Virology tidbits

Virology tidbits

Sunday, 3 August 2014

Ebola: Role of the Glycoprotein in viral entry and ER stress

Ebola viruses (EBOV) are the causative agent of severe hemorrhagic fever in humans with a high mortality rate, with a case fatality rate of up to 90%, with Ebola Zaire the most pathogenic. As belonging tot he Filoviridae, the EBOV genome is a single stranded non-segmented negative sense RNA, containing seven monocistronic genes. 

Genome structure of Ebola and Marburg Virus

Filoviruses have a broad host cell tropism, including dendritic cells (DCs), hepatocytes, fibroblasts, endothelial cells, monocytes, macrophages, and adrenal cortical cells, but excluding lymphocytes. Among the first to be infected are DCs, monocytes as well as macrophages and indeed following experimental infection of guinea pig with Marburg virus, viral replication in macrophages can be detected as early as 24 hrs p.i. whereas in hepatocytes of infected African green monkeys can not be detected until 48 to 72 hrs p.i., suggesting that the early infection of immune cells contributes to viral dissemination. In general, infected macrophage, fibroblasts, and endothelial cells can be detected in all organs in experimentally animals. It should be noted that infected alveolar and bronchial cells only can be occasionally found in infected humans, although aerosol transmission initially has been postulated for Ebola Reston, the main route of transmission is by contaminated blood and other secretions.

                                    Role of EBOV-GP in viral entry

The Glycoprotein (GP) is displayed on the surface of all Filoviruses and as such facilitates viral entry by binding the cellular receptor. via macropinocytosis into macropinocytosis-specific endosomes.  Similar to other viral Class I fusion proteins, GP is a trimer consisting of monomers which are a complex connected by disulphide bonds of the receptor binding GP1 subunit and the fusion GP2 subunit akin to the Coronaviral S1 and S2 subunits of the viral S protein. The structure of the ectodomain consists of glycosylated cap, a head, and a base, also known as a “chalice-like” structure, whose binding to the cellular receptor can be inhibited by monoclonal antibodies. Within the GP1 subdomain a mucin-like, highly glycosylated, domain not only protects the receptor-binding domain from recognition by mAbs but also seems to enhance viral attachment as well as masking immune regulatory molecules on infected cells. 


Studies using vesicular stomatitis virus (VSV) pseudovirions bearing the EBOV-GP (VSV-GP) showed that EBOV-GP although requires acidified endosomes for entry and fusion, the low pH of endosomes is not sufficient for viral entry, a finding supported by observations that mutating the furring cleavage site between the GP1 and GP2 subunits does not ablate infection. Indeed, co-factors needed for successful infection of VSV-GP are Cathepsin B and L, endosomal cysteine proteases which are activated by the low pH of acidic endosomes and that are known to be required for the infection of cells with a number of viruses including SARS-CoV, Reovirus, Hendra Virus, and Influenza Virus. Inhibition of Cathepsin B and L with either chemical inhibitors (CA074Me and Z-FY(t-Bu)-dmk respectively) or siRNA decreases the infectivity of VSV-GP by put o 80% (Cathepsin B) and 46% (Cathepsin L) in Vero cells and in Mouse Embryonic Fibroblasts (MEF) as do class specific protease inhibitor targeting endosomal cysteine proteases (leupeptin/E64d/MG132). Since Cathepsin B and L cleaves GP into its two subdomains -GP1 and GP2- and the cleavage of of viral surface glycoproteins has been reported to be essential for viral infectivity during Influenza Virus and Coronavirus entry (among others), a similar function has  been proposed for cleavage of EBOV-GP order pseudotyped VSV-GP. Indeed, treatment of VSV-GP of Cathepsin B plus Cathepsin L or thymolysin enhanced the infectivity of VSV-GP, partially due to removal of the mucin-like domain. In conclusion, these results lead to model where the initial attachment of the viral particle is mediated by the GP2 subunit, followed by cleavage of GP into GP1 and GP2 by cellular Cathepsin B/L, and subsequent fusion event either facilitated by other host factors in a Bafilomycin A and E64D sensitive manner. 

Cleavage of EBOV-GP is required for NPC1 mediated uptake of GP2 into the endosome

In both cases, the GP1 subunit is digested by Cathepsin, which allows for structural changes of GP2, allowing the fusion of GP2 with the endosome.  One of the potential co-factors necessary for EBOV-GP3 fusion between the vial and endosomal membrane might be Niemann-Pick C1 (NPC1), a protein commonly associated with the regulation of the transport of cholesterol through the late endosome/lysosome en route to other intracellular membranes. Indeed, NPC1 -/- cells and cells expressing soluble NPC1 are resistant to EBOV infection suggesting that NPC1 plays a crucial role in not only fusing the viral membrane to the endosome but also allowing viral escape of the vesicular compartment and subsequent viral replication. In this scenario, following viral entry via macropinocytosis, the GP protein localises to the early endosome where Cathepsin B/L cleave the viral GP protein into the GP1 and GP2 subunits, the former being degraded and the latter binding to NPC1 thus allowing completion of the fusion process and release of the nucleocapsid. In addition to NPC1, this process requires the homotypic fusion and vacuole protein-sorting (HOPS) multisubunit tethering complex (HOPS), which essentially consists of Vps11, Vps18, Vps33, Vps34, Vps41, and Vps33, as well as Guanine Nucleotide Exchange Factors (GEFs) and RabGTPase 5/7. In this extended model, the HOPS complex would be required chiefly for the fusion of the endosome with the lysosome and NPC1 for triggering the fusion of EBOV-GP(2) with the endosomal membrane. The question remains if this complex also translocates the NPC1-GP2 complex to the ER since UVRAG has been reported -in concert with ATG9- to be involved in the retrograde Golgi to ER traffic or if EBOV-GP akin to VSV-G inhibits the formation ATG9 positive vesicles. Alternatively, autophagosomes may be formed which are then undergoing clearance via HOPS - or in the case of EBOV, instead of clearance the viral genome would be released. Indeed, murine leukaemia virus pseudotyped EBOV-GP has been shown to be sensitive to Chloroquine, a drug that stabilises autophagosomes and inhibits the formation of autolysosomes, although more detailed studies are required.

Potential role of UVRAG and HOPS in the localisation of EBOV-GP to acidic vesicles

Alternatively, the HOPS complex might be required for the internalisation of EBOV in a role similar for NPC1 mediated intake of LDL, which is mediated by clathrin coated vesicles. Indeed, clathrin coated vesicles are observed during the entry of Ebola via a Ebs15, DAB2m AP-2 dependent pathway.  

                      Role of EBOV-GP in apoptosis induction

Apoptosis is a major host defense to eliminate virus-infected cells and several viruses have been shown to express proteins that limit or inhibit the apoptotic pathway. As outlined for Japanese Encephalitis Virus, the localisation of viral proteins to the ER can induce apoptosis via the ER stress response (also known as Unfolded Protein Response) by inhibiting Bcl-2 and inducing the activation of caspases. Indeed, full length EBOV-GP -but not a variant lacking the mucin-like domain (MLD)-  localises to the ER in HEK293T cells. So far however it is not clear if EBOV-GP induces a ER stress response akin to JEV, although the MLD is required for the induction of the NF-κB signalling pathway. It seems therefore possible that EBOV-GP induces the expression of Cytokines and Chemokines via PERK akin to SARS-CoV 3a. Activation of this pathway might then be responsible for bystander apoptosis of for instance non-infected cells such s lymphocytes as well as the induction of the immune response and thus to clinical outcome since elevated levels of IL-1α (interleukin 1α), IL-1RA (interleukin 1 receptor antagonist). IL-6 (interleukin 6), IP-10 (interferon γ–inducible protein 10), MCP-1 (monocyte chemo attractant protein 1), MCSF (macrophage colony-stimulating factor), and sCD40L (soluble CD40 ligand) have been shown to be associated with fatal cases. If EBOV-GP would induce PERK indeed then the question remains if the subsequent pathways -ATF6 and IRE1- are also induced or not. Interestingly enough, EBOV-GP does not block apoptosis induced by poly (I:C) but PKR, the latter involving the phosphorylation of eIF2α, similar to PERK induced ER stress. Since PERK also induces the expression of autophagy related genes, one strategy employed by EBOV-GP might be the induction of autophagy to selectively block p-eIF2α induced ER stress. PKR mediated activation of NF-κB signaling can be inhibited by the viral VP35 protein, so it remains to be seen if VP35 -or any other viral protein- can also inhibit ER stress/PERK induced NF-κB signaling. 

Induction of ER stress induced apoptosis by EBOV-GP may be inhibited by inducing the expression of autophagy related genes whereas PKR mediated signalling and chemokine expression may be inhibited by VP35

Further reading

Wool-Lewis RJ, & Bates P (1998). Characterization of Ebola virus entry by using pseudotyped viruses: identification of receptor-deficient cell lines. Journal of virology, 72 (4), 3155-60 PMID: 9525641 

Nanbo A, Watanabe S, Halfmann P, & Kawaoka Y (2013). The spatio-temporal distribution dynamics of Ebola virus proteins and RNA in infected cells. Scientific reports, 3 PMID: 23383374

Tran EE, Simmons JA, Bartesaghi A, Shoemaker CJ, Nelson E, White JM, & Subramaniam S (2014). Spatial localization of the Ebola glycoprotein mucin-like domain using cryo-electron tomography. Journal of virology PMID: 25008940 

Ito H, Watanabe S, Sanchez A, Whitt MA, & Kawaoka Y (1999). Mutational analysis of the putative fusion domain of Ebola virus glycoprotein. Journal of virology, 73 (10), 8907-12 PMID: 10482652

Matsuyama, S., Ujike, M., Morikawa, S., Tashiro, M., & Taguchi, F. (2005). Protease-mediated enhancement of severe acute respiratory syndrome coronavirus infection Proceedings of the National Academy of Sciences, 102 (35), 12543-12547 DOI: 10.1073/pnas.0503203102 

Nanbo, A., Imai, M., Watanabe, S., Noda, T., Takahashi, K., Neumann, G., Halfmann, P., & Kawaoka, Y. (2010). Ebolavirus Is Internalized into Host Cells via Macropinocytosis in a Viral Glycoprotein-Dependent Manner PLoS Pathogens, 6 (9) DOI: 10.1371/journal.ppat.1001121

Chandran, K. (2005). Endosomal Proteolysis of the Ebola Virus Glycoprotein Is Necessary for Infection Science, 308 (5728), 1643-1645 DOI: 10.1126/science.1110656 

Schornberg K, Matsuyama S, Kabsch K, Delos S, Bouton A, & White J (2006). Role of endosomal cathepsins in entry mediated by the Ebola virus glycoprotein. Journal of virology, 80 (8), 4174-8 PMID: 16571833 

Marzi A, Reinheckel T, & Feldmann H (2012). Cathepsin B & L are not required for ebola virus replication. PLoS neglected tropical diseases, 6 (12) PMID: 23236527 

Carette JE, Raaben M, Wong AC, Herbert AS, Obernosterer G, Mulherkar N, Kuehne AI, Kranzusch PJ, Griffin AM, Ruthel G, Dal Cin P, Dye JM, Whelan SP, Chandran K, & Brummelkamp TR (2011). Ebola virus entry requires the cholesterol transporter Niemann-Pick C1. Nature, 477 (7364), 340-3 PMID: 21866103 

Miller EH, Obernosterer G, Raaben M, Herbert AS, Deffieu MS, Krishnan A, Ndungo E, Sandesara RG, Carette JE, Kuehne AI, Ruthel G, Pfeffer SR, Dye JM, Whelan SP, Brummelkamp TR, & Chandran K (2012). Ebola virus entry requires the host-programmed recognition of an intracellular receptor. The EMBO journal, 31 (8), 1947-60 PMID: 22395071 

Garver WS, & Heidenreich RA (2002). The Niemann-Pick C proteins and trafficking of cholesterol through the late endosomal/lysosomal system. Current molecular medicine, 2 (5), 485-505 PMID: 12125814 

Mulherkar, N., Raaben, M., de la Torre, J., Whelan, S., & Chandran, K. (2011). The Ebola virus glycoprotein mediates entry via a non-classical dynamin-dependent macropinocytic pathway Virology, 419 (2), 72-83 DOI: 10.1016/j.virol.2011.08.009 

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Poirier S, Mayer G, Murphy SR, Garver WS, Chang TY, Schu P, & Seidah NG (2013). The cytosolic adaptor AP-1A is essential for the trafficking and function of Niemann-Pick type C proteins. Traffic (Copenhagen, Denmark), 14 (4), 458-69 PMID: 23350547 

Bröcker C, Kuhlee A, Gatsogiannis C, Balderhaar HJ, Hönscher C, Engelbrecht-Vandré S, Ungermann C, & Raunser S (2012). Molecular architecture of the multisubunit homotypic fusion and vacuole protein sorting (HOPS) tethering complex. Proceedings of the National Academy of Sciences of the United States of America, 109 (6), 1991-6 PMID: 22308417

Richardson SC, Winistorfer SC, Poupon V, Luzio JP, & Piper RC (2004). Mammalian late vacuole protein sorting orthologues participate in early endosomal fusion and interact with the cytoskeleton. Molecular biology of the cell, 15 (3), 1197-210 PMID: 14668490 

Lamb CA, Yoshimori T, & Tooze SA (2013). The autophagosome: origins unknown, biogenesis complex. Nature reviews. Molecular cell biology, 14 (12), 759-74 PMID: 24201109 

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 

Takáts S, Pircs K, Nagy P, Varga Á, Kárpáti M, Hegedűs K, Kramer H, Kovács AL, Sass M, & Juhász G (2014). Interaction of the HOPS complex with Syntaxin 17 mediates autophagosome clearance in Drosophila. Molecular biology of the cell, 25 (8), 1338-54 PMID: 24554766 

Olejnik J, Alonso J, Schmidt KM, Yan Z, Wang W, Marzi A, Ebihara H, Yang J, Patterson JL, Ryabchikova E, & Mühlberger E (2013). Ebola virus does not block apoptotic signaling pathways. Journal of virology, 87 (10), 5384-96 PMID: 23468487 

Bhattacharyya S, & Hope TJ (2011). Full-length Ebola glycoprotein accumulates in the endoplasmic reticulum. Virology journal, 8 PMID: 21223600 

McElroy AK, Erickson BR, Flietstra TD, Rollin PE, Nichol ST, Towner JS, & Spiropoulou CF (2014). Ebola hemorrhagic Fever: novel biomarker correlates of clinical outcome. The Journal of infectious diseases, 210 (4), 558-66 PMID: 24526742 

Feng Z, Cerveny M, Yan Z, & He B (2007). The VP35 protein of Ebola virus inhibits the antiviral effect mediated by double-stranded RNA-dependent protein kinase PKR. Journal of virology, 81 (1), 182-92 PMID: 17065211

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