Virology tidbits

Virology tidbits

Tuesday, 12 August 2014

Coxsackievirus B3: autophagy, p62, ER stress, and apoptosis

The induction of autophagy by cells infected with viruses can play an important strategy to prevent viral replication - or alternatively, can be subverted by viral proteins to allow the formation of viral particles as it is the case for many positive strand RNA viruses and discussed previously for Corona- and Arteriviruses. Enteroviruses are no different, and indeed the modulation of autophagy by Poliovirus has been studied extensively. 

Processing of the Coxsackievirus B3 genome involves the viral proteas
2Apro and 3CD

                                Coxsackievirus B3 and autophagy

Similar to to Corona- and Arteriviruses, Coxsackievirus B3 (CVB3) is a positive strand ssRNA virus, which causes serious infections ranging from gastrointestinal to respiratory infections in addition to myocarditis, pericarditis, as well as Diabetes Type 1.The genome itself has a size of ~7.4 kB and encodes for a polyprotein containing the non-structural and structural proteins, whose organisation mirrors those of other enteroviruses. Following translation, the polyprotein is cleaved into four capsid proteins and seven functional proteins including the RNA dependent RNA Polymerase (RdRP) and viral proteases 2Apro and 3Cpro , the latter cleaving not only viral proteins but also cellular proteins.

Following the infection of pancreatic acinar cells with CVB3, the formation of autophagy-like vesicles can be observed which may -akin to Coronavirus RTCs- form a scaffold required for CVB3 replication. Indeed, these structures do not represent mature autophagosomes but GFP-LC3/LAMP1 positive “megaphagosomes” which contain a protein whose size corresponds to the viral (RdRP). Furthermore, both the application of 3-Methyladenine or small interfering RNAs targeting ATG5, ATG7, or Beclin1, decreases viral replication whereas the induction of autophagy by rapamycin or starvation increases viral titers in HeLa cells infected with CVB3. More important however is the observation that treating cells with siRNA against LAMP2 -thus inhibiting the formation of lysosomes- increases viral titers as well, suggesting that then induction of autophagy by CVB3 has not only a positive effect on viral replication but also constitutes an antiviral response by leading to a degradation of viral components. Indeed it has been speculated that the megaphagosomes induced in pancreatic cells are in fact structures destined for degradation rather than replicative structures. To complicate the picture however, mature CVB3 released from the cells contain LC3-II, the lipidated from of LC3-I characteristic of mature autophagosomes. As outlined before, degradation of viral components via autophagy can lead to TLR-7 mediated activation of the innate cellular antiviral response by increased chemokine expression and thus contributing to the inflammation observed in pancreatitis. Indeed, Atg5f/f/Cre+ mice exhibit a less pronounced pancreatitis following CVB3 infection.

It seems therefore that a careful balance is maintained between the induction of autophagosome like structures required for viral replication whilst at the same time it is crucial to prevent the fusion of these structures with endosomal and lysosomal structures. 
The central question therefore is, how does the expression of CVB3 proteins alter the cellular environment to achieve this? Based on results which are discussed in this post, the author of this post favours a model, where the expression of viral proteins combined with the cleavage of a cellular protein, p62/SQSTM1, induces the induction of the ER stress response, that not only induces autophagy -or to be precise the formation of omegasomes which might or might not be EDEM1 positive – but also CHOP dependent apoptosis.                                                                     

                                       p62/SQSTM1, NBR1, and CVB3

p62/SQSTM1 is a Ubiquitin (Ub) binding protein which first identified in cells from patients with hepatic proteinophaties as well as neurodegenerative disorders such as Huntington, which exhibit aggregates of poly-ubiquitinylated proteins. Poly-ubiquitinylated proteins are bound to p62/SQSTM1 via a C-terminal Ubiquitin-binding domain (UBA). In addition, complexes of p62/SQSTM1  and poly-ubiquitinylated proteins form polymers with itself and another Ub binding domain, NBR1 (Neighbour of BRCA1 gene 1). 

Role of p62/SQSTM1 and NBR1 in the formation of autophagosomes

Upon binding mono- and poly-ubiquitinylated proteins, both p62/SQSTM1 and NBR1 bind Atg8/GABARAP (gamma-aminobutyrate receptor-associated protein) and microtubule associated light chain3 (LC3) via specific LC3 interaction domains (LIR) (one in p62/SQSTM1, two in NBR1), thus allowing the formation of autophagosomes and subsequently degradation of proteins via autophagy in a BAG3 dependent manner in addition to be targeted for proteasomal degradation via BAG1. Although NBR1 and p62/SQSTM1 can interact with each other, the deletion of p62/SQSTM1 in p62 -/- MEF or HeLa cells transfected with siRNA against endogenous p62/SQSTM1 does not prevent NBR1 recruitment to autolysosomes, concomitant with a reduction of ubiquitinylated proteins and an increase in endogenous levels of NBR1.

p62/SQSTM1 cleavage products

In the case of CVB3 infected cells and in vitro, both p62/SQSTM1 and NBR1 can be cleaved by two viral proteases, 2Apro and 3Cpro generating fragments which might inhibit central functions of p62/SQSTM1.  

NBR1 cleavage products

HeLa cells transfected with a plasmid encoding the p62/SQSTM1 C terminal fragment, full length p62/SQSTM1 indeed does not form  punctae, suggesting that the expression of the cleaved fragment prevents the degradation of wt p62/SQSTM1 Since p62/SQSTM1-C contains the LIR as well as the UBA, the fragment might sequester LC3-I and/or Ubiquitin in addition to prevent the formation of functional dimers of full length p62/SQSTM1.

Inhibition of NBR1 and p62/SQSTM1 selective autophagy by p62/SQSTM1-C

In any case, autophagy induced by p62/SQSTM1 is inhibited; consequently (in the opinion of the author of these lines) misfolded or damaged proteins might accumulate and initiate a ER stress response characterised by the induction of EDEM-1/-2/-3 and subsequently CHOP, the former might induce the formation of EDEMosomes akin to those found in Coronavirus infected cells. These EDEMosomes in turn might form the scaffold for viral replication.

Expression of cleaved p62/SQSTM1-C fragment may induce ER stress and the formation
of EDEMosomes in addition to induction of the PERK pathway

More importantly, it is possible that the expression of p62/SQSTM1-C inhibits p62/SQSTM1-mediated activation of NF-κB via binding of TRAF6 to the TRAF6 binding domain (TB) domain - in other words, counteracting the antiviral response induced by CVB3.
In addition, cleavage of the IkBα subunit by 3Cpro  has been shown to inhibit NF-κB signaling and reduce apoptosis, suggesting that inhibition of p62/SQSTM1 mediated degradation may enhance the stabilization of the NF-κB/IκB complex and thus decrease the expression of cytokines. 
In contrast to the C-terminal fragment of p62/SQSTM1, expression of the N terminal fragment does not reduce the formation of punctae, which -due to the absence of the LIR-  are negative for LC3 and do not undergo autophagy. Since p62/SQSTM1-N contains the Phox/Bem1p domain, the truncated form can form dimers with full length p62/SQSTM1 but fails to bind constituents of the  NF-κB pathway or ubiquitinylated proteins. One of the remaining questions is however why p62/SQSTM1-C fails to bind and degrade ubiquitinylated proteins. So far the answer is not known, but it might be possible that one of the domains missing in the fragment is necessary to bind ubiquitinylated proteins to the UBA or that the UBA is simply not exposed and thus can not be recognized by ubiquitin.

In addition to inhibiting NF-κB signaling, the expression of p62/SQSTM1-C also may fail to activate caspase-8 if autophagy is inhibited. If this is the case in cells infected with CVB3 remains to be seen, but if so, this would place the formation of p62/SQSTM1 in the center of counteracting antiviral signaling.

mTORC1 activation activates p62/SQSTM1 which in turn induces apoptosis
via Caspase-8 activation, activates NF-κB via TRAF6, and forms protein aggregates.
p62/SQSTM1 may inhibit these processes.

Finally we have to take a look at NBR1. The expression of p62/SQSTM1-C leads not only to an accumulation of p62/SQSTM1 but also NBR1, indicating that NBR1 dependent (selective) autophagy is inhibited as well. Reciprocally, the expression of the 3Cpro NBR1-C fragment but not the 2Apro NBR1-C fragment results in the accumulation of p62/SQSTM, probably due to the presence of a second LIR domain in 3Cpro NBR1-C.

                           Coxsackievirus B3, p62/SQSTM and ER stress

The accumulation of unfolded proteins following the inhibition of p62/SQSTM1 and NBR, may trigger a ER stress response which not only activates the formation of EDEMosomes as part of the ERAD pathway and induces the formation of autophagy like vesicles, but also induces caspase-dependent apoptosis. Cardiomyocyte  and pancreatic cell apoptosis is induced following CVB3 infection as a result of induction and activation of CHOP and Caspase-12, including increased expression of EDEM1, probably via ATF6a mediated downregulation of p58(IPK) and subsequent activation of PERK. The induction of autophagy might therefore enable the cell to survive ER stress induced by CVB3 in addition to the formation of replication centers. As it the case for JEV however, prolonged ER stress induces apoptosis and indeed the release of CVB3 particles has been proposed to be dependent on apoptosis. 

How then is the balance between autophagy and apoptosis maintained? One key protein might be Cathepsin D. In the case of Enterovirus 71 infected cells, the formation of autolysosomes increases Cathepsin D which leads to apoptosis whereas the formation of autophagosomes reduces Cathepsin D levels. Inhibiting lysosomal maturation therefore might have two effects: (1) preventing the activation of endosomal TLR signalling and (2) prevent apoptosis induction by Cathepsin D. 

In the end this might be a common strategy employed not only by Enterovirus 71, but also by CVB3, JEV, and Coronavirus. Cleavage of p62/SQSTM1 might be not the only way to achieve this.

ER stress pathways induced by Coxsackievirus B3: induction of autophagy v. apoptosis

Does the cleavage of p62/SQSTM1 and NBR induce the ER stress response - well, this is  a question which I hope gets answered soon (unfortunate not by me). Given that p62/SQSTM1 also is involved in the maturation of lysosomes via mTORC1, it is conceivable that inhibition of p62/SQSTM1 also effects the formation of the autolysosome akin to Coronavirus nsp-6; so does  the expression of nsp-6 alter the localisation of p62/SQSTM1?

Further reading

Lindberg AM, Stålhandske PO, & Pettersson U (1987). Genome of coxsackievirus B3. Virology, 156 (1), 50-63 PMID: 3027968 

Wong J, Zhang J, Si X, Gao G, Mao I, McManus BM, & Luo H (2008). Autophagosome supports coxsackievirus B3 replication in host cells. Journal of virology, 82 (18), 9143-53 PMID: 18596087

Robinson SM, Tsueng G, Sin J, Mangale V, Rahawi S, McIntyre LL, Williams W, Kha N, Cruz C, Hancock BM, Nguyen DP, Sayen MR, Hilton BJ, Doran KS, Segall AM, Wolkowicz R, Cornell CT, Whitton JL, Gottlieb RA, & Feuer R (2014). Coxsackievirus B exits the host cell in shed microvesicles displaying autophagosomal markers. PLoS pathogens, 10 (4) PMID: 24722773

Alirezaei M, Flynn CT, Wood MR, & Whitton JL (2012). Pancreatic acinar cell-specific autophagy disruption reduces coxsackievirus replication and pathogenesis in vivo. Cell host & microbe, 11 (3), 298-305 PMID: 22423969 

Pankiv S, Clausen TH, Lamark T, Brech A, Bruun JA, Outzen H, Øvervatn A, Bjørkøy G, & Johansen T (2007). p62/SQSTM1 binds directly to Atg8/LC3 to facilitate degradation of ubiquitinated protein aggregates by autophagy. The Journal of biological chemistry, 282 (33), 24131-45 PMID: 17580304 

Kirkin V, Lamark T, Sou YS, Bjørkøy G, Nunn JL, Bruun JA, Shvets E, McEwan DG, Clausen TH, Wild P, Bilusic I, Theurillat JP, Øvervatn A, Ishii T, Elazar Z, Komatsu M, Dikic I, & Johansen T (2009). A role for NBR1 in autophagosomal degradation of ubiquitinated substrates. Molecular cell, 33 (4), 505-16 PMID: 19250911 

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 

Shi J, Wong J, Piesik P, Fung G, Zhang J, Jagdeo J, Li X, Jan E, & Luo H (2013). Cleavage of sequestosome 1/p62 by an enteroviral protease results in disrupted selective autophagy and impaired NFKB signaling. Autophagy, 9 (10), 1591-603 PMID: 23989536 

Moscat J, Diaz-Meco MT, Albert A, & Campuzano S (2006). Cell signaling and function organized by PB1 domain interactions. Molecular cell, 23 (5), 631-40 PMID: 16949360 

Saura, M., Lizarbe, T., Rama-Pacheco, C., Lowenstein, C., & Zaragoza, C. (2007). Inhibitor of NFκB Alpha is a Host Sensor of Coxsackievirus Infection Cell Cycle, 6 (5), 503-506 DOI: 10.4161/cc.6.5.3918

Durán A, Serrano M, Leitges M, Flores JM, Picard S, Brown JP, Moscat J, & Diaz-Meco MT (2004). The atypical PKC-interacting protein p62 is an important mediator of RANK-activated osteoclastogenesis. Developmental cell, 6 (2), 303-9 PMID: 14960283 

Komatsu M, Kageyama S, & Ichimura Y (2012). p62/SQSTM1/A170: physiology and pathology. Pharmacological research : the official journal of the Italian Pharmacological Society, 66 (6), 457-62 PMID: 22841931 

Amitava Mukherjee,, Stefanie A. Morosky,, Elizabeth Delorme-Axford,, Naomi Dybdahl-Sissoko,, M. Steven Oberste,, Tianyi Wang,, & Carolyn B. Coyne (2011). The Coxsackievirus B 3Cpro Protease Cleaves MAVS and TRIF to Attenuate Host Type I Interferon and Apoptotic Signaling PLOS pathogens 

Huang S, Okamoto K, Yu C, & Sinicrope FA (2013). p62/sequestosome-1 up-regulation promotes ABT-263-induced caspase-8 aggregation/activation on the autophagosome. The Journal of biological chemistry, 288 (47), 33654-66 PMID: 24121507 

Zhang HM, Ye X, Su Y, Yuan J, Liu Z, Stein DA, & Yang D (2010). Coxsackievirus B3 infection activates the unfolded protein response and induces apoptosis through downregulation of p58IPK and activation of CHOP and SREBP1. Journal of virology, 84 (17), 8446-59 PMID: 20554776 

Xin L, Xiao Z, Ma X, He F, Yao H, & Liu Z (2014). Coxsackievirus B3 induces crosstalk between autophagy and apoptosis to benefit its release after replicating in autophagosomes through a mechanism involving caspase cleavage of autophagy-related proteins. Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases, 26, 95-102 PMID: 24836289

Duran A, Amanchy R, Linares JF, Joshi J, Abu-Baker S, Porollo A, Hansen M, Moscat J, & Diaz-Meco MT (2011). p62 is a key regulator of nutrient sensing in the mTORC1 pathway. Molecular cell, 44 (1), 134-46 PMID: 21981924

Xi X, Zhang X, Wang B, Wang T, Wang J, Huang H, Wang J, Jin Q, & Zhao Z (2013). The interplays between autophagy and apoptosis induced by enterovirus 71. PloS one, 8 (2) PMID: 23437282

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