Virology tidbits

Virology tidbits

Monday, 8 September 2014

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

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