Coronavirus nsp, ER stress, UPR, and ERAD

As discussed before, the essential step in the intracellular life cycle of many positive strand ssRNA viruses is the generation of double membrane vesicles (DMVs) that in most cases (but not all) contain the viral replication and transcription complexes (RTC) and hence serve as the replication platform. In the case of both the Nidovirales and the Flaviviridae these are formed by subverting the membrane of the ER in the absence of conventional ER and secretory pathway markers. Additionally,  non-structural proteins (nsps or NS) do not contain classic sequences such as the KDEL sequence that direct these proteins to the ER. The analysis of the DMVs/RTCs derived from members of both viral families -in particular Mouse Hepatitis Virus (MHV), Equine Arterivirus (EAV), and Japanese Encephalitis Virus (JEV)- however revealed that the DMVs not only contain viral RNA and proteins but also cellular proteins, more specifically components that form the ER-associated degradation (ERAD) vesicle thereby segregating the ERAD factors EDEM-1, OS-9, and SEL-1 from the ER lumen in addition to displaying the non-lipidiated form of LC3, LC3-I, on their cytoplasmic surface. In non-infected cells, the ER is the site of maturation for membrane and secretory proteins. Unfolded or misfolded proteins are normally dislocated across the ER membrane followed by proteasomal and endolysosomal degradation in a process collectively known as ERAD which involves the recruitment of ERAD associated proteins and the subsequent formation of EDEMosomes which form mature autophagosomes and autolysosomes; alternatively, proteins are targeted for proteasomal degradation. Any viral protein therefore which binds EDEM-1, OS-9, and /or SEL-1 therefore would eventually be degraded unless this pathway is inhibited by viral proteins. In the case of the Coronaviral nsp-6 protein, inhibits the maturation of endosomes into lysosomes by preventing the binding and activation of mTORC-1 to the surface of lysosomes through an unknown mechanism as described elsewhere. It would be expected that inhibiting this pathway induces the accumulation of unfolded or misfolded proteins and subsequently induction of apoptosis. In order to counteract the negative effect of accumulated unfolded or misfolded proteins and subsequent ER stress the host cell however induces a unfolded protein response (UPR), a signaling pathway that starts with the activation of three ER stress transducers, (a) PKR- like ER protein kinase (PERK), (b) Activating Transcriptional Factor- 6 (ATF6) and (c) Inositol-requiring Protein-1 (IRE1), which not only increases the expression of genes encoding for components of the ERAD pathway but also induces apoptosis and upregulates the expression of cytokines such as Interferon-β and Interleukin-6 (which of course can be counteracted by CoV). In terms of infection of cells with a Coronavirus such as the avian Infectious Bronchitis Virus (IBV), the infection induces the PERK and IRE1 dependent pathways (as well as PKR, but this pathway is induced by dsRNA and not via ER stress).

Overview of Coronavirus induced UPR via ER stress by orf8ab, nsp-3/-4/-6, and structural S/M/E proteins

As shown in the figure, EDEM1 expression is induced via the splicing of XBP1 whereas the induction of PERK induces apoptosis and the expression of pro-inflammatory cytokines. Whilst the induction of these pathways following the infection with MHV, IBV, or SARS-CoV has been well established it is not clear if the expression of nsp-6, -3, or -4 ( or a combination of these in the absence or presence of other components of the viral RTCs) is sufficient for the induction of the UPR. In infected cells, the maturation of the viral E, M, and S protein involves glycosylation that might increase ER stress in addition to the formation of DMVs. Also, in cells expressing SARS-CoV orf3a the UPR is induced in a PERK dependent manner. Further evidence that components of the ERAD pathway are required for Coronavirus replication and that ERAD tuning is subverted by Coronaviral proteins stems from observations that silencing of EDEM-1, SEL1L, and LC3 impairs the replication of MHV.

Obvious candidates for viral proteins which are responsible for recruiting components of the ERAD pathway are non-structural proteins which are known to localize to the ER and induce the formation of DMVs - in other words, nsp-3,-4, and -6. In principal, these proteins could be recruited in two ways, either by sequestering EDEM-1 and OS-9 following the insertion into the ER or being sequestered to the ER by EDEM-1 and/OS-9. In both cases, EDEM-1/OS-9 might recognize the carboxyterminal helical domain of the respective nsps’. 

Induction of ERAD pathway via nsps-3/-4/-6: localisation and glycosylation recruits EDEM-1/OS-9 (see next figure for legend)

The author of this post favors a model in which cytosolic ERAD recognizes the carboxyterminal helical domain and translocates nsp-3/-4/-6 to the ER where the protein is inserted into the membrane where (accumulated) cytosolic nsps’ are recognised as part of t ERAD-C pathway. Since the nsp’s in question are not marked for entry into the ER, the cell tags them for degradation via the formation of EDEMosomes. This step might be necessary since none of the nsps in question has an intrinsic KDEL sequence which would be recognised by the Signal Recognition Particle (SRP) and thus allow the formation of the RTC. Following the insertion into the membrane, EDEM-1/OS-9 then recruits SEL1L and subsequently LC3-I, forming EDEMosomes, a step which might be preceded by N-glycosylation of the nsps' in question. Targeting nsp-3/-4/-6 by ERAD-C and localizing to the ER might therefore be a requisite for N-glycosylation. Since the ERAD-C pathway is best characterized in S. Cerevisiae,  investigating this possibility might however be problematic unless coronaviral proteins are glycosylated oat the same sites as in mammalian cells. Also, naturally studies using the replicon systems available would not possible.  

Induction of ERAD pathway via nsps-3/-4/-6: ERAD-C localizes nsps followed by glycosylation  and recruitment of LC3-I by SEL1L

In addition, it might be possible that the recruitment of nsp-6 sequesters PtdIns thus inducing the recruitment of factors necessary for the formation of the omegasome. The induction of PtdInsP3 by recruiting the cellular Vp34 kinase complex might also counteract the induction of apoptosis by prolonged ER stress and thus favour viral replication.

Finally, it should be noted that so far omegasomes have shown not to contain EDEM-1/OS-9 so it might be possible that nsp-6 either does not recruit components of the ERAD pathway, preferentially induces the formation of omegasomes via PtdIns, or that EDEMosomes are degraded (in contrast to omegasomes). Since in MHV infected cells the degradation of EDEMosomes is blocked it is possible that nsp-6 induced vesicles contain EDEM-1 or that nsp-6 by itself does not recruit EDEM-1/OS-9. This however is open for investigation and I hope that somebody is up to challenge.

The basic question of course is why any virus would express proteins that upon localisation to the ER induce a response which is potentially lethal to the host cell? The UPR not only induces apoptosis or the expression of proteins such EDEM-1 that have the potential to degrade viral components but also increases the expression of ER chaperones. This protein family -with BiP, Calnexin, ERp57, and PDI the most abundant members- have key roles in the folding of cellular membrane proteins and therefore may be also used for correct folding of viral proteins located in the envelope of viruses or having other structural finctions. Since these proteins are generally only required relatively late in the replication cycle, viruses need to develop strategies to avoid early apoptosis. One example is African Swine Fever Virus (ASFV) that triggers an UPR and prevents early apoptosis by inducing the ATF6 pathway. The SARS-CoV orf8ab protein in a similar way not only localizes to the ER lumen, but induces UPR by activation of ATF6.  If however the mere expression of nsp-3/-4/-6 causes UPR and subsequently ATF6 - this remains to be seen.   

Further reading

Bernasconi R, & Molinari M (2011). ERAD and ERAD tuning: disposal of cargo and of ERAD regulators from the mammalian ER. Current opinion in cell biology, 23 (2), 176-83 PMID: 21075612

Verchot, J. (2014). The ER quality control and ER associated degradation machineries are vital for viral pathogenesis Frontiers in Plant Science, 5 DOI: 10.3389/fpls.2014.00066 

Noack J, Bernasconi R, & Molinari M (2014). How viruses hijack the ERAD tuning machinery. Journal of virology PMID: 24990995 

Fung TS, & Liu DX (2014). Coronavirus infection, ER stress, apoptosis and innate immunity. Frontiers in microbiology, 5 PMID: 24987391 

Minakshi R, Padhan K, Rani M, Khan N, Ahmad F, & Jameel S (2009). The SARS Coronavirus 3a protein causes endoplasmic reticulum stress and induces ligand-independent downregulation of the type 1 interferon receptor. PloS one, 4 (12) PMID: 20020050 

Reggiori F, Monastyrska I, Verheije MH, Calì T, Ulasli M, Bianchi S, Bernasconi R, de Haan CA, & Molinari M (2010). Coronaviruses Hijack the LC3-I-positive EDEMosomes, ER-derived vesicles exporting short-lived ERAD regulators, for replication. Cell host & microbe, 7 (6), 500-8 PMID: 20542253

Hagemeijer MC, Ulasli M, Vonk AM, Reggiori F, Rottier PJ, & de Haan CA (2011). Mobility and interactions of coronavirus nonstructural protein 4. Journal of virology, 85 (9), 4572-7 PMID: 21345958 

Bernasconi R, Noack J, & Molinari M (2012). Unconventional roles of nonlipidated LC3 in ERAD tuning and coronavirus infection. Autophagy, 8 (10), 1534-6 PMID: 22895348 

Bernasconi R, Galli C, Noack J, Bianchi S, de Haan CA, Reggiori F, & Molinari M (2012). Role of the SEL1L:LC3-I complex as an ERAD tuning receptor in the mammalian ER. Molecular cell, 46 (6), 809-19 PMID: 22633958 

Ast T, Aviram N, Chuartzman SG, & Schuldiner M (2014). A cytosolic degradation pathway, prERAD, monitors pre-inserted secretory pathway proteins. Journal of cell science PMID: 24849653

Sun S, Shi G, Han X, Francisco AB, Ji Y, Mendonça N, Liu X, Locasale JW, Simpson KW, Duhamel GE, Kersten S, Yates JR 3rd, Long Q, & Qi L (2014). Sel1L is indispensable for mammalian endoplasmic reticulum-associated degradation, endoplasmic reticulum homeostasis, and survival. Proceedings of the National Academy of Sciences of the United States of America, 111 (5) PMID: 24453213 
 
Baliji, S., Cammer, S., Sobral, B., & Baker, S. (2009). Detection of Nonstructural Protein 6 in Murine Coronavirus-Infected Cells and Analysis of the Transmembrane Topology by Using Bioinformatics and Molecular Approaches Journal of Virology, 83 (13), 6957-6962 DOI: 10.1128/JVI.00254-09

Clementz MA, Kanjanahaluethai A, O'Brien TE, & Baker SC (2008). Mutation in murine coronavirus replication protein nsp4 alters assembly of double membrane vesicles. Virology, 375 (1), 118-29 PMID: 18295294 

Gadlage MJ, Sparks JS, Beachboard DC, Cox RG, Doyle JD, Stobart CC, & Denison MR (2010). Murine hepatitis virus nonstructural protein 4 regulates virus-induced membrane modifications and replication complex function. Journal of virology, 84 (1), 280-90 PMID: 19846526 

Kanjanahaluethai A, Chen Z, Jukneliene D, & Baker SC (2007). Membrane topology of murine coronavirus replicase nonstructural protein 3. Virology, 361 (2), 391-401 PMID: 17222884 


Axe EL, Walker SA, Manifava M, Chandra P, Roderick HL, Habermann A, Griffiths G, & Ktistakis NT (2008). Autophagosome formation from membrane compartments enriched in phosphatidylinositol 3-phosphate and dynamically connected to the endoplasmic reticulum. The Journal of cell biology, 182 (4), 685-701 PMID: 18725538 

Thakur PC, Davison JM, Stuckenholz C, Lu L, & Bahary N (2014). Dysregulated phosphatidylinositol signaling promotes endoplasmic-reticulum-stress-mediated intestinal mucosal injury and inflammation in zebrafish. Disease models & mechanisms, 7 (1), 93-106 PMID: 24135483 

Galindo, I., Hernáez, B., Muñoz-Moreno, R., Cuesta-Geijo, M., Dalmau-Mena, I., & Alonso, C. (2012). The ATF6 branch of unfolded protein response and apoptosis are activated to promote African swine fever virus infection Cell Death and Disease, 3 (7) DOI: 10.1038/cddis.2012.81 

Sung SC, Chao CY, Jeng KS, Yang JY, & Lai MM (2009). The 8ab protein of SARS-CoV is a luminal ER membrane-associated protein and induces the activation of ATF6. Virology, 387 (2), 402-13 PMID: 19304306

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

Autophagy, Endosomes, and Viral Entry: UVRAG as an interlocutor

Cellular autophagy is an intracellular membrane trafficking pathway which delivers cytoplasmic material to lysosomes where the material is degraded thereby removing not only damaged organelles -as in the case of mitophagy- but also as part of the unfolded protein response misfolded proteins (aggrephagy) or the replication process of viruses (xenophagy). Generally, autophagy is induced under conditions of cellular stress such as nutrient withdrawal, DNA damage, or viral infections. During this process, isolation membranes (the phagosome) derived from the ER are formed and expanded into double-membrane autophagosomes that engulf the cellular cargo. Autophagosomes then fuse either with late Endosomes or Lysosomes forming the autolysosome where the cargo is degraded by lysosomal enzymes. The induction of autophagy is regulated by mammalian target of rapamycin complex-1 (mTORC1) kinase, that regulates a complex consisting of unc-51-like kinase (ULK-1), focal adhesion kinase family interacting protein of 200 kDa (FIP200), Atg13, and Atg101. ULK-1 activates a downstream complex couch as the phsophatidylinositol 3-kinase (PI3-kinase) complex (Atg6/beclin1-Atg14-Vps15-Vps34) and the Atg12 (Atg12-Atg5-Atg16) system as well as the LC-3 ubiquitin-like conjugation system that converts LC3-I to LC3-II in a PI3-kinase dependent manner by recruiting the Atg12-Atg5-Atg16 complex to the phagosome. 

Outline of autophagy, its enzymes and inhibitors

In the absence of starvation, mTORC1 inhibitors such as rapamycin or torin can activate the formation of the autophagosome, whereas PI-3 kinase inhibitors such as Wortmannin can inhibit the recruitment of Beclin1/Atg14/Vps15/Vps34. In general, the engulfment of cytoplasmic components is non-specific but ubiquitinylated proteins can be recruited to the autophagosomes specifically by nuclear or cytosolic p62/Sequestosome-1 (SQSTM1) thus binding polyubiquitinated proteins to membrane-associated LC3-II.

In contrast to intracellular cargo, extracellular cargo that is internalized by endocytosis, endocytosed macromolecules are delivered to lysosomes via endosomes, bypassing the autophagy pathway. This pathway involves a “kiss and run” as well as a complete fusion event, the latter dependent on the presence of a target membrane (t-) SNARE complex and a vesicle membrane (v-) SNARE complex which form a tetrameric transSNARE complex. Late endosomes can either fuse with other late endosomes (“homotypic fusion”) or other lysosomes (“heterotypic fusion”), the former requiring Syntaxin-8, VAMP-8, and Vti-1b and the latter Syntaxin -11, VAMP-7 as well as Vti-1b. Both homotypic and heterotypic fusion are also dependent on Rab5GTPase, Rab7GTPase, N-ethylmaleimide (NEM) sensitive factor (NSF) and its soluble associated proteins (SNAPs).

Both the autophagy and the endocytic pathway are not completely separated but united by a common interlocutor, UV radiation resistance-associated (UVRAG). UVRAG activates autophagy by associating with Beclin-1 (thus extending the phagophore) as well as inducing the maturation of the autophagosome in later stages via binding to C/Vps, and accelerating endocytic transport by activating Rab7 (thus leading to heterotypic fusion of the late endosome with lysosomes).  In addition to the role in autophagy and fusion of endosomes with lysosomes, UVRAG also plays a role in the integrity of the ER and the Golgi as well in the DNA damage response.

UVRAG as the interlocutor between autophagy and homotypic endosome fusion

Although the role of various autophagy related proteins in viral infections is well established, the role of UVRAG in particular in mediating viral entry has been elusive. A potential role for UVRAG in mediating viral entry can be postulated from the observation that in UVRAG deficient cells cell surface receptor degradation is downregulated. Since UVRAG overexpression is known to target viral proteins to autophagosomes (and thus lead to potential degradation of viral components) and cell surface receptors to lysosomal degradation, findings that suggest that UVRAG overexpression leads to increased viral replication of two negative strand RNA viruses -Influenza A and Vesicular Stomatitis Virus (VSV) - seem at first counterintuitive. Both viruses however encode proteins that inhibit the autophagy pathway (VSV-G and Influenza Virus M2 protein) thus counteracting the antiviral autophagy response, either by inhibiting Akt kinase mediated activation of mTOR (VSV-G) or the degradation of the autophagosome (Influenza Virus M2). On the other hand, UVRAG seems to be required for the replication of VSV and Influenza Virus, suggesting that UVRAG targets internalized viral particles to structures, that prevent them from being degraded and/or recognised by Pattern Recognition Receptors (PRRs) which as we have seen in a different post are an essential part of the cellular antiviral response. By infecting HeLa cells with DiI-labelled VSV,  it was shown that viral particles localize to (acidic) late endosomes in a UVRAG dependent manner. Mutational analysis of UVRAG determined that UVRAG mediated virus entry is dependent on the interaction between the C2 and CDD domain of UVRAG and C/Vps as well as Vps-16 and -18. Successful targeting of VSV to endosomes is in addition dependent on VAMP-8. Indeed, VAMP-8 is recruited to VSV-G and Influenza Virus M protein positive vesicles. Since the endosome is involved in recognizing viral RNA via Toll-like receptors, it might be interesting to determine if these endosomes are positive for TLRs or if viral proteins inhibit the antiviral signaling.

VSV-G and UVRAG

It remains to be seen if the infection of cells with positive strand RNA viruses such as Coronaviruses or Enteroviruses or the infection with DNA viruses induces the formation of similar structures or if these are limited to negative sense RNA viruses. As discussed before, positive strand RNA viruses induce the formation of replication transcription complexes and induce autophagy via viral nsps. Targeting viral particles to late endosomes by recruiting UVRAG however might be required early in the replication cycle prior to the formation of RTCs. In this context it might be possible that these early (hypothetical) structures are transported to the site of replication via the cytoskeleton. Indeed, late endosomes have been shown to be transported towards the perinuclear MTOC in a Rab7GTPase dependent manner and VSV-G co-localises with acidic endosomal vesicles in the perinuclear region in a Nocadozole sensitive manner.  

Finally, it remains to be seen if the endosomal structures induced by the interaction between VSV-G and Influenza Virus respectively, can mature into LC3-II positive autophagosomes via ATG9; it might be possible however that both VSV-G and Influenza Virus M2 proteins. The finding that the localisation of VSV and Influenza Virus to acidic vesicles is dependent on UVRAG is contrasted by Lassa Virus (LASV) which also localises to acidic vesicles. In contrast to VSV-G, the LASV glycoprotein (LASV-GP) mediates viral entry by triggering a receptor switch from glycosylated α-dystroglycan (α-DG) to LAMP1 in a sialyltransferase ST3GAL4 dependent manner upon infection of chicken embryonic fibroblasts, human HAP1, and HEK293T cells. LASV and other Arenavirus' do form cytoplasmatic RTCs but these might not derive from autophagic vesicles akin to the RTC of positive strand RNA viruses.

Autophagosomes are known to be induced upon entry of various other viruses. Food Mouth and Disease Virus (FMDV) and Vaccinia Virus (VACV) induces the formation of autophagosomes in a PI3-Kinase independent pathway (maybe UVRAG and/or ATG9 dependent?), and Echovirus 7 utilizes the autophagy pathway for its entry by an yet unidentified mechanism (although ATG16L is required) ditto for Dengue Virus. African Swine Fever Virus (AFSV) particles localize to the late endosome, suggesting that UVRAG might be important as well. 

In conclusion, UVRAG might be an universal connector between viral entry the induction of autophagy.

Further reading

Dreux, M., & Chisari, F. (2011). Impact of the Autophagy Machinery on Hepatitis C Virus Infection Viruses, 3 (12), 1342-1357 DOI: 10.3390/v3081342 

Pryor PR, Mullock BM, Bright NA, Lindsay MR, Gray SR, Richardson SC, Stewart A, James DE, Piper RC, & Luzio JP (2004). Combinatorial SNARE complexes with VAMP7 or VAMP8 define different late endocytic fusion events. EMBO reports, 5 (6), 590-5 PMID: 15133481 

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