Epstein-Barr virus BALF0/1 protein in patients with nasopharyngeal carcinoma

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EBV-associated human diseases

EBV is a ubiquitous lymphocrytovirus which persistently infects more than 95% of population worldwide. EBV is mainly transmitted by saliva and establishes lifelong infection. Whereas EBV persistent infection is usually symptomless, it has also been associated with several lymphoproliferative diseases and with a number of human malignancies of lymphoid and epithelial origins. Based on ex vivo data as well as medical and epidemiological evidences, EBV is currently considered as carcinogenic to humans by the International Agency for Research on cancer (IARC) (group 1).

Infectious mononucleosis

Infectious mononucleosis (IM) is an acute and self-limiting infectious disease with clinical symptoms including fever, marked fatigue, lymphadenopathy and pharyngitis, that is accompanied by a large number of atypical lymphocytes in the blood [116]. Primary infection by EBV is the major cause of IM. Whereas primary infection with EBV is asymptomatic when occurring in childhood, it might be responsible for IM in adolescents or young adults. During acute illness, high viral loads are detectable both in the oral cavity and blood, that is accompanied by a massive expansion of CD8+ T cells directed against EBV-infected B cells and the production of immunoglobulin M antibodies against VCA, whereas the number of CD8+ T cells decreases to normal level and antibodies develop against EBNA-1 in convalescence [117,118].

X‑linked lymphoproliferative disease

X‑linked lymphoproliferative disease (XLP) was first reported in 1975 as “Duncan’s disease” of 18 boys in Duncan kindred [119]. XLP is an inherited immunodeficiency, which in the majority of cases exacerbates following the primary infection with EBV, resulting in fatal IM, hypogammaglobulinemia, and malignant lymphoma [120,121]. The genetic defect responsible for XLP has been identified as a mutation in the SH2 domain containing 1A (SH2D1A) gene of the X chromosome, which encodes for a defective signaling lymphocyte activation molecule (SLAM)-associated protein (SAP) leading to inability to regulate immune responses to control B-cell proliferation caused by EBV infections [122,123].

Post-transplant lymphoproliferative disease

In immunocompetent individuals, EBV-induced B-cell transformation is controlled by EBV-specific T-cell response. Conversely impaired T-cell response occurring during acquired or innate immunodeficiency can lead to the unregulated EBV-driven B-cell proliferation and transformation. After transplantation of solid organs or hematopoietic stem cells, latently infected B-cells may proliferate and be the cause of post-transplant lymphoproliferative diseases (PTLD) [124]. Following solid organ and hematopoietic stem cell transplantation, PTLD is thought to derive from lymphoid cells of the recipient or from the donor [125,126]. This severe and life-threatening disease is characterized by clinical symptoms including fever, lymphadenopathy, fulminant sepsis, and mass lesions in lymph nodes, spleen, or central nervous system [127], which is associated with EBV infection displaying a latency III pattern of gene expression.

Bcl-2 family proteins and viral Bcl-2 homologs

Apoptosis is a process of fundamental importance for cellular and tissue homeostasis, pathophysiological processes and development. Bcl-2 (B cell lymphoma 2) has been identified as the first inhibitor of apoptosis [184]. The activation of Bcl-2 resulted from a t(14;18) chromosomal translocation in follicular lymphoma, a malignant B-cell lymphoma. Following this recombination, Bcl-2 expression was placed under the control of the immunoglobulin transcription enhancer which lead to its over-expression [185–187]. Subsequent studies have determined that Bcl-2 plays an important role in the tumorigenesis by inhibiting apoptosis rather than promoting proliferation [188,189]. The Bcl-2 family proteins, including Bcl-2 and its homologs, have been characterized by the presence of conserved short regions termed as Bcl-2 homology (BH) domains
[190]. Based on their structure and function, the Bcl-2 family proteins can be classified into three groups: the anti-apoptotic proteins, the pro-apoptotic proteins and the BH3-only proteins (Figure 11). Most of the Bcl-2 family proteins contain an α-helix with high hydrophobicity at their C-terminus. This region is considered as a potential transmembrane (TM) domain that may target Bcl-2 related proteins to cellular membranes, such as nuclear outer membrane, endoplasmic reticulum membrane, and mitochondrial membranes [191,192]. In mammals, the anti-apoptotic group consists of Bcl-2, Bcl-xL, Bcl-w, Mcl-1, A1/Bfl-1, Boo/Diva/Bcl-2-L-10, Bcl-B and NR-13 whereas pro-apoptotic group contains Bax Bak Bok and Bcl-Xs [193–195]. The BH3-only proteins only bear the BH3 domains and include Bid, Bad, Noxa, Puma, Bmf, BimL/Bod, Bik/Nbk, Blk, Hrk/DP5, Bnip3 and Bnip3 L [193,194].
Activation of Bax and Bak in the mitochondrial outer membrane (MOM) results in the MOM permeabilization (MOMP), which leads to the release of cytochrome c and other pro-apoptotic factors from the mitochondrial intermembrane space to initiate the apoptotic cascades [193,196] (Figure 12). In response to apoptotic stimuli, BH3-only proteins bind to and neutralize the pro-survival proteins, thereby activate Bax/Bak and trigger apoptosis [197]. Alternatively, it has been shown that BH3-only proteins can initiate apoptosis through direct interaction with pro-apoptotic proteins [197]. In addition, the BH3-only protein BID is activated by proteolytic cleavage to form tBid (truncated Bid). This releases its BH3 domain that becomes available for interaction [196,198–200].

Herpesvirus-encoded Bcl-2 homologs

A multitude of Herpesviridae members encode for Bcl-2 like proteins, including α-herpesvirinae (herpesvirus of turkeys [HVT]), β-herpesvirinae (HCMV), γ-herpesvirinae (KSHV, EBV, herpesvirus saimiri [HVS] and murine γ-herpesvirus 68 [MHV-68]).
Amongst the α-herpesviruses, vnr-13 encoded by HVT shares 80% homology with Nr-13, an apoptosis inhibitor in avian cells, and inhibits apoptosis after serum deprivation [205]. HCMV encodes vMIA, which is targeted to mitochondria and inhibits oligomerization of pro-apoptotic Bcl-2 family members Bax and Bak. vMIA appears to be distinct both in sequence and structure from Bcl-2 proteins [206]. Ks-Bcl-2, a vBcl-2 protein encoded by KSHV, is able to bind Bim, Bid, Bik, Bmf, Hrk, Noxa, and Puma. Ks-Bcl-2 plays a key role in the completion of the lytic cycle during viral infection [193,207,208]. EBV encodes for two vBcl-2 proteins, namely BHRF1 and BALF0/1 [102–105] that will be more discussed later in following sections. Oncogenic HVS also encodes for a Bcl-2 homolog named ORF16, which protects cells from heterologous virus-induced apoptosis [209]. Finally, M11 encoded by MHV-68 has been identified as an inhibitor of Fas- and TNF-induced apoptosis [193,210–212].

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The autophagic machinery and its regulation pathways

The identification of ATG in yeast led to the discovery of their homologs in mammalian cells, whose functions have been extensively studied [239,257]. The ATG proteins play essential roles in the initiation of autophagy and the formation of autophagosome, herein referred to as the autophagic machinery [255]. In mammalian cells, the core autophagic machinery consists of several complexes, including the Unc-51-like kinase 1 (ULK1) initiation complex, the class III phosphoinositide 3-kinase (PI3K) nucleation complex, the phosphatidylinositol 3-phosphate (PI3P)-binding complex, the ATG12 conjugation system and the microtubule-associated protein 1 light chain 3/gamma-aminobutyric receptor-associated protein (MAP1LC3/GABARAP) conjugation system, as well as ATG9 (Figure 22).

Physiological and pathological roles of autophagy

The genetic studies in yeast have identified a set of ATG genes, and most of them have highly conserved functional homologs in the mammalian systems [305]. The analysis of autophagy-defective organisms revealed various physiological roles and pathological effects of autophagy at both the cellular and whole-organism levels [306] (Figure 28).
Autophagy occurs constitutively at basal level under normal conditions to maintain homeostasis by recycling intracellular long-lived proteins, lipids, and damaged organelles. It is stimulated under starvation for replenishing energy stores as well as by various stress such as hypoxia, ER stress, the presence of free radicals, and genotoxic stress [244,307]. A reduced autophagic activity is associated with normal and pathological aging whereas pharmacological or genetic methods stimulating autophagy give rise to the extended lifespan of model organisms from flies to mice [308,309]. Autophagy also plays an important role in the preimplantation development, survival during neonatal starvation, and cell differentiation during erythropoiesis, lymphopoiesis and adipogenesis [310]. Additionally, defects in autophagy may be involved in the pathogenesis of multiple diseases, including Crohn’s disease [311,312], type 2 diabetes [313,314], Huntington’s disease [296], Alzheimer’s disease [315] and Parkinson’s disease [316]. The implication of autophagy in tumorigenesis is complex and likely dependent on the stage of tumor development [317]. At early tumor stages, autophagy rather suppress tumorigenesis. Conversely, once the tumor is formed, autophagy might promote tumor progression and metastasis [318].
It is to note that the autophagic pathways play an essential role in innate and adaptive immunity. Autophagic vesicles allow the direct elimination of intracellular microbes by engulfing and targeting them to lysosomal degradation [319,320]. Virus can be eliminated by autophagy, a process called virophagy [321]. In addition, peptides generated from the autophagic pathways can be processed and matured for major histocompatibility complex (MHC) class I and class II presentation [236,322].

Table of contents :

Abstract
Résumé
Table of Contents
Abbreviations
List of Illustrations
1. Introduction
1.1 Epstein-Barr virus
1.1.1 Discovery of Epstein-Barr virus
1.1.2 Classification of EBV
1.1.3 Structure of EBV virion and genome
1.2 EBV infection
1.2.1 Transmission
1.2.2 Primary infection and viral persistence
1.2.3 Viral entry
1.2.4 Immune response
1.3 Life cycle of EBV
1.3.1 Latency
1.3.2 Lytic cycle
1.3.3 Reactivation
1.4 EBV-associated human diseases
1.4.1 Infectious mononucleosis
1.4.2 X‑linked lymphoproliferative disease
1.4.3 Post-transplant lymphoproliferative disease
1.4.4 Burkitt’s lymphoma
1.4.5 Hodgkin’s lymphoma
1.4.6 NK/T-cell lymphoma
1.4.7 Nasopharyngeal carcinoma
1.4.8 Gastric carcinoma
1.5 EBV viral Bcl-2 homologs
1.5.1 Bcl-2 family proteins and viral Bcl-2 homologs
1.5.2 Herpesvirus-encoded Bcl-2 homologs
1.5.3 EBV vBcl-2s
1.6 BHRF1
1.6.1 Expression
1.6.2 Subcellular localization
1.6.3 Function
1.7 BAL0/1
1.7.1 Expression
1.7.2 Subcellular localization
1.7.3 Function
1.8 Autophagy
1.8.1 Different types of autophagy
1.8.2 A multistage process
1.8.3 The autophagic machinery and its regulation pathways
1.8.4 Physiological and pathological roles of autophagy
1.8.5 Selective autophagy
1.9 EBV and autophagy
1.9.1 Autophagy and EBV latency
1.9.2 Autophagy modulation during EBV lytic cycle
1.10 Herpesvirus and autophagy: a lesson to explore the contribution of autophagy to EBV infection
1.10.1 Subversion of autophagy by Herpesviruses
1.10.2 Herpesvirus-encoded vBcl-2s inhibit autophagy
1.11 Aims and objectives
2. Results
2.1 Detection of IgG directed against a recombinant form of Epstein-Barr virus BALF0/1 protein in patients with nasopharyngeal carcinoma
2.2 Epstein-Barr virus BALF0 and BALF1 modulate autophagy
2.3 BHRF1, a Bcl-2 viral homolog, disturbs mitochondrial dynamics and stimulates mitophagy to dampen type I IFN induction
3. Discussion
3.1 The existence of EBV BAFL0 and BALF1 in vivo
3.2 EBV vBcl-2s modulate autophagy 􀀂 VI
3.3 The interplay between vBcl-2s of EBV
4. Take home message
5. References

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