Virology tidbits

Virology tidbits

Monday, 10 November 2014

Beclin-1 and Viruses: Activation and Inhibition of Autophagy by Viral Proteins

Autophagy is an essential process that allows the degradation of cellular components such as damaged organelles (e.g. Mitochondria via mitophagy or Peroxisomes via pexophagy) or misfolded proteins, as well as bacteria or viral proteins. Generally, the initial step in autophagy is sequestering of organelles or proteins within the phagophore -an isolation membrane derived from the plasma membrane, ER, Golgi, or endosome. Following the formation of the phagophore the edges of the membrane are fused, forming a phagosome, a double membrane vesicle, that matures into the autophagosome and subsequently fuses with the lysosome to form the autolysosome. Therefore, the process can be divided into six steps, (1) Initiation, (2) Nucleation, (3) Elongation, (4) Closure, (5) Maturation, (6) Degradation.

This post will focus on the interaction of viral proteins with two steps -Initiation and Degradation-, in particular highlighting the role of the interaction of viral proteins with Beclin-1, using the M2 protein of Influenza A Virus and the coronaviral ER associated papain like protease (PLP-2/PLPro) as examples.

In mammalian cells not undergoing autophagy, Beclin-1 forms a complex with Bcl-2 and Bcl-xL at the ER, thus rendering Beclin-1 inactive. This complex however dissociates following phosphorylation of Beclin-1 by AMBRA1 or death-associated protein kinase (DAPK) at Thr-119 within the BH3-domain of Beclin-1 or phosphorylation of Bcl-2/Bcl-xL by c-Jun N-terminal kinase 1 (JNK1), thus allowing the formation of the phagophore by recruitment of Beclin-1 to Atg14L, Vps34, and Vps15/p150. This complex in turn recruits LC3-I - the non-lipidated form of LC-3- as well as a complex consisting of ATG5, ATG12, and ATG16L allowing the subsequent formation of LC3-I positive autophagic vesicles that mature into autophagosomes by lipidation of LC3-I in a process similar to ubiquitination. LC3-II positive vesicles subsequently fuse with the lysosome to form the autolysosome where the cargo is degraded by lysosomal enzymes. This process is not only dependent on  the Rab7, Rab8B and Rab24 GTPases, but also on Beclin-1 as cells deficient for Beclin-1 exhibit increased autophagic flux. While the RabGTPases -in particular Rab7- regulate the transport of mature autophagosomes to the lysosome via lysosome bound Rab7 that binds  to LC3-II  via FYCO, thus tethering lysosomes to autophagosomes and allowing the fusion of mature autophagosomes with lysosomes. In the case of Beclin-1 the precise mechanism is less clear.
Similar to the aforementioned process of initiating the formation of the autophagosome, in the case of facilitating the formation of the autolysosome, Beclin-1 forms a complex with Vps34 as well as Vps15, the latter anchoring the complex at the lysosome (similar to Rab7) . Different from the initiation complex however, instead of ATG14L, this complex contains UVRAG (UV radiation resistance-associated gene protein); indeed ATG14L and UVRAG bind Beclin-1 in mutually exclusive manner. Furthermore, a subpopulation of this complex associates with a negative regulator of autophagy, RUBICON (RUN domain and cysteine-rich domain containing, Beclin-1-interacting protein), thus leading to the existence of three different populations of Beclin-1 associated complexes, two which increase autophagic flux, one inhibiting autophagy.

Invitation of autophagosome formation and fusion with the lysosome involves Beclin-1
Complexes formed by Beclin-1

Viruses and Beclin-1: Influenza A Virus and Coronavirus

Activation of Beclin-1 and subsequent induction of autophagy by pathogens can be considered to be part of the innate immune response especially if the autophagic particle engulfs and degrades the infectious particle, as it is not only the case for bacteria but also for viruses such as Herpes Simplex Virus 1 (HSV 1). Consequently, all three subfamilies of the Herpesviridae encode for proteins that target Beclin-1, in the case of HSV 1 the ICP 34.5 protein, and thus inhibit autophagy by inhibiting the formation of the phagophore; indeed, in cells infected with a viral Δ ICP 34.5 mutant, HSV 1 particles are detectable in autophagosomes. In the case of positive strand RNA viruses however the inhibition of the formation of autophagosome might prevent the formation of the viral replication centers. In this case, some viral proteins may induce the formation of the phagophore whilst inhibiting the fusion with the lysosome and thus degradation of viral components (proteins and RNA). As discussed before, the coronaviral nsp-6 protein is an example and below recent results suggesting a similar role for the coronaviral PLP2/PLPro is discussed below.  In the case of Retroviridae, the HIV Nef protein has been demonstrated to block the fusion of the autophagosome with the lysosome and thus prevent the degradation of virions.

Influenza A Virus M2 protein: the more complex story

Influenza A Virus’ are negative strand RNA viruses, which utilise both the nucleus and the cytoplasm for its replication but autophagosome -or autophagosome like structures such as omegasomes-, are not used as replication centers in opposition to those induced positive strand RNA viruses. Following the infection of various cell lines including CV-1, MDCK, A549, and MEF cells with different strains of Influenza A Virus both of human and avian origin (A/Chicken/Beijing/04 (H9N2), A/PR8, and A/WSN/33), an increase in GFP-LC3 positive punctae can be observed concomitant with an increase in LC3-II levels, suggesting that the infection of (mammalian) cells with Influenza A induces the formation of mature autophagosomes. This increase in autophagy however does not result in an increase in autophagic flux since the application of a lysosome inhibitor -E64- does not increase LC3-II levels (although p62/SQSTM1 is degraded in infected primary human blood macrophages). Therefore, one or more proteins expressed by Influenza A has the capacity to induce autophagy but inhibit the fusion of the mature autophagosome with the lysosome. As discussed above, in uninfected cells this due to binding of Rubicon to Beclin-1 and indeed in cells infected with Influenza A virus the viral M2 and proteolytically cleaved HA proteins have been shown to prevent the fusion step in a similar manner, by binding to Beclin-1 (in the case o f M2 at least) independent of the ability to form proton channels. Paradoxically however, inhibiting the fusion of the autophagosome with the lysosome also induces apoptosis and it has been shown that in infected MEF cells autophagy is only induced when Bax mediated, mitochondrial, apoptotic pathways are blocked; the induction of autophagy and subsequent inhibition of the fusion of the autophagosome with the lysosome therefore represents an alternative cell death pathway, and inhibition of autophagy consequently does not increase viral titers. M2 however also binds LC3 via a LC3-Interacting domain (similar to optineurin or p62/SQSTM1) and relocates LC3 positive structures to the plasma membrane. These structures have been proposed to contain mature virions and contribute tot eh spread of (filamentous) Influenza A virus particles. Consequently, in the case of Influenza A virus, the expression of M2 is a mixed blessing: on one hand, it induces apoptosis independent of caspase activation via the mitochondrial pathway, on the other hand, it facilitates the spread of filamentous viral particles by interacting with LC3.  Since induction of M2 mediated autophagy can only be observed late in the infection, and M2 mediated apoptosis is only observed in BaxKO cells or cells treated with Caspase inhibitors, the predominant function of M2 regarding autophagy might be inducing the formation of mature autophagosomes containing filamentous virions, followed by preventing degradation of these and subsequent (re-)localisation of these to the cell surface via interaction with LC3 (personal opinion).

Influenza A Virus M2, Beclin-1, and LC3: induction of autophagy via biding to Beclin-1 and inhibition
fusion with the lysosome by binding to LC3 via M2-LIR

Coronavirus PLP2/PLPro, Beclin-1, and autophagy

As discussed in previous posts, the infection of cells with different members of the Coronaviridae as well as the expression of various coronaviral proteins -in particular the non-structural proteins nsp-3, -4, and -6 - induces the formation of double membrane vesicles (that form the coronaviral replication center) similar but distinct from autophagosomes. The precise mechanism of the formation of these vesicles is not known, although it has been shown that both nsp-3 and -4 have the ability to induce membrane curvature and that the EDEM-1 might play a role as well. In any case, these vesicles most likely do not represent mature autophagosomes but omegasomes (in the case of vesicles formed by nsp-6) or EDEMosomes; furthermore, the expression of nsp-6 inhibits the formation of mature lysosomes probably by inhibiting mTORC2 localisation to the lysosomal surface. In general however the requirement of the autophagic machinery for the replication of Coronavirus’ is still disputed since both Mouse Hepatitis Virus (MHV) and SARS-CoV replicate in primary ATG5-/- Mouse Embryonic Fibroblasts albeit with reduced viral titres.

As one of the non-structural proteins required for the formation of the RTCs and replication of the viral genome (for detailed review see previous posts here and here), the coronaviral genome encodes for nsp-3 which contains a membrane associated papain-like protease activity (PLP2-TM), whose main function is to cleave the viral orf1a polyprotein, generating a plethora of viral proteins, including nsp-4 and -6. 

MHV PLP2 is part of nsp-3

Intriguingly, PLP2 derived from various Coronavirus’, HCoV-NL63, Porcine Epidemic Diarrhoea Virus (PEDV), as well as the SARS-CoV PLPro, co-localise with LC3 in HEK293T, HeLa, as well as MCF-7 cells and increase the formation of LC3 positive punctae that resemble mature autophagosomes based on increased levels of LC3-II. These results indicate that the expression of PLP2 and PLPro respectively induces the formation of truly autophagic vesicles and further analysis showed that this process is independent of the catalytic activity of PLP2. As outlined above, the formation of the phagosome depends on the activation of Beclin-1 by dissociation of Beclin-1 from Bcl-2/Bcl-xL at the ER. In the case of PLP2, experimental data point towards the ability of PLP2 to bind Beclin-1 and thus promote the formation of the phagophore, although the domain responsible for such binding has not been identified. Rather than binding directly to Beclin-1, the author of this post favours a model where the expression of PLP2 (or PLPro in the case of SARS-CoV) induces the ER stress response with concomitant activation of JNK which then leads to the dissociation of Beclin-1 and subsequent induction of autophagy. The dissociation of Beclin-1 then allows the formation of a complex with PLP2/PLPro, a process that favours the formation of the autophagosomes -similar to the expression of Vacuole Membrane Protein 1 (VMP1).

The expression of PLP2 and PLPro not only leads to the accumulation of mature autophagosomes but also to decreased autophagic flux as measured by turnover of p62/SQSTM1. It has been proposed that the interaction of HCoV-NL63 PLP2 with Beclin-1 prevents the recruitment of lysosomes to the mature LC3-II positive autophagosome and thus the formation of the autolysosome, although a soluble form of PLP2 lacking the transmembrane domain fails to do so, indicating to the author of these lines that the transmembrane domain of PLP2 might be responsible for recruiting Beclin-1 and preventing the degradation of autophagosomes.

CoV PLP2 and Beclin-1: binding of Beclin-1 

Functionally, the association of PLP2 with Beclin-1 might prevent the induction of antiviral signalling namely inhibiting the activation of IRF3 via STING, although the precise mechanism is not known. Indeed expression of PLP2-TM in cells deficient for Beclin-1 fails to inhibit the Interferon response, suggesting that the induction of autophagy -albeit not leading to degradation- prevents antiviral signalling. It remains to be seen what the precise mechanism is, but the author of these lines favours a model in which the association of PLP2/PLPro with both STING and Beclin-1 triggers not only the formation of autophagosomes but also STING being localised to these autophagosomes. In other words, are the autophagosomes induced by PLP/PLPro expression also positive for STING?

CoV-PLP2, Beclin-1, and STING: sequestering of STING in autophagosomes?
Also it remains to be seen if the DUB domain of PLP2/PLPro is important for the induction of autophagy or if PLP2/PLPro or any other viral protein such as nsp-6 recruits Rubicon to autophagosomes. Also, is Beclin-1 being ubiquitinylated prior to associating with PLP/PLPro ?
As always, much to do and I hope that somebody is going to investigate these questions - or give me the opportunity and to do it.

Coronaviral proteins and autophagy: nsp-6 and PLP2 promote formation
of double membrane vesicles yet inhibit fusion with the lysosome

Interestingly, in the case of Influenza A Virus, the GFP-LC3 positive structures observed following the expression of M2 have been reported to accumulate in the perinuclear region prior to their transport to the plasma membrane. This raises the question if the inhibition of autophagosome-lysosome fusion by coronaviral proteins not only prevents the degradation of viral components as well as innate immune signalling but also ensures that the viral RTC can be transported to the ER-Golgi Intermediate Compartment and interact with the intracellular cargo receptor ERGIC-53.

Further reading

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 

Ao X, Zou L, & Wu Y (2014). Regulation of autophagy by the Rab GTPase network. Cell death and differentiation, 21 (3), 348-58 PMID: 24440914 

Pankiv S, Alemu EA, Brech A, Bruun JA, Lamark T, Overvatn A, Bjørkøy G, & Johansen T (2010). FYCO1 is a Rab7 effector that binds to LC3 and PI3P to mediate microtubule plus end-directed vesicle transport. The Journal of cell biology, 188 (2), 253-69 PMID: 20100911 

Gu W, Wan D, Qian Q, Yi B, He Z, Gu Y, Wang L, & He S (2014). Ambra1 is an essential regulator of autophagy and apoptosis in SW620 cells: pro-survival role of Ambra1. PloS one, 9 (2) PMID: 24587252 

Zhong Y, Wang QJ, Li X, Yan Y, Backer JM, Chait BT, Heintz N, & Yue Z (2009). Distinct regulation of autophagic activity by Atg14L and Rubicon associated with Beclin 1-phosphatidylinositol-3-kinase complex. Nature cell biology, 11 (4), 468-76 PMID: 19270693

Matsunaga K, Saitoh T, Tabata K, Omori H, Satoh T, Kurotori N, Maejima I, Shirahama-Noda K, Ichimura T, Isobe T, Akira S, Noda T, & Yoshimori T (2009). Two Beclin 1-binding proteins, Atg14L and Rubicon, reciprocally regulate autophagy at different stages. Nature cell biology, 11 (4), 385-96 PMID: 19270696 

Molejon MI, Ropolo A, Re AL, Boggio V, & Vaccaro MI (2013). The VMP1-Beclin 1 interaction regulates autophagy induction. Scientific reports, 3 PMID: 23316280 

Münz C (2011). Beclin-1 targeting for viral immune escape. Viruses, 3 (7), 1166-78 PMID: 21994775  

Dumit VI, & Dengjel J (2012). Autophagosomal protein dynamics and influenza virus infection. Frontiers in immunology, 3 PMID: 22566925 

Gannagé M, Dormann D, Albrecht R, Dengjel J, Torossi T, Rämer PC, Lee M, Strowig T, Arrey F, Conenello G, Pypaert M, Andersen J, García-Sastre A, & Münz C (2009). Matrix protein 2 of influenza A virus blocks autophagosome fusion with lysosomes. Cell host & microbe, 6 (4), 367-80 PMID: 19837376 

Hull JD, Gilmore R, & Lamb RA (1988). Integration of a small integral membrane protein, M2, of influenza virus into the endoplasmic reticulum: analysis of the internal signal-anchor domain of a protein with an ectoplasmic NH2 terminus. The Journal of cell biology, 106 (5), 1489-98 PMID: 2836432 

Zhirnov OP, & Klenk HD (2013). Influenza A virus proteins NS1 and hemagglutinin along with M2 are involved in stimulation of autophagy in infected cells. Journal of virology, 87 (24), 13107-14 PMID: 24027311
Law AH, Lee DC, Yuen KY, Peiris M, & Lau AS (2010). Cellular response to influenza virus infection: a potential role for autophagy in CXCL10 and interferon-alpha induction. Cellular & molecular immunology, 7 (4), 263-70 PMID: 20473322 

McLean JE, Datan E, Matassov D, & Zakeri ZF (2009). Lack of Bax prevents influenza A virus-induced apoptosis and causes diminished viral replication. Journal of virology, 83 (16), 8233-46 PMID: 19494020 

Beale R, Wise H, Stuart A, Ravenhill BJ, Digard P, & Randow F (2014). A LC3-interacting motif in the influenza A virus M2 protein is required to subvert autophagy and maintain virion stability. Cell host & microbe, 15 (2), 239-47 PMID: 24528869

Dumit VI, & Dengjel J (2012). Autophagosomal protein dynamics and influenza virus infection. Frontiers in immunology, 3 PMID: 22566925 

Maier HJ, & Britton P (2012). Involvement of autophagy in coronavirus replication. Viruses, 4 (12), 3440-51 PMID: 23202545 

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 

Chen X, Wang K, Xing Y, Tu J, Yang X, Zhao Q, Li K, & Chen Z (2014). Coronavirus membrane-associated papain-like proteases induce autophagy through interacting with Beclin1 to negatively regulate antiviral innate immunity. Protein & cell PMID: 25311841

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