Marburg Virus and Keap1: induction of antioxidant response whilst inhibiting selective autophagy ?

Under normal conditions, Keap1 interacts with Nrf2 via the Nrf2-ECH homology domain 2 (Neh2) located within the N-terminal end of Nrf2 and ubiquitinates Nrf2 in a Cullin-3 dependent manner., leading to the proteasomal degradation of Nrf2 via the 26S proteasome independent of autophagy. Inactivation of Keap1 by binding p62/SQSTM1 via the C-terminal KELCH domain of Keap1 and the Keap1 interacting domain (KIR) of p62/SQSTM1 induces the degradation of Keap1 via selective autophagy, thus releasing Nrf2. In the case of oxidant independent activation of Nrf2, this translocation is preceded by phosphorylation of p62/SQSTM1 at Ser-351 of the KIR domain of p62/SQSTM1 whereas in the case of reactive oxygen species, nitric oxide, or electrophiles cysteine residues (Cys-151, Cys-273, and Cys-288 being the critical residues) of Keap1 are modified whereas Cadmium or Arsenic binds Keap1 at these residues while at the same time prevents Keap1 from interacting with p62/SQSTM1 and thus stabilising Keap1.

Domains of p62/SQSTM1/ Keap1, and Nrf2

Since both Keap1 and LC-3B are competing for binding to p62/SQSTM1 the ability of p62/SQSTM1 to form dimers with either NBR1 or with itself is crucial for the degradation of  p62/SQSTM1 complexes with Keap1 and ubiquitinylated proteins by selective autophagy; indeed, overexpression of mCherry-Keap1 has been shown to decrease the autophagic degradation of  p62/SQSTM1, but not abolish it, and endogenous as well overexpressed Keap1 co-localises with GFP-p62/SQSTM1 in p62/SQSTM1 and Ubiquitin  positive foci. 

Stabilised Nrf2 translocates to the nucleus where it forms a heterodimer with small musculoaponeurotic fibrosarcoma (MAF) proteins. This complex then binds to the Antioxidant response element (ARE) and thus induces the expression of cytoprotective genes, among them p62/SQSTM1 itself. Other target genes involve those in eliminating ROS (thioredoxin reductase1 and peroxiredoxin 1), detoxification of xenobiotics (NAD(P)H Dehydrogenase Quinone1, Glutathione S-Transferase), drug transport (multidrug resistance associated proteins), and glutathione synthesis (Glutamate-Cysteine Ligase). Regarding antiviral signalling, the infection of human alveolar epithelial cells with Influenza A/PR8 increases the production of ROS and thus activates the antioxidant response via the Keap1-Nrf2 pathway and thus increases the expression of ARE target genes inhibiting viral replication whilst being cytoprotective, in particular heme oxygenase -1 (HO-1), myxovirus resistance-1 (Mx1) and  2'-5'-oligoadenylate synthetase 1 (OAS1), in a Nrf2 dependent/Interferon independent manner.

The antioxidant response promotes selective autophagy by Nrf2 dependent upregulation of
p62/SQSTM1 expression and binding of Keap1 to p62/SQSTM1 as well as ubiquination of misfolded proteins 

Marburg (MARV) and Ebola (EBOV) VP24 and the antioxidant response pathway

Marburg (MARV) and Ebola (EBOV) viruses are both members of the Filoviridae, and are zoonotic viruses which utilize bats as a reservoir host species and cause highly fatal hemorrhagic fever in humans.  As mentioned in a previous post, the genomes of both MARV and EBOV are similar in structure, with both encoding for VP24, a multifunctional protein involved in viral RNA synthesis, formation of the viral nucleocapsid as well as the release of infectious viral particles. As such, VP24 derived from both MARV and EBOV localises in ring like structures in the cytoplasm of cells transfected with VP24 or infected with MARV or EBOV respectively, co-localising with viral RNA in infected cells. Accordingly, EBOV VP24 deletion mutants exhibit impaired viral replication due to impaired formation of the viral nucleocapsid, replication of the viral genome and increased interferon antiviral signalling. In contrast to EBOV VP24, MARV VP24 does not interact with Karyopherin-α and thus not block the translocation of tyrosine phosphorylated STAT-1 into the nucleus of infected cells.

MARV VP24 binds Keap1 and thus releases Nrf2 

Unlike EBOV VP24, MARV VP24 however does associate with Keap1 via the Kelch domain of both human and bat derived Keap1. Consequently, Nrf2 translocates into the nucleus where it activates genes under the control of the ARE element, including HO-1, NAD(P)H Dehydrogenase Quinone1 (NQO1) and Glutamate-cysteine ligase (GCLM). Interestingly, MARV VP24 does not upregulate the expression of p62/SQSTM1, whereas EBOV VP24 downregulates p62/SQSTM1 expression at 12 and 24 h p.i. , suggesting that both EBOV VP24 and MARV VP24 inhibit selective autophagy, allowing the accumulation of ubiquitinylated proteins. In the context of viral replication, in the opinion of the author of these lines, preventing the degradation of ubiquitinylated proteins via p62/SQSTM1 would prevent MARV VP40 from being degraded. MARV VP40 itself is required for viral budding, which is dependent on the ability of the PPPY motif to bind members of the HECT and/or Nedd4-like ubiquitin ligase. Therefore it might be possible that inhibiting selective autophagy by MARV VP24 might prevent the degradation of MARV VP40 and thus favours budding of the mature virions. Stabilising MARV VP40 by preventing autophagic degradation might also contribute to the inhibiting the Interferon signalling by inhibiting Jak1 signaling. Expression of MARV VP24 and subsequent induction of the antioxidant response pathway in the absence of inducing ROS might therefore not only prevent apoptosis but also inhibit antiviral signalling pathways. 

Model of inhibition of p62/SQSTM1 dependent autophagy by EBOV/MARV VP35 and/or MARV VP24 

In addition to VP40, both the EBOV and MARV VP35 proteins might inhibit selective, p62/SQSTM1 dependent, autophagy by preventing the phosphorylation of  p62/SQSTM1 at Ser403 via sequestering of TANK-Binding Kinase 1 (TBK1) since binding TBK1 by both EBOV and MARV VP35 has been implicated in inhibiting the phosphorylation of Interferon Regulatory Factor -3 (IRF-3).  If however, this interaction also inhibits or decreases the phosphorylation of p62/SQSTM1 at Ser403 and subsequently increases ubiquitin positive foci has not been demonstrated.

Further reading 

Ishimura R, Tanaka K, & Komatsu M (2014). Dissection of the role of p62/Sqstm1 in activation of Nrf2 during xenophagy. FEBS letters, 588 (5), 822-8 PMID: 24492006 

Jain A, Lamark T, Sjøttem E, Larsen KB, Awuh JA, Øvervatn A, McMahon M, Hayes JD, & Johansen T (2010). p62/SQSTM1 is a target gene for transcription factor NRF2 and creates a positive feedback loop by inducing antioxidant response element-driven gene transcription. The Journal of biological chemistry, 285 (29), 22576-91 PMID: 20452972 

Fan W, Tang Z, Chen D, Moughon D, Ding X, Chen S, Zhu M, & Zhong Q (2010). Keap1 facilitates p62-mediated ubiquitin aggregate clearance via autophagy. Autophagy, 6 (5), 614-21 PMID: 20495340 

Komatsu M, Kurokawa H, Waguri S, Taguchi K, Kobayashi A, Ichimura Y, Sou YS, Ueno I, Sakamoto A, Tong KI, Kim M, Nishito Y, Iemura S, Natsume T, Ueno T, Kominami E, Motohashi H, Tanaka K, & Yamamoto M (2010). The selective autophagy substrate p62 activates the stress responsive transcription factor Nrf2 through inactivation of Keap1. Nature cell biology, 12 (3), 213-23 PMID: 20173742

Amman BR, Carroll SA, Reed ZD, Sealy TK, Balinandi S, Swanepoel R, Kemp A, Erickson BR, Comer JA, Campbell S, Cannon DL, Khristova ML, Atimnedi P, Paddock CD, Crockett RJ, Flietstra TD, Warfield KL, Unfer R, Katongole-Mbidde E, Downing R, Tappero JW, Zaki SR, Rollin PE, Ksiazek TG, Nichol ST, & Towner JS (2012). Seasonal pulses of Marburg virus circulation in juvenile Rousettus aegyptiacus bats coincide with periods of increased risk of human infection. PLoS pathogens, 8 (10) PMID: 23055920 

Baird L, & Dinkova-Kostova AT (2011). The cytoprotective role of the Keap1-Nrf2 pathway. Archives of toxicology, 85 (4), 241-72 PMID: 21365312 

He X, & Ma Q (2010). Critical cysteine residues of Kelch-like ECH-associated protein 1 in arsenic sensing and suppression of nuclear factor erythroid 2-related factor 2. The Journal of pharmacology and experimental therapeutics, 332 (1), 66-75 PMID: 19808700 

Wu KC, Liu JJ, & Klaassen CD (2012). Nrf2 activation prevents cadmium-induced acute liver injury. Toxicology and applied pharmacology, 263 (1), 14-20 PMID: 22677785 Ma, Q. (2013). Role of Nrf2 in Oxidative Stress and Toxicity Annual Review of Pharmacology and Toxicology, 53 (1), 401-426 DOI: 10.1146/annurev-pharmtox-011112-140320 

Mateo M, Carbonnelle C, Martinez MJ, Reynard O, Page A, Volchkova VA, & Volchkov VE (2011). Knockdown of Ebola virus VP24 impairs viral nucleocapsid assembly and prevents virus replication. The Journal of infectious diseases, 204 Suppl 3 PMID: 21987766 

Mateo M, Reid SP, Leung LW, Basler CF, & Volchkov VE (2010). Ebolavirus VP24 binding to karyopherins is required for inhibition of interferon signaling. Journal of virology, 84 (2), 1169-75 PMID: 19889762 

Noda, T., Ebihara, H., Muramoto, Y., Fujii, K., Takada, A., Sagara, H., Kim, J., Kida, H., Feldmann, H., & Kawaoka, Y. (2006). Assembly and Budding of Ebolavirus PLoS Pathogens, 2 (9) DOI: 10.1371/journal.ppat.0020099 

Edwards MR, Johnson B, Mire CE, Xu W, Shabman RS, Speller LN, Leung DW, Geisbert TW, Amarasinghe GK, & Basler CF (2014). The Marburg virus VP24 protein interacts with Keap1 to activate the cytoprotective antioxidant response pathway. Cell reports, 6 (6), 1017-25 PMID: 24630991 

Bamberg S, Kolesnikova L, Möller P, Klenk HD, & Becker S (2005). VP24 of Marburg virus influences formation of infectious particles. Journal of virology, 79 (21), 13421-33 PMID: 16227263

Mateo M, Carbonnelle C, Martinez MJ, Reynard O, Page A, Volchkova VA, & Volchkov VE (2011). Knockdown of Ebola virus VP24 impairs viral nucleocapsid assembly and prevents virus replication. The Journal of infectious diseases, 204 Suppl 3 PMID: 21987766 

Urata S, Noda T, Kawaoka Y, Morikawa S, Yokosawa H, & Yasuda J (2007). Interaction of Tsg101 with Marburg virus VP40 depends on the PPPY motif, but not the PT/SAP motif as in the case of Ebola virus, and Tsg101 plays a critical role in the budding of Marburg virus-like particles induced by VP40, NP, and GP. Journal of virology, 81 (9), 4895-9 PMID: 17301151 

Martin-Serrano J, Eastman SW, Chung W, & Bieniasz PD (2005). HECT ubiquitin ligases link viral and cellular PPXY motifs to the vacuolar protein-sorting pathway. The Journal of cell biology, 168 (1), 89-101 PMID: 15623582 

Kosmider B, Messier EM, Janssen WJ, Nahreini P, Wang J, Hartshorn KL, & Mason RJ (2012). Nrf2 protects human alveolar epithelial cells against injury induced by influenza A virus. Respiratory research, 13 PMID: 22672594 

Niture SK, & Jaiswal AK (2011). Inhibitor of Nrf2 (INrf2 or Keap1) protein degrades Bcl-xL via phosphoglycerate mutase 5 and controls cellular apoptosis. The Journal of biological chemistry, 286 (52), 44542-56 PMID: 22072718     

Prins KC, Cárdenas WB, & Basler CF (2009). Ebola virus protein VP35 impairs the function of interferon regulatory factor-activating kinases IKKepsilon and TBK-1. Journal of virology, 83 (7), 3069-77 PMID: 19153231 

Pilli M, Arko-Mensah J, Ponpuak M, Roberts E, Master S, Mandell MA, Dupont N, Ornatowski W, Jiang S, Bradfute SB, Bruun JA, Hansen TE, Johansen T, & Deretic V (2012). TBK-1 promotes autophagy-mediated antimicrobial defense by controlling autophagosome maturation. Immunity, 37 (2), 223-34 PMID: 22921120

Coronavirus antivirals: RNA replication machinery as an antiviral target?

The genome of Coronaviruses encodes not only structural proteins required for the formation of viral particles, but also for non-structural proteins (nsp’s) which are required for viral replication. Some of the latter are involved in the formation of viral replication centers (RTCs), particularly nsp-3/-4/-6, while others are involved in processing the viral orf1ab polyprotein, interfering with the cellular antiviral response, or with facilitating the replication of the viral RNA.

During viral replication, following the cleavage of the viral orf1ab polyprotein by viral proteases, the viral nsp’s assemble at the ER into a multienzyme complex which is associated with viral RNA and double membrane vesicles that derive from the ER and whose formation is induced by the viral nsp-3, -4, and -6 proteins as described before. As described before, CoV replicate in the cytoplasm of infected cells although an involvement of the nucleus cannot be ruled out. Consequently, the viral positive strand ssRNA genome is transcribed into a negative sense RNA and subsequently into full length and subgenomic RNAs by the viral RNA dependent RNA polymerase (RdRP) in the cytoplasm and thus (1) not protected from degradation by cellular 5’ to 3” exoribonucleases, (2) subject to recognition by cellular proteins such as Toll-like receptors which are part of the innate immune response and (3) are not efficiently translated. Furthermore, due to the lack of RNA helicases, dsRNA intermediates formed would prevent translation of viral subgenomic RNAs. Consequently, a subset of the nsp’s encoded are exhibiting RNA helices, 3’ to 5’ exoribnuclease (ExoN) activity as well as Methyltransferase (MT) activity. Whilst a number of RNA viruses express proteins with ExoN and MT activity are well characterised, in the case of CoV the roles of the viral nsp-14 and -15 proteins only begin to emerge. CoV nsp-14 is a bifunctional protein, where the 3’ to 5’ ExoN activity is localised within the N-terminus and the guanine-N7-methyltransferase (N7-MTase) activity is located with the C-terminal domain; both activities however depend on each other, i.e. mutations rendering the ExoN domain inactive also inhibit the N7-MTase activity. Functionally, nsp-14 has been postulated to remove excise 3′-end mismatched nucleotides from the dsRNA intermediate synthesised by the viral RdRP (nsp-12), which is enhanced by binding of a co-factor, nsp-10, and nsp-10 mutant which do not bind nsp-14 fail to stimulate nsp-14 activity. Aside from the proofreading mechanism, the 3′–5′ ExoN domain is also involved in the degradation of viral dsRNA replication intermediates and thus may inhibit the induction of the cellular type I Interferon response akin to Lassa Fever virus nucleoprotein. In contrast to the ExoN activity, N7-MTase activity is not activity is not affected by nsp-10.

In addition to nsp-14, nsp-10 also binds to nsp-16. In contrast to nsp-14, nsp-16 however is solely involved in RNA capping, more precisely in converting the ^7MeGpppN cap  (cap 0) generated by the N7-MTase activity of nsp-14 into a cap-1 structure via a 2􏰃O-Methyltransferase activity, a step that enhances the translation efficiency of the viral RNA. Indeed, mutations of nsp-10 preventing the interaction with nsp-14 inhibit the ExoN activity of nsp-14 whereas failure to interact with nsp-16 only has moderate effect on viral replication. nsp-14 also interacts with a complex of nsp-7 and -8, thus forming a tripartite complex, which in turn binds to nsp-14 as described above. Functionally, the nsp-7/-8 complex is a primate, i.e. a primer independent RNA Polymerase synthesising primer sequences utilised by the viral RdRP.

Coronavirus replication complex 

Both nsp-7 and -8 also bind to nsp-12/RdRP and again this tripartite complex interacts with nsp-14, thus leading to the assembly of a complex which not only allows synthesis of the viral RNA but also a proofreading mechanism as well as capping the nascent RNA and thus protecting viral RNA from being degraded and recognised by the cellular pattern recognition receptors.

Consequently, mutations of the conserved D/ExD/E site rendering ExoN inactive result in viable mutant which exhibit a substantial increase in the mutation rate of the viral genome with a decrease in viral titers. Mutations of nsp-16 or nsp-10 likewise lead to a decrease in viral titers (albeit less pronounced than nsp-14 mutants) or non-viable viruses. Furthermore antiviral drugs targeting conserved residues within the proteins that form part of the complex might provide treatment not only during infections with currently circulating Coronaviruses but also for novel and emerging Coronaviruses in humans as well as animals.

Finally, how is the replication complex target to the double membrane vesicles induced by the expression of nsp-3, -4, and-6? In murine DBT cells transfected with both nsp-4 and nsp-8 both proteins localise to the ER and SARS-CoV nsp8 co-localises with p6 in the perinuclear region (presumably the ER). In addition, nsp-4 co-localises with nsp-8 in cells infected with MHV-A59. These results suggest that nsp-8 is recruited to the ER by interacting with nsp-4 and maybe SARS-CoV p6. nsp-8 then might recruit nsp-7, nsp-12, nsp-10, and nsp-14 (and viral RNA) prior to the formation of the RTC; whether viral RNA is required or not - that remains to be seen.

Recruitment of nsp-7/-8 by nsp-4 and /or p6 initiates the replication of viral RNA

Further reading 

Prentice E, McAuliffe J, Lu X, Subbarao K, & Denison MR (2004). Identification and characterization of severe acute respiratory syndrome coronavirus replicase proteins. Journal of virology, 78 (18), 9977-86 PMID: 15331731


Jin X, Chen Y, Sun Y, Zeng C, Wang Y, Tao J, Wu A, Yu X, Zhang Z, Tian J, & Guo D (2013). Characterization of the guanine-N7 methyltransferase activity of coronavirus nsp14 on nucleotide GTP. Virus research, 176 (1-2), 45-52 PMID: 23702198 

Bouvet M, Lugari A, Posthuma CC, Zevenhoven JC, Bernard S, Betzi S, Imbert I, Canard B, Guillemot JC, Lécine P, Pfefferle S, Drosten C, Snijder EJ, Decroly E, & Morelli X (2014). Coronavirus Nsp10, a Critical Co-factor for Activation of Multiple Replicative Enzymes. The Journal of biological chemistry, 289 (37), 25783-96 PMID: 25074927 

Bouvet M, Imbert I, Subissi L, Gluais L, Canard B, & Decroly E (2012). RNA 3'-end mismatch excision by the severe acute respiratory syndrome coronavirus nonstructural protein nsp10/nsp14 exoribonuclease complex. Proceedings of the National Academy of Sciences of the United States of America, 109 (24), 9372-7 PMID: 22635272 

Bouvet M, Debarnot C, Imbert I, Selisko B, Snijder EJ, Canard B, & Decroly E (2010). In vitro reconstitution of SARS-coronavirus mRNA cap methylation. PLoS pathogens, 6 (4) PMID: 20421945 

Decroly E, Imbert I, Coutard B, Bouvet M, Selisko B, Alvarez K, Gorbalenya AE, Snijder EJ, & Canard B (2008). Coronavirus nonstructural protein 16 is a cap-0 binding enzyme possessing (nucleoside-2'O)-methyltransferase activity. Journal of virology, 82 (16), 8071-84 PMID: 18417574

Zhou H, & Perlman S (2007). Mouse hepatitis virus does not induce Beta interferon synthesis and does not inhibit its induction by double-stranded RNA. Journal of virology, 81 (2), 568-74 PMID: 17079305 

Menachery VD, Debbink K, & Baric RS (2014). Coronavirus Non-Structural Protein 16: Evasion, Attenuation, and Possible Treatments. Virus research PMID: 25278144 

Hastie KM, Kimberlin CR, Zandonatti MA, MacRae IJ, & Saphire EO (2011). Structure of the Lassa virus nucleoprotein reveals a dsRNA-specific 3' to 5' exonuclease activity essential for immune suppression. Proceedings of the National Academy of Sciences of the United States of America, 108 (6), 2396-401 PMID: 21262835 

Qi X, Lan S, Wang W, Schelde LM, Dong H, Wallat GD, Ly H, Liang Y, & Dong C (2010). Cap binding and immune evasion revealed by Lassa nucleoprotein structure. Nature, 468 (7325), 779-83 PMID: 21085117 

Martínez-Sobrido L, Giannakas P, Cubitt B, García-Sastre A, & de la Torre JC (2007). Differential inhibition of type I interferon induction by arenavirus nucleoproteins. Journal of virology, 81 (22), 12696-703 PMID: 17804508 

Xiao, Y., Ma, Q., Restle, T., Shang, W., Svergun, D., Ponnusamy, R., Sczakiel, G., & Hilgenfeld, R. (2012). Nonstructural Proteins 7 and 8 of Feline Coronavirus Form a 2:1 Heterotrimer That Exhibits Primer-Independent RNA Polymerase Activity Journal of Virology, 86 (8), 4444-4454 DOI: 10.1128/JVI.06635-11 

Li S, Zhao Q, Zhang Y, Zhang Y, Bartlam M, Li X, & Rao Z (2010). New nsp8 isoform suggests mechanism for tuning viral RNA synthesis. Protein & cell, 1 (2), 198-204 PMID: 21203988 

Lundin A, Dijkman R, Bergström T, Kann N, Adamiak B, Hannoun C, Kindler E, Jónsdóttir HR, Muth D, Kint J, Forlenza M, Müller MA, Drosten C, Thiel V, & Trybala E (2014). Targeting membrane-bound viral RNA synthesis reveals potent inhibition of diverse coronaviruses including the middle East respiratory syndrome virus. PLoS pathogens, 10 (5) PMID: 24874215

Subissi L, Posthuma CC, Collet A, Zevenhoven-Dobbe JC, Gorbalenya AE, Decroly E, Snijder EJ, Canard B, & Imbert I (2014). One severe acute respiratory syndrome coronavirus protein complex integrates processive RNA polymerase and exonuclease activities. Proceedings of the National Academy of Sciences of the United States of America, 111 (37) PMID: 25197083 

te Velthuis AJ, van den Worm SH, & Snijder EJ (2012). The SARS-coronavirus nsp7+nsp8 complex is a unique multimeric RNA polymerase capable of both de novo initiation and primer extension. Nucleic acids research, 40 (4), 1737-47 PMID: 22039154 

Eckerle LD, Lu X, Sperry SM, Choi L, & Denison MR (2007). High fidelity of murine hepatitis virus replication is decreased in nsp14 exoribonuclease mutants. Journal of virology, 81 (22), 12135-44 PMID: 17804504 

Eckerle LD, Becker MM, Halpin RA, Li K, Venter E, Lu X, Scherbakova S, Graham RL, Baric RS, Stockwell TB, Spiro DJ, & Denison MR (2010). Infidelity of SARS-CoV Nsp14-exonuclease mutant virus replication is revealed by complete genome sequencing. PLoS pathogens, 6 (5) PMID: 20463816 

Beachboard DC, Lu X, Baker SC, & Denison MR (2013). Murine hepatitis virus nsp4 N258T mutants are not temperature-sensitive. Virology, 435 (2), 210-3 PMID: 23099203 

Oostra M, te Lintelo EG, Deijs M, Verheije MH, Rottier PJ, & de Haan CA (2007). Localization and membrane topology of coronavirus nonstructural protein 4: involvement of the early secretory pathway in replication. Journal of virology, 81 (22), 12323-36 PMID: 17855519 

Kumar P, Gunalan V, Liu B, Chow VT, Druce J, Birch C, Catton M, Fielding BC, Tan YJ, & Lal SK (2007). The nonstructural protein 8 (nsp8) of the SARS coronavirus interacts with its ORF6 accessory protein. Virology, 366 (2), 293-303 PMID: 17532020