Measles Virus: a lot of autophagy

In non-infected cells, autophagy is associated with the degradation of proteins in particular upon induction of cell stress such as starvation or oxidative stress. Upon infection of cells with viruses however the induction of autophagy leads to the degradation of viral components or virions and therefore constitutes an antiviral response. In addition, the autophagic pathway can deliver viral genomes to TLR containing endosomes and thus induce a antiviral type I Interferon response, whereas viral proteins undergoing autophagy can contribute to virus derived peptide presentation on MHC- Class I and Class II molecules respectively. Thus autophagy constitutes an essential part of the antiviral response and a number of viral proteins- such as HSV-I ICP 34.5 or the CoV nsp-6 -have indeed been shown to inhibit autophagy flux. The induction of autophagy however also counteracts cellular apoptosis induced by the expression of viral proteins and also can provide a compartment for viral replication such as the replication centers observed in cells infected with Chikungunya Virus or Infectious Bronchitis Virus.

Measles is a highly contagious disease cause by the infection with Measles Virus (MeV), a Morbillivirus belong to the order of the Paramyxoviridae, of the respiratory tract characterised by a distinctive maculapapular rash and fever among other symptoms, causing also severe complications such as post-infectious encephalitis. MeV is a negative strand (non-segmented) enveloped RNA virus encoding for six structural and two non-structural proteins. So far three host cell receptors for MeV have been identified, CD46 (all human somatic cells), SLAM/CD150 (immature thymocytes, activated B-/T-Lymphocytes, macrophages and mature dendritic cells), and NECTIN-4 (epithelial cells) with clinical/virulent strains attaching to SLAM/CD150 and NECTIN-4 and vaccine/attenuated laboratory strains attaching to either SLAM/CD150, CD46 or NECTIN-4.

                         MeV and the autophagic pathway

Upon infection of the host cell with MeV, two phases of autophagy can be observed, the first upon entry and involving the surface receptor (CD46) and the scaffold protein (GOPC) independent of viral replication with the second phase being part and dependent of viral replication and initiated by viral C protein. Apart from these two phases, two more can be distinguished which are however part of the first two: one initiated by and part of the antiviral signalling which involves mitochondrial signalling events, and another which facilitates the formation of syncytia, resulting in cell-cell fusion and a sustained autophagy independent of the C protein. 

                         Viral entry of MeV and autophagy

As mentioned above, both the cellular SLAM/CD150 and CD46 proteins are receptors for clinical and attenuated MeV strains. Coexpression of both proteins in Vero cells and subsequent infection with MeV induces the formation of LC3 positive punctae, indicative of mature autophagosomes. Indeed, SLAM/CD150 has been shown to recruit the intracellular PI-3K kinase Vps34 as well as Vps15 and Beclin-1 and thus induce the formation of the phagophore. As in the case of CD46, infection with MeV induces the formation of autophagosomes and mature autolysosomes in a ATG5 and Beclin-1 dependent manner -albeit independent of the scaffold protein GOPC and Cyt-1- suggesting that the engagement of both receptors by MeV -probably the viral hemagglutinin (H) and Fusion (F) proteins- allows the entry of the viral particle via autophagy. For productive infection however degradation of the viral particle via the autolysosome would result in an abortive infection. 
So how to reconcile these results obtained from different studies? As discussed in a prior post, the entry of various viruses and subsequent release of the viral genome requires the localisation of the viral particle to the endosome, a process which in some viruses involves subverting components of the autophagic pathway, including Beclin-1 and UVRAG. In my opinion the problem with the studies postulating the induction of autolysosomes is, that the authors only showed the formation of acidic vesicles by using GFP-RFP-LC3 tandem construct, yet failed to identify the nature of these vesicles by labeling them for lysosomal and endosomal markers, which would have confirmed that the viral proteins undergo degradation and/or localisation to endosomes. In short, it is possible that the entry of MeV induces the recruitment of components of the autophagic pathway to SLAM/CD150 and CD46 and exposes the viral RNA to TLR located within the endosome. Indeed, SLAM/CD150 dependent entry of E.coli localises internalised bacteria to EEA-1 positive endosomes prior degradation in the lysosome, a process that involves the recruitment of Vps34 and Beclin-1.

Induction of autophagy leads to the formation of amphisomes prior to
the formation autolysosomes

The delivery of viral particles to the endosome might also trigger a TLR dependent induction of the Interferon response, which might be counteracted via proviral IRGM signalling and subsequent mitophagy.

MeV, IRGM, Mitochondria and autophagy: inhibition of antiviral signalling

As outlined before, antiviral signalling induced by binding of viral ds or ssRNA to Pattern Recognition Receptors (PRR)  such as RIG-1 or RIG-1 like receptors - in particular MDA5 and PACT- involves the binding to to the mitochondrial MAVS via CARD domains, which ultimately induces the type I Interferon response via a TRAF6 dependent signalling pathway. Additionally, MAVS mediated signalling induces the formation of a MAM complex by binding to ER resident proteins which in turn facilitates mitophagy.

In addition to the MAM complex, the immunity-related GTPase family M (IRGM) protein has been shown to interact with cellular proteins that are part of the complex allowing the formation of autophagosomes, namely ATG5, ATG10, (MAP)LC3C, and SH3 domain GRB2-like endophilin B1 (SH3GLB1)/Bif-1. Interestingly, one of these proteins, SH3GLB1/Bif-1, has been reported to associate with Bax/Bak and implicated in activating mitochondrial depolarization and cytochrome c release, although the results are conflicting.

IRGM activation counteracts the IFN response induced by RIG-1
via the induction of autophagy

The presence of these proteins and the induction of autophagy inhibits the induction of the Interferon response following the translocation of the dsRNA/RIG-1 complex to the mitochondrial membrane by inducing mitophagy and thus constitutes a proviral signalling pathway. Indeed, interactome analysis revealed that IRGM1 interacts not only with 44 autophagy associated proteins but also with 83 viral proteins from different RNA viruses, with 12 viral proteins belonging to just five different viruses - Chikungunya Virus (CHIKV) Measles Virus (MeV, Mumps Virus (MuV), HIV-1, and Hepatitis C Virus (HCV). With the exception of MuV, so far all viruses have been shown to interfere with the autophagic pathway.  The importance of IRGM for MeV induced autophagy is highlighted that in Hela or Huh7.5 cells treated with siRNA targeting ATG5 or IRGM1, the formation of autophagosomes is reduced as are viral titres; similar results were obtained for HCV and HIV but not i(n the case of siRNA IRGEM1) Influenza A/New Caledonia (H1N1).  

Proteins from different viruses whose autophagy is dependent on IRGM

For MeV, the viral C protein has been shown to co-localise with the IRGM in the absence of other viral proteins or viral RNA, suggesting that C binds and recruits IRGM to the perinuclear region into aggregates that might or might not represent aggregates of mitochondria. Both HIV-Nef and HCV NS3A co-localise with IRGM1 in a similar way and show IRGM dependent autophagy. Infection of non-small cell lung cancer (NSCLC) cells with the Edmonston strain of MeV (MV-Edm) suggest that indeed the induction of mitophagy mitigates the RIG-1 response which also involves p62/SQSTM1; if this however is dependent on IRGEM1 remains to be seen. Since the expression of the viral Capsid protein induces the formation of aggregates containing IRGM1, the interaction of both proteins might form a complex that targets mitochondria to the perinuclear region -maybe the ER?- prior to mitophagy, similar to the formation of MAM complexes by EMCV. If this is the case, then sustained mitophagy would deplete RIG-1 and thus prevent antiviral signalling.

Induction of mitophagy via the binding of C to IRGM and/or formation of
a MAM complex

        MeV: syncytia formation, cell to cell spread and autophagy

The importance for sustained autophagy during viral replication was first highlighted in VerodogSLAMtag cells infected with a relative of MeV, Canine Distemper Virus (CDV). Treatment of VerodogSLAMtag with Chloroquine or transfection with siATG7 prior to the infection with CDV reduces the formation of syncytia with neighbouring and cell to spread, indicating that sustained autophagy induced during viral replication is required for syncytia formation (but not for viral replication). Moreover, cells infected with MeV or CDV exhibit an increase in mature autophagosomes that are not degraded in lysosomes at 20 h p.i. and do not represent viral replication centers. Further analysis confirmed previous results that both the viral H- and F- glycoproteins are required for induction of autophagy, in a process that also contributes to the downregulation of the cell surface expression of the SLAM/CD150 receptor. Indeed, H, F, and SLM/CD150 co-localise at the ER, suggesting that the formation of autophagosomes via the interaction between these proteins induce the formation of autophagosomes similar to the processes observed during viral entry.  

Autophagy induced by the H/F proteins however is not sufficient to induce the formation of syncytia per se. On the other hand, fusion with neighbouring cells is necessary to prevent the degradation of the autophagosomes as the treatment with FIP, a synthetic permitted inhibiting cell-cell fusion, decreases the number of GFP-LC3 positive punctae in cells expressing the H/F proteins indicating the necessity of membrane fusion. The problem is that in some experiments cells expressing the H and F protein still require PEG for cell-cell fusion and it has been suggested that the viral C protein might be required as well.

Induction of autophagy by the viral H and F as well as the cellular SLAM/CD150
may be induced by ER stress and p62/SQSTM1 dependent 

In the opinion of the author of this post, one of candidates required for inducing syncytia formation upon induction of secondary autophagy might be connexin43. In this case, the formation of the autophagosome and subsequent localisation to the cell-cell interface involves the induction of autophagy via connexin 43 in a ATG14L sensitive manner.

Since as we have observed above, the initial infection might induce the formation of endosomes, the transferred viral particle might be able to initiate replication in neighbouring cells without the requirement of binding to cell surface receptors. More importantly, neighbouring cells might be “primed or sensitised” to viral infection by downregulating the Interferon response. This might also explain the severity of secondary infections with bacterial or viral pathogens following the initial infection with MeV.

Cell to cell spread might be facilitated by activation of connexin 43 mediated autophagy

Interestingly, the second wave of autophagy is dependent on the non-structural MeV C protein and probably on the interaction with IRGM1 since the infection of MeV C deficient strain, Schwarz (Sch)-MeV Δ C, does not induce autophagy 24 h p.i. .

One of the questions which remains to be covered is if the mitophagy induced upon MeV infection induces apoptosis. In the case of oncolytic MeV, apoptosis has been reported to be induced upon mitophagy induction by MeV in cancer cells. If this is the case in primary cells remains to be seen. 

Apart from Measles Virus, the results obtained from MeV might also be applicable to other viruses such as Nipah or Hendra Virus, and the envelope proteins of both viruses have been shown to induce autophagy and membrane fusion.

The role of IRGM in viral infections might not only be limited to the iduciton of mitophagy and inhibiting the Interferon response. In cells infected with Coxsackievirus B, the IRGM isoform 3 has been reported to relieve ER stress by inducing autophagy in PI-3-K dependent manner. If this is the case in cells infected with MeV or transfected with the viral C prtein remains to be seen, but is not impossible since the C protein being an ER resident protein might induce ER stress as it is the case for the viral H and F proteins. Sustained autophagy might therefore serve multiple purposes - counteracting ER stress, enabling cell to cell spread, and counteract antiviral signalling. 

Further reading


Ader N, Brindley M, Avila M, Örvell C, Horvat B, Hiltensperger G, Schneider-Schaulies J, Vandevelde M, Zurbriggen A, Plemper RK, & Plattet P (2013). Mechanism for active membrane fusion triggering by morbillivirus attachment protein. Journal of virology, 87 (1), 314-26 PMID: 23077316

Bose S, Song AS, Jardetzky TS, & Lamb RA (2014). Fusion activation through attachment protein stalk domains indicates a conserved core mechanism of paramyxovirus entry into cells. Journal of virology, 88 (8), 3925-41 PMID: 24453369 

Joubert PE, Meiffren G, Grégoire IP, Pontini G, Richetta C, Flacher M, Azocar O, Vidalain PO, Vidal M, Lotteau V, Codogno P, Rabourdin-Combe C, & Faure M (2009). Autophagy induction by the pathogen receptor CD46. Cell host & microbe, 6 (4), 354-66 PMID: 19837375 

Ludlow M, Allen I, & Schneider-Schaulies J (2009). Systemic spread of measles virus: overcoming the epithelial and endothelial barriers. Thrombosis and haemostasis, 102 (6), 1050-6 PMID: 19967134 

Grégoire IP, Richetta C, Meyniel-Schicklin L, Borel S, Pradezynski F, Diaz O, Deloire A, Azocar O, Baguet J, Le Breton M, Mangeot PE, Navratil V, Joubert PE, Flacher M, Vidalain PO, André P, Lotteau V, Biard-Piechaczyk M, Rabourdin-Combe C, & Faure M (2011). IRGM is a common target of RNA viruses that subvert the autophagy network. PLoS pathogens, 7 (12) PMID: 22174682 

Er, E., Oliver, L., Cartron, P., Juin, P., Manon, S., & Vallette, F. (2006). Mitochondria as the target of the pro-apoptotic protein Bax Biochimica et Biophysica Acta (BBA) - Bioenergetics, 1757 (9-10), 1301-1311 DOI: 10.1016/j.bbabio.2006.05.032 

Deretic V (2012). Autophagy as an innate immunity paradigm: expanding the scope and repertoire of pattern recognition receptors. Current opinion in immunology, 24 (1), 21-31 PMID: 22118953

Richetta C, Grégoire IP, Verlhac P, Azocar O, Baguet J, Flacher M, Tangy F, Rabourdin-Combe C, & Faure M (2013). Sustained autophagy contributes to measles virus infectivity. PLoS pathogens, 9 (9) PMID: 24086130 

Delpeut S, Rudd PA, Labonté P, & von Messling V (2012). Membrane fusion-mediated autophagy induction enhances morbillivirus cell-to-cell spread. Journal of virology, 86 (16), 8527-35 PMID: 22647692 

Berger SB, Romero X, Ma C, Wang G, Faubion WA, Liao G, Compeer E, Keszei M, Rameh L, Wang N, Boes M, Regueiro JR, Reinecker HC, & Terhorst C (2010). SLAM is a microbial sensor that regulates bacterial phagosome functions in macrophages. Nature immunology, 11 (10), 920-7 PMID: 20818396 

Welstead GG, Hsu EC, Iorio C, Bolotin S, & Richardson CD (2004). Mechanism of CD150 (SLAM) down regulation from the host cell surface by measles virus hemagglutinin protein. Journal of virology, 78 (18), 9666-74 PMID: 15331699 

Xia M, Meng G, Li M, & Wei J (2014). Mitophagy in Viral Infections. DNA and cell biology PMID: 25050805 

Xia M, Meng G, Jiang A, Chen A, Dahlhaus M, Gonzalez P, Beltinger C, & Wei J (2014). Mitophagy switches cell death from apoptosis to necrosis in NSCLC cells treated with oncolytic measles virus. Oncotarget, 5 (11), 3907-18 PMID: 25004098 

Kang R, Zeh HJ, Lotze MT, & Tang D (2011). The Beclin 1 network regulates autophagy and apoptosis. Cell death and differentiation, 18 (4), 571-80 PMID: 21311563 

Bejarano E, Yuste A, Patel B, Stout RF Jr, Spray DC, & Cuervo AM (2014). Connexins modulate autophagosome biogenesis. Nature cell biology, 16 (5), 401-14 PMID: 24705551 

Xia M, Gonzalez P, Li C, Meng G, Jiang A, Wang H, Gao Q, Debatin KM, Beltinger C, & Wei J (2014). Mitophagy enhances oncolytic measles virus replication by mitigating DDX58/RIG-I-like receptor signaling. Journal of virology, 88 (9), 5152-64 PMID: 24574393

Encephalomyocarditis virus (EMCV) and autophagy: anti - and proviral effects

Encephalomyocarditis virus (EMCV) is small non-enveloped ssRNA with a genome of 7.8 kb and thus considerably smaller than CoV. Being the causative agent of myocarditis, encephalitis, reproductive disorders, diabetes, and neurological diseases, EMCV pathogenesis is viral strain and host specific. Although EMCV infection in humans are only associated with low morbidity, the use porcine xenografts may cause future problems.

                                          EMCV and the autophagy pathway

Permissive rodent cell lines derived from hamster, rat and mice infected with EMCV exhibited an increase in LC3-II positive double or single membrane vesicles in the perinuclear region indicating that the infection of cells with EMCV induces autophagy, although in BHK-1 cells the kinetics are slower compared to rat H9c2 or murine BV cells. Furthermore, the induction of autophagosomes is dependent on viral replication as UV inactivated virus fails to increase the levels of LC3-II. The formation of autophagosomes increases over time as a cumulative amount of LC3-II increases as the infection progresses., although the increase of LC3-II itself however only indicates that autophagosomes are accumulating but not necessarily subject to lysosomal degradation. Treatment of infected cells with E64d -an inhibitor of lysosomal hydrolases- therefore should increase the number of LC3-II positive punctae, as it indeed it is the case in EMCV infected BHK-1 cells; EMCV infection therefore enhances the autophagic flux. Interestingly, the treatment of EMCV infected cells also increases the levels of the viral VP1 protein, suggesting that the induction of autophagy might be a cellular antiviral response rather than beneficial for viral replication.  Concomitant with the increase in the formation of autophagosomes is a degradation of p62/SQSTM1, which initially suggested that EMCV might induce selective autophagy. In the light of recent results indicating that the Coxsackievirus B3 viral proteases 3C^Pro and 2A^Pro  cleave cellular p62/SQSTM1 and that the p62/SQSTM1 C-terminal fragment inhibits the formation of p62/SQSTM1 positive punctae, this result needs to be revisited and  it is the opinion of the author that EMCV might not induce selective autophagy per se - which of course does not rule out that one or more viral protein might do so. Indeed, recent results suggest that the overexpression of the viral protein 2C and 3D in the absence of the viral proteases induces the degradation of p62/SQSTM1 by selective autophagy. 

The notion that the induction of autophagy by EMCV constitutes an antiviral response is further supported by finding that in infected MEF treated with Bafilomycin A or siRNA against Beclin-1 or ATG5 viral replication increases rather than decreased. This picture is however is complicated by finding that the effect of Bafilomycin A treatment on viral yields depends on when the virus was harvested -with viral yields higher at 7 h p.i. in BafA1 treated compared to untreated cells whereas at 12 and 16 h p.i. autophagy inhibition reduces the viral yield. In other words, at the early stages of infection the increase of autophagy induces an antiviral response whereas later in infection the formation of autophagosomes favours viral replication. How can these conundrums being solved? When we look closer at the experiments conducted with E64d, which suggest that the autophagosomes formed are degraded within the lysosome then we see that cell extracts from cells at more than 16 h p.i. were not analysed. It may therefore possible that from 16 h p.i. onwards, the autophagosomes formed are not degraded but part of the viral replication centers.  In a further twist, the kinetics might differ between the viral strain and the cell line used as well as dependent on the MOI.

So if the induction of autophagy early in the replication cycle can be considered an anti - rather than a pro-viral pathway, then we have to ask, how is this pathway induced? Being a Picornavirus, EMCV has a ssRNA genome. As discussed for the Coronaviridae, ssRNA viruses produce dsRNA intermediates that alongside the viral ssRNA can be recognised by cellular pathogen recognition receptors (PRR), which in turn induce the expression of cytokines, including Interferon-γ (IFN-γ).  Among other genes, IFN-γ induces the expression of 2,5 oligoadenylate synthetase (2,5-OAS) in a STAT1 and BRCA1 dependent manner. 2,5-OAS in turn activates RNaseL, thus degrading viral RNA and damaging components of the host cell -including ribosomes- required for viral replication. RNaseL has also been shown to induce autophagy and it is indeed this mechanism which might induce autophagy early in infection by phosphorylating Bcl-2 in  N terminal JNK dependent manner, thus dissociating BcL-2 from Beclin-1. Interestingly, expression and activation of RNaseL also degrades p62/SQSTM1, suggesting that at least early in infection it is RNaseL and not the viral 3C^Pro protease that cleave p62/SQSTM1, which is further supported by results that in cells expressing an inactive R667A RNaseL mutant p62/SQSTM1 is not degraded at 16 h p.i. Although so far the precise mechanism has not been elucidated, EMCV and other Picornaviruses have been postulated to inhibit RNaseL later in infection.

In any case, the activation of RNaseL via 2,5-OAS binding to viral RNA not only degrades viral RNA and inhibits viral replication, but also allows the recognition of these RNA fragments by PACT and MDA5. Both proteins bind to the mitochondrial-associated adaptor molecule MAVS and thus activating a pathway which ultimately increases the expression of IFN-β and other chemokines. In addition - and this is the important point in the context discussed in this post- PPRs such as RIG-1 or  the RIG-1 like protein MDA5 both of which contain a MAVS domain also bind to the ER where they form a mitochondrial-associated membrane (MAM) complex via MAVS, forming an immune synapse. Recruitment of ATG14L via Syntaxin 17 and ATG5 initiates then the formation of autophagosomes and the degradation of viral RNA via autophagy. Consequently, in the context of EMCV, during the early stages of the infection cycle, the viral RNA not only activates the RNaseL system, but also indirectly increases autophagy. If this requires the MAVS signalling pathway and the inhibition of this pathway increases viral yield at early timepoints remains to be seen. In the case of Hepatitis C Virus, the viral NS3/4A protease cleaves MAVS and prevents the formation of MAM complexes by targeting the synapse. In the case of EMCV, the viral 3C^Pro protease might target the synapse as well - only later in infection. 

Induction of 2,5OAS/RNaseL signalling and MAM
by EMCV viral RNA induces autophagy

In relation to the potential of inducing the formation of LC3 positive vesicles, a number of viral proteins have been shown to co-localise with LC3 in vesicular structures, in particular VP1, VP4, 2B, 3A, 2C, and 3D. With the exception of the viral 2C and 3D proteins however none of them induces the formation of autophagosomes as determined by the conversion of LC3-I to LC3-II, suggesting that LC3-I positive vesicles containing these proteins might play a role as transport vesicles. The question remains however, if the expression of these proteins does inhibit the formation of autophagosomes under conditions of starvation or amino-acid withdrawal. Also, it might be interesting to see whether these vesicles are positive for LC3-C, analogous to LC3-C positive vesicles containing the Capsid protein of Chikungunya Virus. Although it has been postulated that EMCV replication takes place on LC3 positive membranes, it is not clear if these represent mature autophagosomes - which is rather unlikely, unless fusion with the lysosome is prevented or if these “replication vesicles” fuse with components of the endosomal system instead. In the case of the viral 2C and 3D proteins, the formation of autophagosomes and subsequent fusion with lysosomes seems not to be inhibited, a process that also degrades p62/SQSTM1; indeed, autophagosomes containing 2C and 3D are positive for p62/SQSTM1 and induce the degradation of the latter. Presently, it is not clear however if p62/SQSTM1 is required for 2C and 3D to undergo autophagic degradation. 

2C and 3D induce the formation of mature autophagsomes

The expression of the EMCV 2C and 3D proteins has been shown to induce all three branches of the ER stress response, PERK, ATF6α and IRE1, as characterised by the phosphorylation of eIF2α, cleavage of XBP1, and increase of CHOP. Unfortunately so far the expression levels of autophagy-related genes have not been examined. If the switch coincides with an accumulation of the viral 3C^Pro protease is not known: as outlined above, 3C^Pro  might cleave the MAM complex akin to Hepatitis C Virus NS3/4. Also, cleavage of the MAVS might lead to mitochondrial fission in similar way as Respiratory Syncytial Virus NS1/NS2 proteases. As it is the case in RSV infected cells, this would inhibit antiviral signalling by the MAVS/TRAF3/TRAF6 pathway, suppressing the   type I interferon  response and thus favour viral replication.

Formation of the MAM complex at the ER

The induction of the ER stress response itself is probably a result of lipid repletion induced by the excessive formation of membranous vesicles - not only by viral proteins directly but also the induction of autophagy by RNaseL and indeed the induction of MAM induces the transfer of lipids to Mitochondria. 

Consequently, the MAM complex induced by the viral RNA leads to the induction of autophagy, a process that ultimately degrades the viral RNA as well as MDA5. Inducing autophagy early in the infection therefore has an antiviral effect which in turn depletes the cell of MDA5, which combined with the inhibiton of host cell translation would create favourable conditions for viral replication. Indeed, in murine L cells infected with EMCV, host cell translation decreases by 50% within the first 6 h p.i. .

Connecting the dots:RNaseL activation induces the formation of the
Mitoxosome and subsequent mitophagy

However, since only a relative small number of studies addressed the formation of the replication complexes of EMCV directly, the details of the induction of autophagosomes or autophagosome like structures during EMCV infection are not very well characterised. What emerges however, is a picture where EMCV similar to other RNA viruses hijacks the autophagic pathway in order to facilitate the formation of replication centers or replication compartments. One question which needs to be answered is the role of EMCV 2C in inhibiting the viral protease early in the infection cycle and thus preventing the degradation of RIG-1. If this is the case, then expressing 2C in trans might prevent autophagy induced by RNaseL and increase virus yield. Finally, is the induction of autophagy in EMCV infected cells by viral proteins required to prevent the induction of apoptosis which would be expected following the activation of RNaseL? One question I personally would like to address is if the infection of cells with EMCV re-localises mitochondria to MAM and thus induces mitophagy. 
Targeting mitochondria dependent antiviral signalling however is not confined to cells infected with EMCV or Hepatitis C Virus. In the case of SARS-CoV, the viral orf9b protein suppresses MAVS dependent signalling and induces ATG5 dependent mitophagy, suggesting again that different viruses subvert common pathways.

As for JEV, Coxsackievirus B3, or CoV replication, the ER stress response is central for EMCV replication. Personally, the author of these lines hopes to contribute to solving some of the puzzles.





Further reading

Carocci, M., & Bakkali-Kassimi, L. (2012). The encephalomyocarditis virus Virulence, 3 (4), 351-367 DOI: 10.4161/viru.20573 

  
Hou, L., Ge, X., Xin, L., Zhou, L., Guo, X., & Yang, H. (2014). Nonstructural proteins 2C and 3D are involved in autophagy as induced by the encephalomyocarditis virus Virology Journal, 11 (1) DOI: 10.1186/1743-422X-11-156 

Zhang Y, Li Z, Ge X, Guo X, & Yang H (2011). Autophagy promotes the replication of encephalomyocarditis virus in host cells. Autophagy, 7 (6), 613-28 PMID: 21460631 

Shi J, Fung G, Piesik P, Zhang J, & Luo H (2014). Dominant-negative function of the C-terminal fragments of NBR1 and SQSTM1 generated during enteroviral infection. Cell death and differentiation, 21 (9), 1432-41 PMID: 24769734

Maurel M, Chevet E, Tavernier J, & Gerlo S (2014). Getting RIDD of RNA: IRE1 in cell fate regulation. Trends in biochemical sciences, 39 (5), 245-54 PMID: 24657016 

Silverman RH (2007). Viral encounters with 2',5'-oligoadenylate synthetase and RNase L during the interferon antiviral response. Journal of virology, 81 (23), 12720-9 PMID: 17804500 
  
Siddiqui MA, & Malathi K (2012). RNase L induces autophagy via c-Jun N-terminal kinase and double-stranded RNA-dependent protein kinase signaling pathways. The Journal of biological chemistry, 287 (52), 43651-64 PMID: 23109342 

Chakrabarti A, Ghosh PK, Banerjee S, Gaughan C, & Silverman RH (2012). RNase L triggers autophagy in response to viral infections. Journal of virology, 86 (20), 11311-21 PMID: 22875977

Mullan PB, Hosey AM, Buckley NE, Quinn JE, Kennedy RD, Johnston PG, & Harkin DP (2005). The 2,5 oligoadenylate synthetase/RNaseL pathway is a novel effector of BRCA1- and interferon-gamma-mediated apoptosis. Oncogene, 24 (35), 5492-501 PMID: 15940267 

Hamasaki M, Furuta N, Matsuda A, Nezu A, Yamamoto A, Fujita N, Oomori H, Noda T, Haraguchi T, Hiraoka Y, Amano A, & Yoshimori T (2013). Autophagosomes form at ER-mitochondria contact sites. Nature, 495 (7441), 389-93 PMID: 23455425 

Jen G, & Thach RE (1982). Inhibition of host translation in encephalomyocarditis virus-infected L cells: a novel mechanism. Journal of virology, 43 (1), 250-61 PMID: 6287000 

Horner SM, Liu HM, Park HS, Briley J, & Gale M Jr (2011). Mitochondrial-associated endoplasmic reticulum membranes (MAM) form innate immune synapses and are targeted by hepatitis C virus. Proceedings of the National Academy of Sciences of the United States of America, 108 (35), 14590-5 PMID: 21844353 

Banerjee R, Weidman MK, Echeverri A, Kundu P, & Dasgupta A (2004). Regulation of poliovirus 3C protease by the 2C polypeptide. Journal of virology, 78 (17), 9243-56 PMID: 15308719

Shi CS, Qi HY, Boularan C, Huang NN, Abu-Asab M, Shelhamer JH, & Kehrl JH (2014). SARS-Coronavirus Open Reading Frame-9b Suppresses Innate Immunity by Targeting Mitochondria and the MAVS/TRAF3/TRAF6 Signalosome. Journal of immunology (Baltimore, Md. : 1950) PMID: 25135833