Chikungunya Virus and NDP52: a deadly association?

Chikungunya virus (CHIKV) is the causative agent of an arthropod (mosquito) transmitted disease which is characterised by a high fever, rash, joint pain, and arthritis which was reported in 1952 in Tanzania but has spread since to Europe, the Americas, Asia, and Australia and currently epidemic in the Americas.

Confirmed cases of Chikungunya Virus infections in the Caribbean and continental
US as August 22nd 2014

Being a member of the Alphaviridae genus of the family Togaviridae related to Ross River virus, O'nyong'nyong virus, and Semliki Forest Virus (SFV), CHIKV has a positive strand ssRNA genome of 11.6 kb in size which encodes for both the structural and non-structural (nsP) proteins. In accordance with other prototype members of the Alphaviridae, the 5’ two thirds encodes for the four viral nsP (nsP 1-4) whereas the structural proteins are encoded within a subgenomic m 26S RNA, which in turn derives from a precursor 42S RNA.

Alphavirus genome, particle, and proteins

Both in vitro and in vivo, CHIKV can infect a wide variety of cells, including primary fibroblast cells, macrophages, astrocytes as well as hepatic cells. Similar to other alphaviruses, CHIKV infection has the capacity to induce caspase dependent apoptosis especially late infection. This process is preceded by autophagy (as discussed below) which might not only favour viral replication but also prevent apoptosis. Viral replication itself takes place in perinuclear replication centers (RCs) derived from endosomal and lysosomal membranes containing the three nsP in addition to the viral RNA dependent RNA Polymerase, akin to to the RTC induced by the Nidovirales, although viral RCs have also been observed close to the plasma membrane. Similar to the coronaviral N protein, the nsP2 of SFV also localises to the nucleolus where it associates with the ribosomal protein S6 (RpS6), thus might contribute to the shut off of host cell translation.

                          Induction of ER stress and apoptosis

Following the infection of glioblastoma cells with CHIKV, apoptosis as evidenced by DNA fragmentation, cleavage of PARP, nuclear condensation, loss of mitochondrial membrane potential, and activation of caspases can be observed at 48 h p.i. . Akin to cells infected with Japanese Encephalitis Virus (JEV), apoptosis is induced by the ER stress response, which in the case of SFV is characterised by the induction of IRE1 and ATF6 (but not PERK) pathways, involving the splicing of XBP1 as well as ATF6 mediated phosphorylation of eIF2α (subsequently increasing the expression of CHOP) as well up regulation of proteins inducting the antiviral response such as proteins of the RIG-I like receptor (RLR) pathway as well pro-inflammatory cytokines namely CXCL9, IL-6, TNF-α, and IL-1β within 24 h p.i. Interestingly, in contrast to Sindbis Virus (SINV), in CHIKV infected cells, the viral RdRP, nsP4, inhibits the phosphorylation of eIF2α following treatment with tunicamycin not only in mosquito (Aedes albopictus) C6/36 cells,  but also in HEK293T,  MRC-5 and BHK-21 cells,  similar to vaccinia virus K3L or Hepatitis C Virus NS5A. In the context of viral replication, the data support a model in which CHIKV nsP4 delays the activation of PERK mediated phosphorylation of eIF2α by at least 21 h p.i. compared to cells infected with SINV, although it has been not demonstrated that a recombinant SINV expressing CHIKV nsP4 has delayed pattern of eIF2α  phosphorylation similar to CHIKV .

CHIKV and the ER stress response: inhibition of PERK by nsP4 and
BiP by preE1/E2

In general the ER stress response is triggered however not by the accumulation of non-structural proteins at the ER, but by the envelope glycoproteins, namely the E1 and E2 proteins in a process that depends on a cellular BiP. In the case of SINV both the precursor of E2, PE2, and E1 are co-translationally inserted into the ER membrane prior to their transport to the cell surface via the Golgi where they are inserted into the plasma membrane by palmitoylated residues. The insertion into the ER membrane therefore does not induce the formation of the replication complex but is only transient in nature. Both proteins however might induce the ER stress pathways by two mechanisms, either depletion of lipid due to the formation of vesicles and/or via BiP. BiP is a molecular chaperone, which not only associates with unfolded proteins within the ER and facilitates correct folding but is also involved in transporting proteins across the ER membrane. In the case of SINV E2 and E1 proteins, BiP associates with both E2 and E1 within the ER and is implicated in their transport across the ER membrane. More importantly however (at least in terms of the UPR), in SINV infected cells, BiP also induces the ATF6 and IRE1 induced ER stress response, problem by releasing BiP from its interaction with ATF6, IRE1, and Caspase-4/-12.  As discussed in prior posts, the induction of both the ATF6 and IRE1 dependent ER stress response not only induces apoptosis, but also autophagy. Indeed cells infected with SFV, CHKV, or SINV exhibit the induction of the autophagy pathway prior to the induction of apoptosis and autophagy has been shown to be required for productive infection and CHIKV induced autophagy has been shown to delay caspase induced apoptosis.

                                       CHIKV and autophagy

In the case of CHIKV, autophagy not only has a cytoprotective function but also enhances viral replication and the switch from autophagy to viral assembly late in infection increases apoptosis induced by CHIKV.

In human and murine cells infected with CHIKV, the viral ubiqutinated capsid protein co-localises and co-immunoprecipates with p62/SQSTM1 mediated the UBA domain and subsequently is degraded via the formation of autophagosome and targeting to LAMP1 positive lysosomal structures; indeed, both the depletion of p62 and the expression of a p62/SQSTM1 ΔUBA mutant stabilise the capsid protein as well as treatment of infected with Bafilomycin A as well as increasing viral replication as measured by viral RNA and viral titers.

The viral capsid protein is degraded by p62/SQSTM1 and localised
to viral replication centers by NPD52

In cells infected with CHIKV or other members of the Alphaviridae, the nsPs associate with viral RNA to form the viral replication complexes (RC), which in turn are associated double membrane vesicles (DMV) -termed cytopathic vacuoles- located in the perinuclear region. The DMVs not only contain nsPs, viral RNA, and dsRNA intermediates, but also (in the case of SINV and SFV) but also markers of the endo-and lysosomal system such as TGN-46. As part of this complex, nsP2 in addition to the role in shutting off host cell translation and inducing apoptosis, also binds the viral RNA and forms autophagy like vesicles containing the viral RNA that are localised in the perinuclear region and positive for TGN-46. The latter structure is not only positive for nsP2, but also for the viral capsid protein and nsP3, thus representing the viral RC. The process of the localisation of nsP2-RNA complex is not only dependent on the nuclear localisation and the presence of the C-terminal domain, but also on a cellular protein, Nuclear Dot Protein (NDP52). NDP52 has been described as a receptor for xenophagy and extensive studied in cells infected with bacteria, in particular Listeria and Salmonella. In contrast to p62/SQSTM1, NDP52 lacks a UBA and therefore does not bind ubiqutinated proteins. In the case of CHIKV infected cells, NDP52 forms structures akin to but different from “classic” autophagosomes the viral capsid protein as well as nsP2, and dsRNA intermediates and LC3-C  -but not LC3-B- via binding to the non-canonical LIR of NDP52.

p62/SQSTM1 and NDP52

Binding to NDP52 therefore is being postulated to be required for viral assembly late in infection and contributes to the cytoxicity observed in cells in the late stages of viral infection. In the early stages of replication however, the viral proteins might preferentially degraded via selective, p62/SQSTM1 dependent, autophagy and thus allowing survival of the infected cells. Interestingly, the time at which this switch occurs might be different for CHIKV and SINV infected cells and thus might contribute to the pathology of the disease.

Targeting of viral nsP and structural proteins to TGN-46
positive structures may involve NDP52 and LC3-C 

Further to the role in targeting viral proteins to the RC, in the opinion of the author of these lines, NDP52 might be implicated in the clearance of damaged mitochondria by mitophagy at early timepoints p.i. . The induction of oxidative stress -characterised by increased levels in reactive oxygen species (ROS)- due to a decrease of antioxidant enzymes and an increase in the fold change of pro-inflammatory cytokines as a result of ER stress as well induction of mitochondrial damage leading to the release of Cytochrome C, not only induces apoptosis but also increases autophagy via inhibition of mTOR.

Switch between p62/SQSTM1 and NDP52 binding to structural proteins
may determine the localisation of these proteins and the induction of apoptosis as a
result of ER stress induced by the formation of vesicular structures

In summary, at early timepoints, the expression of the structural proteins of CHIKV, SINV, or SFV, induces ER  stress which in turn induce selective –p62/SQSTM1 dependent- autophagy, whereas at later timepoints the formation of replication centers in the perinuclear region is favoured by the localisation of viral proteins in a NDP52/LC3-C dependent pathway. Interestingly, microscopic studies using PALM, suggest that the capsid protein is located inside of double membrane structures, suggesting that the LC3-C/NDP52 coated vesicles fuse with the TGN-46 positive replication compartment.  

Apart from CHIKV, does NDP mediated localisation of viral proteins to their respective replication centers or complexes play a role during the replication of other viruses? The answer is…..we do not know. As always, the relevant experiments need to be done, but is it possible that both LC3-C and NDP52 are involved in localising components of the CoV replication centers to the ERGIC. In the case of MERS-CoV, this might explain why the virus is not cytotoxic in bats: similar to CHIKV, where the murine NDP52 does not bind viral proteins, bat derived bNDP52 (if it exists) might not bind MERS-CoV and thus target viral proteins for degradation in a p62/SQSTM1 dependent or independent manner. So if anyone in the US has a place for me to study this and other questions, I am up for it. Are you?

Further reading

Jose J, Snyder JE, & Kuhn RJ (2009). A structural and functional perspective of alphavirus replication and assembly. Future microbiology, 4 (7), 837-56 PMID: 19722838 

Froshauer S, Kartenbeck J, & Helenius A (1988). Alphavirus RNA replicase is located on the cytoplasmic surface of endosomes and lysosomes. The Journal of cell biology, 107 (6 Pt 1), 2075-86 PMID: 2904446 

Spuul P, Balistreri G, Kääriäinen L, & Ahola T (2010). Kujala P, Ikäheimonen A, Ehsani N, Vihinen H, Auvinen P, & Kääriäinen L (2001). Biogenesis of the Semliki Forest virus RNA replication complex. Journal of virology, 75 (8), 3873-84 PMID: 11264376 

Abraham R, Mudaliar P, Padmanabhan A, & Sreekumar E (2013). Induction of cytopathogenicity in human glioblastoma cells by chikungunya virus. PloS one, 8 (9) PMID: 24086645 

Laakkonen P, Ahola T, & Kääriäinen L (1996). The effects of palmitoylation on membrane association of Semliki forest virus RNA capping enzyme. The Journal of biological chemistry, 271 (45), 28567-71 PMID: 8910486 

Barry G, Fragkoudis R, Ferguson MC, Lulla A, Merits A, Kohl A, & Fazakerley JK (2010). Semliki forest virus-induced endoplasmic reticulum stress accelerates apoptotic death of mammalian cells. Journal of virology, 84 (14), 7369-77 PMID: 20427528 

Rikkonen M, Peränen J, & Kääriäinen L (1992). Nuclear and nucleolar targeting signals of Semliki Forest virus nonstructural protein nsP2. Virology, 189 (2), 462-73 PMID: 1386484 

Montgomery SA, Berglund P, Beard CW, & Johnston RE (2006). Ribosomal protein S6 associates with alphavirus nonstructural protein 2 and mediates expression from alphavirus messages. Journal of virology, 80 (15), 7729-39 PMID: 16840351 

Rathore AP, Ng ML, & Vasudevan SG (2013). Differential unfolded protein response during Chikungunya and Sindbis virus infection: CHIKV nsP4 suppresses eIF2α phosphorylation. Virology journal, 10 PMID: 23356742 

Migliaccio G, Pascale MC, Leone A, & Bonatti S (1989). Biosynthesis, membrane translocation, and surface expression of Sindbis virus E1 glycoprotein. Experimental cell research, 185 (1), 203-16 PMID: 2806407 

Wang M, Wey S, Zhang Y, Ye R, & Lee AS (2009). Role of the unfolded protein response regulator GRP78/BiP in development, cancer, and neurological disorders. Antioxidants & redox signaling, 11 (9), 2307-16 PMID: 19309259 

Mulvey M, & Brown DT (1995). Involvement of the molecular chaperone BiP in maturation of Sindbis virus envelope glycoproteins. Journal of virology, 69 (3), 1621-7 PMID: 7853497 

Krejbich-Trotot P, Gay B, Li-Pat-Yuen G, Hoarau JJ, Jaffar-Bandjee MC, Briant L, Gasque P, & Denizot M (2011). Chikungunya triggers an autophagic process which promotes viral replication. Virology journal, 8 PMID: 21902836 

Eng KE, Panas MD, Murphy D, Karlsson Hedestam GB, & McInerney GM (2012). Accumulation of autophagosomes in Semliki Forest virus-infected cells is dependent on expression of the viral glycoproteins. Journal of virology, 86 (10), 5674-85 PMID: 22438538 

Mostowy S, Sancho-Shimizu V, Hamon MA, Simeone R, Brosch R, Johansen T, & Cossart P (2011). p62 and NDP52 proteins target intracytosolic Shigella and Listeria to different autophagy pathways. The Journal of biological chemistry, 286 (30), 26987-95 PMID: 21646350 

Randow F (2011). How cells deploy ubiquitin and autophagy to defend their cytosol from bacterial invasion. Autophagy, 7 (3), 304-9 PMID: 21193841 Xie Z, & Klionsky DJ (2007). Autophagosome formation: core machinery and adaptations. Nature cell biology, 9 (10), 1102-9 PMID: 17909521

Lippai M, & Lőw P (2014). The role of the selective adaptor p62 and ubiquitin-like proteins in autophagy. BioMed research international, 2014 PMID: 25013806 

von Muhlinen N, Akutsu M, Ravenhill BJ, Foeglein Á, Bloor S, Rutherford TJ, Freund SM, Komander D, & Randow F (2012). LC3C, bound selectively by a noncanonical LIR motif in NDP52, is required for antibacterial autophagy. Molecular cell, 48 (3), 329-42 PMID: 23022382 

Joubert PE, Werneke SW, de la Calle C, Guivel-Benhassine F, Giodini A, Peduto L, Levine B, Schwartz O, Lenschow DJ, & Albert ML (2012). Chikungunya virus-induced autophagy delays caspase-dependent cell death. The Journal of experimental medicine, 209 (5), 1029-47 PMID: 22508836

Judith D, Mostowy S, Bourai M, Gangneux N, Lelek M, Lucas-Hourani M, Cayet N, Jacob Y, Prévost MC, Pierre P, Tangy F, Zimmer C, Vidalain PO, Couderc T, & Lecuit M (2013). Species-specific impact of the autophagy machinery on Chikungunya virus infection. EMBO reports, 14 (6), 534-44 PMID: 23619093 

Münz C (2013). Macroautophagy--friend or foe of viral replication? EMBO reports, 14 (6), 483-4 PMID: 23661081 

Barry G, Fragkoudis R, Ferguson MC, Lulla A, Merits A, Kohl A, & Fazakerley JK (2010). Semliki forest virus-induced endoplasmic reticulum stress accelerates apoptotic death of mammalian cells. Journal of virology, 84 (14), 7369-77 PMID: 20427528

Coronavirus proteases, p62/SQSTM1, and Deubiquitinases

Ubiquitin is a small protein of 9kDa inside present in all eukaryotic cells and involved in the degradation of proteins by covalently binding target proteins which requires different enzymes, the E1 activating enzyme, the E2 conjugation enzymes, and the E3 ubiquitin ligase. Deubiquitinating enzymes (DUBs) can reverse the ubiquitination of substrates thus preventing the degradation of proteins and can be classified into two main classes, cysteine proteases and metalloproteases.

Basic outline of the ubiquitination machinery

The first evidence that ubiquitination plays a role in the induction of autophagy came from studies that identified p62/SQSTM1, NDP52, and NBR1 as adaptors for poly- and monoubiquitylated proteins to autophagosomes. 

As discussed in previous posts, both p62/SQSTM1 and NBR1 bind cargo via a ubiquitin-associated (UBA) domain and recruit LC3-I via a LC3-I interacting region (LIR); in a further step, ALFY (autophagy-linked FYVE domain) then recruits this complex to phosphatidylinositol 3-phosphate (PtdIns(3)P) and the ATG12-ATG5-ATG16 complex. Deubiqutination of substrates therefore can prevent the induction of (selective) autophagy and indeed the expression of the deubiquitinating enzyme USP36 has been demonstrated to regulate p62/SQSTM1 dependent selective autophagy in Drosophila and human cells. As the name implies, deubiquitinating enzymes remove Ubiquitin from poly- and monoubiquitylated proteins and thus antagonise E3 ligases, leading to the accumulation of proteins which otherwise would be subject to degradation via the proteasome or selective autophagy. Deubiquitinases (DUBs) often contain multiple domains, some of which contribute to the recognition of the substrate and others that facilitate the proteolytic cleavage of the Ubiquitin residues. In the case of dUSP36, the inactivation of dUSP36 (as well as hUSP36) induces the formation of nuclear and cytoplasmic aggregates of ubiquitinated proteins, the latter being degraded by selective autophagy.  

Deubiqutinases inhibit selective autophagy via binding to ubiqutinated substrates  to

a Uba domain or by binding substrates to a a Ubl domain

Although the role of USP36 in viral infected cells has not been studied, the role of DUBs in general has been, although the role of viral DUBs in regulating selective (p62/SQSTM1 dependent) autophagy is not known. In case of bacterial infections however the situation is different and although this post covers viral DUBs, a short look at cells infected with Salmonella is warranted.

Salmonella enterica serovar Typhimurium (S. Typhimurium) SseL 1

In epithelial cells, replicating Salmonella are localised in clusters close to the MTOC and the Golgi, leading to the accumulation of mono- and polyubiquitinated proteins. In contrast to wt strains however, cells infected with a ssaV null mutant (ΔssaV) strain that lacks a functional SPI-2 T3SS less than 10% of infected cells do not accumulate ubiquitinated proteins. One of the effectors of SPI-2 T3SS acts as a deubiquitinase (SseL), while others (SspH1, SspH2 and SlrP) are E3 ligases, suggesting that the formation of the clusters depends on the action of E3 ligase and a DUB. In HeLa cells infected with a Salmonella strain mutant for SseL or complemented with an inactive SseLC262A (SseLC/A) mutant (catalytic inactive mutant), induces the formation of ubiquitinated inclusions which are positive for p62/SQSTM1 as well as LC3, suggesting that ubiquitinated proteins are targeted for selective autophagy. In the context of the intracellular replication of Salmonella, targeting bacteria to the autophagic machinery would be considered the bacterial equivalent of the degradation of viral components; the expression of SseL therefore does favour bacterial replication by stabilising the “bacterial replication centre”. 

Viral DUBs: Coronavirus proteases 3CL^pro and 3C^pro

Although a number of viral deubiquitinases have been characterised, non has been shown to inhibit selective autophagy. Since in various past posts I have discussed the impact of coronaviral non-structural proteins (nsp) on the induction of the autophagic pathway in addition to the role the cleavage of p62/SQSTM1 by Coxsackievirus B3 proteases (and the potential impact on selective autophagy), I want to propose a model in which the coronaviral 3CL^pro and 3C^pro proteases binds and deubiquitinate mono- and polyubiquitinated proteins, thus inhibiting p62/SQSTM1 dependent autophagy. In the case of 3C^pro/PLP^pro, the N-terminal Ubl domain binds ubiquitinated substrates, thus preventing the substrate from being recognised by p62/SQSTM1, followed by either proteolytic cleavage via the cysteine protease activity or -alternatively- by delivering them to the proteasome in a mechanism resembling the cellular Rad23/ Rhp23 system. In both cases, selective autophagy mediated by p62/SQSTM1 is bypassed but not necessarily inhibited. 

CoV PLPpro might sequester and cleave substrates via the Ubl
domain bypassing p62/SQSTM1

If however, ubiquitinated substrates are not degraded following binding to the viral Ubl domain, these proteins might accumulate either in p62/SQSTM1 positive -akin to in cells expressing the bacterial SseLC262 - or negative structures, the latter accumulating proteins in aggresome like structures.  In a similar way, the viral main protease, 3CL^pro , may bind substrates and prevent the degradation via selective autophagy or promote the recruitment of LC3-I via sequestering p62/SQSTM1. If the localisation of substrates to the viral proteases is inducing the proteolytic cleavage of these proteins instead of autophagy, also remains to be seen and can not ruled out at present. 

In the context of viral replication, this system might be used to recruit viral proteins to replication enters whilst preventing them from being degraded. Alternatively -or additionally?-, the recruitment of  p62/SQSTM1 by this non-canonical system might explain why Atg5 is not required for the replication of the murine CoV, MHV. The expression of the viral 3CL^pro and 3C^pro proteases however might not only prevent selective autophagy but also the formation of EDEMosomes in viral infected cells and thus the ERAD pathway. 

Sequestering of p62/SQSTM1 by CoV PLPpro might inhibit the formation of the EDEMosome
 and promote LC3-i recruitment

Central to this question is if p62/SQSTM1 is required for the replication of CoV. A simple way to investigate this would be to infect p62 -/- MEF with MHV. Further questions to be answered are of course related to the localisation of p62/SQSTM1 in cells expressing the viral proteases both in the absence and presence of the orf1a polyprotein as well as nsp-3/-4/-6, in addition to the questions relating to the accumulation of mono- and polyubiquitinated proteins. To extent this beyond CoV, one has to look at the arteriviral equivalents of the coronaviral proteins as well as other viral DUBs such as HAUSP.

As always, who is up to the challenge?

Further reading

Taillebourg E, Gregoire I, Viargues P, Jacomin AC, Thevenon D, Faure M, & Fauvarque MO (2012). The deubiquitinating enzyme USP36 controls selective autophagy activation by ubiquitinated proteins. Autophagy, 8 (5), 767-79 PMID: 22622177 


Birmingham CL, & Brumell JH (2006). Autophagy recognizes intracellular Salmonella enterica serovar Typhimurium in damaged vacuoles. Autophagy, 2 (3), 156-8 PMID: 16874057 

Mesquita FS, Thomas M, Sachse M, Santos AJ, Figueira R, & Holden DW (2012). The Salmonella deubiquitinase SseL inhibits selective autophagy of cytosolic aggregates. PLoS pathogens, 8 (6) PMID: 22719249 

González CM, Wang L, & Damania B (2009). Kaposi's sarcoma-associated herpesvirus encodes a viral deubiquitinase. Journal of virology, 83 (19), 10224-33 PMID: 19640989 

van Kasteren PB, Bailey-Elkin BA, James TW, Ninaber DK, Beugeling C, Khajehpour M, Snijder EJ, Mark BL, & Kikkert M (2013). Deubiquitinase function of arterivirus papain-like protease 2 suppresses the innate immune response in infected host cells. Proceedings of the National Academy of Sciences of the United States of America, 110 (9) PMID: 23401522 

Wu X, Zhang M, & Sun SC (2011). Mutual regulation between deubiquitinase CYLD and retroviral oncoprotein Tax. Cell & bioscience, 1 PMID: 21824392 
  
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 

Ratia K, Saikatendu KS, Santarsiero BD, Barretto N, Baker SC, Stevens RC, & Mesecar AD (2006). Severe acute respiratory syndrome coronavirus papain-like protease: structure of a viral deubiquitinating enzyme. Proceedings of the National Academy of Sciences of the United States of America, 103 (15), 5717-22 PMID: 16581910 

Báez-Santos YM, Mielech AM, Deng X, Baker S, & Mesecar AD (2014). Catalytic Function and Substrate Specificity of the PLpro Domain of nsp3 from the Middle East Respiratory Syndrome Coronavirus (MERS-CoV). Journal of virology PMID: 25142582 

Clementz MA, Chen Z, Banach BS, Wang Y, Sun L, Ratia K, Baez-Santos YM, Wang J, Takayama J, Ghosh AK, Li K, Mesecar AD, & Baker SC (2010). Deubiquitinating and interferon antagonism activities of coronavirus papain-like proteases. Journal of virology, 84 (9), 4619-29 PMID: 20181693