeed inhibiting the exchange of Rab5-GTPase for Rab7-GTPase  by expressing a Rab5S34N mutant or a constitutively active Rab5Q79L mutant inhibits the  formation of lysosomes. Alternatively, nsp-6 might activate AMP kinase which in turn induces the dissociation of mTORC1.

Model for nsp-6 induced formation of hybrid endosomes and inactivation of mTORC-1

Further indication that nsp-6 - and the arteriviral nsp-7 - inhibits the maturation of omegasomes to autophagosomes and subsequently autolysosomes comes from the observation that following starvation, nsp-6 induced punctae are smaller in size than conventional autophagosomes (approx. 0.5μM compared to approx. 1.0μM) but bigger in number. In the context of infected cells, it has been shown that both EDEM-1 and OS-9 positive RTCs are not effectively cleared in cells infected with MHV, suggesting that this defect is or might dependent on nsp-6. If this is also the case for substrates targeted via the p62/SQSTM-1 remains to be investigated although nsp-6 does not impair the ability to transfer of substrate it is very limey that the degradation of these substrates is impaired.

Finally, the importance of a functional nsp-6 for Coronavirus replication is highlighted by  that a novel antiviral compound, K22, targets the transmembrane domains VI and VII of nsp-6 derived from HCoV-229E, feline FCoV, MHV-A59, SARS-CoV, IBV, and MERS-CoV. Indeed, resistant mutants of HCoV-229E nsp-6 render HCoV-229E insensitive to K22.

Sites crucial residues required for K22 sensitivity of HCoV-229E nsp-6. Both potential conformations of nsp-6 are shown.

It should be noted however that the most pronounced effect was seen in cells expressing nsp-6 from members of the α- Coronaviruses and the avian Infectious Bronchitis Virus (a γ- Coronavirus) whereas β-Coronaviruses (with the exception of MERS-CoV) are only moderate sensitive to K22. 
It remains to be seen if the individual composition of the RTC - in addition to nsp-6,  -3, and -4 these include the viral replication complex - confer resistance or not. In addition, host cell proteins resident in the ER such EDEM-1 or OS-9 which are part of the EDEMosome might play a role as well. Finally, it remains to be seen, if viruses containing a mix of nsp-3,-4, and -6 derived from different viruses display different patterns of sensitivity or resistance.  

Further reading

Tooze, S., & Yoshimori, T. (2010). The origin of the autophagosomal membrane Nature Cell Biology, 12 (9), 831-835 DOI: 10.1038/ncb0910-831

Cook KL, Soto-Pantoja DR, Abu-Asab M, Clarke PA, Roberts DD, & Clarke R (2014). Mitochondria directly donate their membrane to form autophagosomes during a novel mechanism of parkin-associated mitophagy. Cell & bioscience, 4 (1) PMID: 24669863

Fujita N, Itoh T, Omori H, Fukuda M, Noda T, & Yoshimori T (2008). The Atg16L complex specifies the site of LC3 lipidation for membrane biogenesis in autophagy. Molecular biology of the cell, 19 (5), 2092-100 PMID: 18321988

Liu, D., Fung, T., Chong, K., Shukla, A., & Hilgenfeld, R. (2014). Accessory proteins of SARS-CoV and other coronaviruses Antiviral Research DOI: 10.1016/j.antiviral.2014.06.013

Maier HJ, Cottam EM, Stevenson-Leggett P, Wilkinson JA, Harte CJ, Wileman T, & Britton P (2013). Visualizing the autophagy pathway in avian cells and its application to studying infectious bronchitis virus. Autophagy, 9 (4), 496-509 PMID: 23328491

Cottam EM, Whelband MC, & Wileman T (2014). Coronavirus NSP6 restricts autophagosome expansion. Autophagy, 10 (8) PMID: 24991833

Sancak Y, Bar-Peled L, Zoncu R, Markhard AL, Nada S, & Sabatini DM (2010). Ragulator-Rag complex targets mTORC1 to the lysosomal surface and is necessary for its activation by amino acids. Cell, 141 (2), 290-303 PMID: 20381137

Flinn RJ, Yan Y, Goswami S, Parker PJ, & Backer JM (2010). The late endosome is essential for mTORC1 signaling. Molecular biology of the cell, 21 (5), 833-41 PMID: 20053679

Bar-Peled L, Schweitzer LD, Zoncu R, & Sabatini DM (2012). Ragulator is a GEF for the rag GTPases that signal amino acid levels to mTORC1. Cell, 150 (6), 1196-208 PMID: 22980980

Kitano M, Nakaya M, Nakamura T, Nagata S, & Matsuda M (2008). Imaging of Rab5 activity identifies essential regulators for phagosome maturation. Nature, 453 (7192), 241-5 PMID: 18385674

Li L, Kim E, Yuan H, Inoki K, Goraksha-Hicks P, Schiesher RL, Neufeld TP, & Guan KL (2010). Regulation of mTORC1 by the Rab and Arf GTPases. The Journal of biological chemistry, 285 (26), 19705-9 PMID: 20457610

Zhang CS, Jiang B, Li M, Zhu M, Peng Y, Zhang YL, Wu YQ, Li TY, Liang Y, Lu Z, Lian G, Liu Q, Guo H, Yin Z, Ye Z, Han J, Wu JW, Yin H, Lin SY, & Lin SC (2014). The Lysosomal v-ATPase-Ragulator Complex Is a Common Activator for AMPK and mTORC1, Acting as a Switch between Catabolism and Anabolism. Cell metabolism PMID: 25002183 

Lundin A, Dijkman R, Bergström T, Kann N, Adamiak B, Hannoun C, Kindler E, Jónsdóttir HR, Muth D, Kint J, Forlenza M, Müller MA, Drosten C, Thiel V, & Trybala E (2014). Targeting membrane-bound viral RNA synthesis reveals potent inhibition of diverse coronaviruses including the middle East respiratory syndrome virus. PLoS pathogens, 10 (5) PMID: 24874215