**Roles of VP35, VP40 and VP24 Proteins of Ebola Virus in Pathogenic and Replication Mechanisms**

Rahma Ait Hammou, Yassine Kasmi, Khadija Khataby, Fatima Ezzahra Laasri, Said Boughribil and My Mustapha Ennaji

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/63830

#### **Abstract**

Ebola epidemic is a fatal disease due to Ebola virus belonging to Filoviridae; currently the viral evolution caused more than 50% of death worldwide. Among the eight proteins of ZEBOV, there are four structural proteins VP35, VP40, VP24, and NP, which have important functions in the intercellular pathogenic mechanisms. The multi‐functionali‐ ty of Ebola's viral proteins allows the virus to reduce its protein number to ensure its proper functioning and keeping the compact structure of the virus. Therefore, the aim of this chapter is to study the mechanism of replication and the roles of VP30, VP35, NP, and L in this process. We provide as well to highlight the influence of the virus on the immune system and on the VP24.

**Keywords:** Ebola, VP35, VP40, VP24, pathogenic, replication, mechanisms, immune system

#### **1. Introduction**

Ebola is an acute viral disease that has appeared in 1976 in two simultaneous outbreaks, Nzara, South Sudan, and the other in Yambuku, Democratic Republic of Congo. The latter occurred in a village near the Ebola River, from which the disease takes its name "Ebola virus" which is an endemic virus of Africa. However, Ebola virus is a member of the filovirus family, character‐ ized by multifunctional proteins. From the appointment of this family, these viruses are filamentous, and they present various forms such as (U), (L) and (6) under electronic micro‐

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

scope (**Figure 1**)[1].Thus, viral propagation wasdue to the varianttrips of populations through countries.

Although the multi‐functionality of these proteins, each type has a specific role such as, GP protein that ensure important functions in the extracellular environment; otherwise, the VP35, VP40, and VP24 proteins have intracellular roles. eVP35 is usually used as symbol for "EBO‐ LA's VP35 protein," one of the most important structural proteins of ZEBOV having diverse functions in pathogenesis mechanism and viral cycle [2]; it is an indispensable co‐factor of replication transcription and an essential member of the replication complex. The virus has two other proteins, which play roles in immune response in intracellular stage.

Thus, the VP24 is a structural protein, that has the ability to internalize the cell nucleus, and known as a minor matrix protein and membrane‐associated protein. Then, the latest protein "VP40" is known as a viral matrix protein, and it is the most abundant protein in Ebola's viral structure.

**Figure 1.** Marburg virus particles purified from the blood of infected guinea pigs, stained by negative contrast medi‐ um. Different forms of the virion are shown: 1, rod shaped; 2, ring shaped; 3, mace or (6); 4, (L) form, and 5, (U) form. Shaped '10.000' the virus was purified and concentrated by A, B, et al.; photo by E. Kandrushin, Center for Virology and Biotechnology ''Vector," Koltsovo, Russia) [1].

Ebola is a zoonosis disease. The bats are the main natural reservoir of the virus, while also chimpanzee and some other animals could transmit EBOV virus to human. Transmission modes are diverse and not manageable: contact with fluids of infected persons, possibility of aerosol transmission [3, 4] and contact with infected animals [5]; here we must mention that the religious, cultural and traditional practices help the large propagation of virus among African population and that simple actions can limit the propagation of the virus. Epidemio‐ logical studies of WHO and CDC have shown that adults are more subjected to infection than children. Furthermore, Ebola virus can infect both men and women [6]. The virus has the ability to replicate in monocyte‐derived dendritic cells without engendering an inflammatory response [7].

The transmission of Ebola virus to the human body is done by blood and spread in most cells, including vital organs, the infections in brain, liver and the heart disrupted the best functioning of these organs and thus occur as a direct result of death [8]. Time of replication *in vitro* is about 12 hours for Ebola virus on E6 cells [9].

#### **2. How is the Filoviridae evolving?**

scope (**Figure 1**)[1].Thus, viral propagation wasdue to the varianttrips of populations through

Although the multi‐functionality of these proteins, each type has a specific role such as, GP protein that ensure important functions in the extracellular environment; otherwise, the VP35, VP40, and VP24 proteins have intracellular roles. eVP35 is usually used as symbol for "EBO‐ LA's VP35 protein," one of the most important structural proteins of ZEBOV having diverse functions in pathogenesis mechanism and viral cycle [2]; it is an indispensable co‐factor of replication transcription and an essential member of the replication complex. The virus has

Thus, the VP24 is a structural protein, that has the ability to internalize the cell nucleus, and known as a minor matrix protein and membrane‐associated protein. Then, the latest protein "VP40" is known as a viral matrix protein, and it is the most abundant protein in Ebola's viral

**Figure 1.** Marburg virus particles purified from the blood of infected guinea pigs, stained by negative contrast medi‐ um. Different forms of the virion are shown: 1, rod shaped; 2, ring shaped; 3, mace or (6); 4, (L) form, and 5, (U) form. Shaped '10.000' the virus was purified and concentrated by A, B, et al.; photo by E. Kandrushin, Center for Virology

Ebola is a zoonosis disease. The bats are the main natural reservoir of the virus, while also chimpanzee and some other animals could transmit EBOV virus to human. Transmission modes are diverse and not manageable: contact with fluids of infected persons, possibility of aerosol transmission [3, 4] and contact with infected animals [5]; here we must mention that the religious, cultural and traditional practices help the large propagation of virus among African population and that simple actions can limit the propagation of the virus. Epidemio‐ logical studies of WHO and CDC have shown that adults are more subjected to infection than children. Furthermore, Ebola virus can infect both men and women [6]. The virus has the ability to replicate in monocyte‐derived dendritic cells without engendering an inflammatory

two other proteins, which play roles in immune response in intracellular stage.

countries.

102 Ebola

structure.

response [7].

and Biotechnology ''Vector," Koltsovo, Russia) [1].

We can find the answer of this question in phylogenic studies. Generally, the RNA viruses are characterized by the accumulation of many mutations during their evolution—these muta‐ tions are not predictable. Therefore, the Filoviridae is divided into genders that are distinct based on the numbers of mRNA encoding by GP gene. A study, demonstrate that the viral genome of this family is very similar. New phylogenetic analysis demonstrated that a few mutations in Reston genome can transfer the virus from non‐Human pathogen to a Human pathogen, essentially in the VP24 gene: three VP24 SDPs (T131S, M136L, and Q139R) are likely to impair VP24 binding to human karyopherin alpha5 (KPNA5) and therefore inhibition of interferon signaling [10].

The human body mainly and the general superiors mammals consisting by several systems that achieve different functions to respond in the needs of body and assurance their life, among these systems, there are the immune system which has vital roles and performs important functions in the protection of the body and the eliminations of hazard. The immune system consists of several mechanisms and factors to ensure the proper functioning of the system; they can be subdivided into two under‐system: innate immune and adaptive immune. The primary defenses against viral infections are the physical and chemical barriers: skin, pH, acidity, secretion, etc. First, the constituents of the innate response ensure the immune responses against viral agents, before request of the specific answer, another response corresponding to an innate response that is mediated by interferon (mainly interferon‐γ), which promotes the activation of NK cells and CD 8 lymphocytes that recognize and destroy cells infected by the virus.

The aim of this chapter is to study the proteins VP35 and VP24 overlooking the immune system. In this chapter, we focus on the structures of those proteins, their roles, and their influence on immune responses.

#### **3. Do the structural proteins have a role in the bending process of Filoviridae without breaking?**

More than 50% of the virions grown in cells are polyploidy. Most families of viruses have a single copy of the genome by particle. However, polyploidy is relatively rare in the viral world [11]. Among these families is the *Paramyxoviridae* [12]; so the first assumption is that the Filoviridae are of polyploidy. In addition, the second is the flexibility of the nucleocapsid (RNA, VP30, VP35, N, and L) with the intervention of the VP24 protein [13].

#### **4. Structural and genomic information**

The genome of the Filoviridae is rather similar, with seven genes that encoding for the seven proteins or eighth for Ebola. The genes contain the respective open reading frame (ORF) flanked by unusually long non‐translated sequences, ranging from 57 to 684 nucleotides [14]. The VP24 protein is expressed by the region 9886–11497, and the region 11498–11501 is an intergenic region, which ensures essential roles for the virus: It is immunosuppressive [15] that allows the virus to control the innate immune system [16]; it binds directly with STAT1 causing antagonize interferon [17]. VP40 has various roles; unmodified polypeptide may assemble into different structures for different functions [18]. The rates of conservation of Filoviridae proteins are 33% for VP35, 27% for VP40, 34% for GP, 33% for VP30, and 37% for VP 24. The board 3' and 5' regions ensures important functions in replication, transcription of the genome, and its control.VP35 gene advance by a conserved transcription start and stop signals, "CUACUU‐ CUAAUU" for start and "UAAUUCU" as stop transcription signal. However, the coding start of VP40 is "CUACUUCUAAUU," and then signal stop is "UAAUUCU." For the viral protein 24 (VP24), the signal start "CUACUUCUAAUU" is sited in position 9886–9897 from Genome's Ebola. However, the VP24 has two stop signals "UAAUUCU" [19]. The genome of EBOV is schematically shown in **Figure 2**.

**Figure 2.** The full‐length genome of Ebola is about 19,000 nucleotide, where L gene coding for the RNA polymerase, it is the length gene and more conserved gene in the Filoviridae, then the VP40 is the more polymorphism gene.

The VP35 gene located among the position 3032 and 4407 of Ebola genome, coding for alone mRNA with same length, though the regulatory region is located at the nucleotide 3032 and 3048 "CUACUUCUAAUU" which is a transcription start signal. For the VP40, it is sited at the 4390th nucleotide and 5894th, with one mRNA at the same length, and thus the start signal is on position 4390th to 4401th; besides region 4397–4407and 5883–5894 in genome are two poly‐ adenine signal sequences [20].

A single mutation in the central basic patch residue R322 or end‐cap residue F239 to alanine capable to disrupt the dsRNA binding and alters VP35 inhibitor of RIG1 [21, 22]. These mutants retain modest inhibitory activity relative to the empty vector control. Thus, they exhibited reduced suppression of SeV‐induced IFNα/β production [23].

The dsRNA‐binding cluster is centered on Arg‐312, a highly conserved residue required for IFN inhibition. Importantly, the stability of the β‐sheet subdomain structure of VP35 requests an interaction between the side chains of Pro‐315, Pro‐318, and Lys‐339 residues and conserved Trp‐324, as well as the Ile‐340 residue, which make bond with Phe‐239, Leu‐242, and Ile‐278 (**Figure 3**). These residues are highly conserved in Ebola genome, and that demonstrates the importance of these residues in the stability and the good functioning of the VP35 protein [24].

**4. Structural and genomic information**

104 Ebola

schematically shown in **Figure 2**.

adenine signal sequences [20].

The genome of the Filoviridae is rather similar, with seven genes that encoding for the seven proteins or eighth for Ebola. The genes contain the respective open reading frame (ORF) flanked by unusually long non‐translated sequences, ranging from 57 to 684 nucleotides [14]. The VP24 protein is expressed by the region 9886–11497, and the region 11498–11501 is an intergenic region, which ensures essential roles for the virus: It is immunosuppressive [15] that allows the virus to control the innate immune system [16]; it binds directly with STAT1 causing antagonize interferon [17]. VP40 has various roles; unmodified polypeptide may assemble into different structures for different functions [18]. The rates of conservation of Filoviridae proteins are 33% for VP35, 27% for VP40, 34% for GP, 33% for VP30, and 37% for VP 24. The board 3' and 5' regions ensures important functions in replication, transcription of the genome, and its control.VP35 gene advance by a conserved transcription start and stop signals, "CUACUU‐ CUAAUU" for start and "UAAUUCU" as stop transcription signal. However, the coding start of VP40 is "CUACUUCUAAUU," and then signal stop is "UAAUUCU." For the viral protein 24 (VP24), the signal start "CUACUUCUAAUU" is sited in position 9886–9897 from Genome's Ebola. However, the VP24 has two stop signals "UAAUUCU" [19]. The genome of EBOV is

**Figure 2.** The full‐length genome of Ebola is about 19,000 nucleotide, where L gene coding for the RNA polymerase, it is the length gene and more conserved gene in the Filoviridae, then the VP40 is the more polymorphism gene.

The VP35 gene located among the position 3032 and 4407 of Ebola genome, coding for alone mRNA with same length, though the regulatory region is located at the nucleotide 3032 and 3048 "CUACUUCUAAUU" which is a transcription start signal. For the VP40, it is sited at the 4390th nucleotide and 5894th, with one mRNA at the same length, and thus the start signal is on position 4390th to 4401th; besides region 4397–4407and 5883–5894 in genome are two poly‐

A single mutation in the central basic patch residue R322 or end‐cap residue F239 to alanine capable to disrupt the dsRNA binding and alters VP35 inhibitor of RIG1 [21, 22]. These mutants retain modest inhibitory activity relative to the empty vector control. Thus, they exhibited

The dsRNA‐binding cluster is centered on Arg‐312, a highly conserved residue required for IFN inhibition. Importantly, the stability of the β‐sheet subdomain structure of VP35 requests an interaction between the side chains of Pro‐315, Pro‐318, and Lys‐339 residues and conserved

reduced suppression of SeV‐induced IFNα/β production [23].

**Figure 3.** The surface area between the VP35 IID subdomains is hydrophobic. (A and B) Electrostatic representations of the inter‐subdomain interaction surface for the α‐helical subdomain (A) and the β‐sheet subdomain (B) reveal hydro‐ phobic surfaces buried between the two subdomains. Red, white, and blue represent negative, neutral, and positive electrostatic potentials, respectively (range -5 to +5 kT). (C) Stereographic image showing the Trp‐324 side chain mak‐ ing important hydrophobic contacts with residues in β4 strand, α5 helix, and PPII [24].

**Figure 4.** A crystal structure of VP35 from RCSB databank number 4IBB, obtunded X‐RAY DIFFRACTION method with a resolution of 1.84 Å R‐Value Free: 0.233 and R‐Value Work: 0.177. The VP35 has two sub units with 4 β‐sheets and 4 α helices [25].

The work of Mateo et al. 2010 [25] has dismiss that the amino acids in position 1–20 of VP24 are not important in the functioning of VP24 to inhibit IFN‐β‐induced gene expression. However, mutations in position 44 influence on the function of VP24 and have a critical role in the inhibition of IFN‐β‐induced gene expression. However, residues 142–146 are important to inhibit ISG54 activation by IFN‐β. Therefore, mutations at residues 142–146 are able to increase the expression of ISG54 reporter up to 90%, thus drastically reducing VP24 activity [26]. Structure of VP24 dimer is shown as **Figure 5**.

**Figure 5.** *(A) Ebola virus VP24 structure (4M0Q) from RCSB with (B) resolution of 1.92 Å, then b. is Protein Feature View— UniProtKB AC: Q05322. The VP35 has two subunits with 8 β‐sheet and 5 α helices. The length amino acids chain is 251AA [27]. (C) Crystal image of VP40 Hexamer [29].*

Residues 213–326 are essential for VP40 to associate with liposomes; 309–317 has a critical role in the associated with the DSM fraction; the truncation of 18 C‐terminal residues resulted in predominantly oligomeric protein that mainly associated with the DSM fraction [28].

#### **5. Ebola virus replication**

The work of Mateo et al. 2010 [25] has dismiss that the amino acids in position 1–20 of VP24 are not important in the functioning of VP24 to inhibit IFN‐β‐induced gene expression. However, mutations in position 44 influence on the function of VP24 and have a critical role in the inhibition of IFN‐β‐induced gene expression. However, residues 142–146 are important to inhibit ISG54 activation by IFN‐β. Therefore, mutations at residues 142–146 are able to increase the expression of ISG54 reporter up to 90%, thus drastically reducing VP24 activity

**Figure 5.** *(A) Ebola virus VP24 structure (4M0Q) from RCSB with (B) resolution of 1.92 Å, then b. is Protein Feature View— UniProtKB AC: Q05322. The VP35 has two subunits with 8 β‐sheet and 5 α helices. The length amino acids chain is 251AA [27].*

Residues 213–326 are essential for VP40 to associate with liposomes; 309–317 has a critical role in the associated with the DSM fraction; the truncation of 18 C‐terminal residues resulted in

predominantly oligomeric protein that mainly associated with the DSM fraction [28].

[26]. Structure of VP24 dimer is shown as **Figure 5**.

106 Ebola

*(C) Crystal image of VP40 Hexamer [29].*

The replication process in the mono‐nonsense‐negative genome is almost similar; the first step is the transfer from negative sense genome to positive sense genome. The positive sense genome (call also anti‐genome or complementary genome) is the complement RNA sequence, direct sense of transcription. From the positive sense genome, two processes are done: the first is replication and getting the negative sense genome for formation of new virion, the second calling the cellular ribosome for the translate process for making new viral proteins (**Figure 6**).

**Figure 6.** The transcription and replication process, the virus pass from negative sense to positive and from positive to negative sense [30].

The Ebola virus life cycle can be spread over following stages: the first stages are adsorption and penetration in the cell, followed by de‐capsulation, transfer genome from negative to positive, primary and syntheses transcription of functional proteins, second transcription replication and assembly of virus.

For Ebola virus, after the liberation of VP30, VP35, L, VP40, and VP24 with the genome in cytoplasm, the first step is the formation of replication complex composed by L (RNA polymerase), VP35, NP, and VP30. The VP30 is an indispensable co‐factor of transcription, even the VP30 is part of this complex as transcription activator, and it is a highly phosphory‐ lated [31]. The L is the polymerase protein of EBOV; it is the large protein in genome and the most conserved protein among Filoviridae. In addition to the transcription and replication functions, it can connect the VP35 and NP where NP‐RNA helices associate with VP35. Ebola virus VP35 is essential for nucleocapsid formation, together with NP and VP24 [32].

The initiation of transcription requires a VP30 signal, and this signal takes place after attach‐ ment of zinc molecule in zinc‐binding Cys3‐His motif comprising amino acids 68–95 [33]. The phosphorylation site is a conserved site, where a simple mutation can get negative effects on the incorporation of VP30 with the other viral particles and therefore affect the efficiency of the recovery of the viruses [31]. The frequency of transcription of an mRNA is different following the position of the gene in genome, the genes proximal to 3' are more translated than those in 5', thanks to the second mRNA produce and that contains essentially [34]. The interagency regions ensure a role in the control and the regulation of replication and tran‐ scription of virus [35].

**Figure 7.** Two secondary structures predicted of ZEBOV genomic RNA. The interactions made by (a) a hairpin struc‐ ture (b) and panhandle structure format [33].

The RNA bonds with the complex of VP35‐NP‐L to initiate the replication and transcription of viral genome. Translation of viral protein is ensured by liberation ribosome in cytoplasm. The replication mechanism is not manageable enough; however, there are estimates of the true mechanism of replication.

*Weik* and their collaborators in 2005 [36] demonstrate that the nucleotides 5–44 of the EBOV leader are involved in RNA secondary‐structure formation; the alteration of 36 nucleotides spanning the region 55–90 did not affect replication. However, when the random sequence was elongated by four additional nucleotides, replication activity could not be detected. Bipartite promoters localized in 3' of gene, and then the second signal is in the beginning of the next gene; it is a succession of eight UN5 hexamer repeats (**Figure 7**) [36], other research shows that EBOV NP is inactive in Marburg the vice‐versa, and this suggests that they present a specific motifs by the complex of replication for each gender. The region of the EBOV promoter start signal of the NP gene (12 nucleotides) and the following 13 nucleotides has been shown to form a stem‐loop structure, which is involved in regulation of VP30‐dependent transcription [1]. Other results of Brauburger et al. 2014 [37] reflect fundamental differences in the control of polymerase behavior by cis‐acting sequences between viruses with conserved and variable gene borders and suggest an important role of conserved IRs in transcription regulation, while the function of variable IRs remains less clear.

#### **6. Ebola and immune system**

The initiation of transcription requires a VP30 signal, and this signal takes place after attach‐ ment of zinc molecule in zinc‐binding Cys3‐His motif comprising amino acids 68–95 [33]. The phosphorylation site is a conserved site, where a simple mutation can get negative effects on the incorporation of VP30 with the other viral particles and therefore affect the efficiency of the recovery of the viruses [31]. The frequency of transcription of an mRNA is different following the position of the gene in genome, the genes proximal to 3' are more translated than those in 5', thanks to the second mRNA produce and that contains essentially [34]. The interagency regions ensure a role in the control and the regulation of replication and tran‐

**Figure 7.** Two secondary structures predicted of ZEBOV genomic RNA. The interactions made by (a) a hairpin struc‐

The RNA bonds with the complex of VP35‐NP‐L to initiate the replication and transcription of viral genome. Translation of viral protein is ensured by liberation ribosome in cytoplasm. The replication mechanism is not manageable enough; however, there are estimates of the true

*Weik* and their collaborators in 2005 [36] demonstrate that the nucleotides 5–44 of the EBOV leader are involved in RNA secondary‐structure formation; the alteration of 36 nucleotides spanning the region 55–90 did not affect replication. However, when the random sequence was elongated by four additional nucleotides, replication activity could not be detected. Bipartite promoters localized in 3' of gene, and then the second signal is in the beginning of the next gene; it is a succession of eight UN5 hexamer repeats (**Figure 7**) [36], other research shows that EBOV NP is inactive in Marburg the vice‐versa, and this suggests that they present a specific motifs by the complex of replication for each gender. The region of the EBOV

scription of virus [35].

108 Ebola

ture (b) and panhandle structure format [33].

mechanism of replication.

The Ebola virus has the ability to flare the immune system by several modes. Furthermore, the virus uses the immune system as tools to fix and internalize in cell, thanks to the link between GP and the antibody as demonstrated in the chapter of Glycoprotein. Mahanty and others [38] illustrate in the **Figure 8** some immune evasion tools [38].

**Figure 8.** A model of the pathogenesis of filoviral hemorrhagic fever, based on studies of Zaire Ebola virus infection. Infection causes lysis of monocytes/macrophages, dendritic cells, and hepatocytes and suppresses innate immune re‐ sponses in these cells, aiding further dissemination. Direct injury to infected cells is accompanied by indirect effects that are mediated by pro‐inflammatory and anti‐inflammatory effector molecules, including interleukin 1 (IL1), inter‐ leukin 6 (IL6), TNF, interleukin 10 (IL10), and type I interferons (IFN). The severe illness results from the combined effects of widespread viral cytolysis and massive release of pro‐inflammatory mediators. Pro‐inflammatory cytokines and chemokines are also produced by activated endothelial cells, resulting in a feedback loop to the monocytes/macro‐ phages. Lymphocyte apoptosis is also apparently brought about through effects of pro‐inflammatory mediators; it may contribute to immunosuppression by weakening adaptive immune responses. The cell‐surface expression of tissue fac‐ tor by virus‐infected monocytes/macrophages induces disseminated intravascular coagulation. MCP monocyte chemo‐ attractant protein; IL1RA interleukin‐1 receptor antagonist [39].

Here, we discuss the intracellular mechanism to escape the immune system via structural proteins and their roles in inhibition of the interferon's expression (**Figure 9**).

**Figure 9.** The crystal structure of VP35 bond to dsRNA [41].

The VP35 implicated in modulation of the host immune response. Studies show that the region C‐terminal‐binding site with the dsRNA in VP 35 is being demonstrated as responsible of antagonism region's interferon and immune evasion. The VP35 bond specifically with specifically with poly(rI) poly(rC), poly(rA), poly(rU) [39].

The VP35 through PACT has the ability to inhibit the retinoic acid inducible Gene‐I (RIG‐I) to bind with the dsRNA, and this action inhibits the transfer of hazardous signal to the interferon promoter simulator I by RIG‐I in first time. Therefore, if the signal has transferred by the RIG‐ I, the VP35 binds to the Tank‐binding kinase‐1 interferon kinases (TBK‐1/IKKε) and inhibiting the phosphorylation of IRF‐3/7 [40–43]. Consequently, the translocation nucleus of signal and the expression of INF‐β will be inhibited (**Figure 10**). More recent study suggests that other filoviral proteins, including EBOV VP30 and VP40, also counter the RNAi pathway [44].

Roles of VP35, VP40 and VP24 Proteins of Ebola Virus in Pathogenic and Replication Mechanisms http://dx.doi.org/10.5772/63830 111

Here, we discuss the intracellular mechanism to escape the immune system via structural

The VP35 implicated in modulation of the host immune response. Studies show that the region C‐terminal‐binding site with the dsRNA in VP 35 is being demonstrated as responsible of antagonism region's interferon and immune evasion. The VP35 bond specifically with

The VP35 through PACT has the ability to inhibit the retinoic acid inducible Gene‐I (RIG‐I) to bind with the dsRNA, and this action inhibits the transfer of hazardous signal to the interferon promoter simulator I by RIG‐I in first time. Therefore, if the signal has transferred by the RIG‐ I, the VP35 binds to the Tank‐binding kinase‐1 interferon kinases (TBK‐1/IKKε) and inhibiting the phosphorylation of IRF‐3/7 [40–43]. Consequently, the translocation nucleus of signal and the expression of INF‐β will be inhibited (**Figure 10**). More recent study suggests that other filoviral proteins, including EBOV VP30 and VP40, also counter the RNAi pathway [44].

proteins and their roles in inhibition of the interferon's expression (**Figure 9**).

110 Ebola

**Figure 9.** The crystal structure of VP35 bond to dsRNA [41].

specifically with poly(rI) poly(rC), poly(rA), poly(rU) [39].

**Figure 10.** The inhibition of the recognition of PAMPs by the RLR, due to the inhibition of RIG‐1 by bonding of VP35 to dsRNA through the CBP.

However, Luther and their collaborator's in 2013 [45] had shown that the PACT also implicated in inhibition of RIG‐I. Other studies shown that the mVP35 and eVP35 effects differently the RIG‐I, ebolaVP35 blocked the RIG‐I then Marburg VP35 decreased the affinity and the activity of RIG‐I [46].

The same context of INF‐β's inhibition, the VP40 interacted with the Janus kinase/tyrosine kinase II (JAK I/TRK II) for block their phosphorylation and as results, inhibition of the activation of STAT heterodimer kinase require the phosphorylation of JAK‐I/TRK II [47, 48].

In the other hand, VP24 bonds with Karyopherin‐α by the residues in activation of sites 26–50 and 142–146 and they are demonstrated to be the most important residues for this activity (**Figure 11**) [46].

**Figure 11.** The binding between VP24 and KPNA5, *and* this link in the active site of STAT homodimer. It prevents the adhesion of the SATA homodimer phosphorylated and thus lack the protein's ability to enter the cell nucleus.

#### **7. Inhibition strategies of Ebola virus**

The involvement of VP35 in diverse parts of the infection (replication, inhibition of RIG1, TAK, and inhibition of interferon) made it a principal target of several medicines to inhibit it. Thus, all the Ebola proteins are crystallized and available in databases as RCSB, addition to the full‐ length genome sequenced is available in NCBI databases make research and information about the virus more accessible (**Figure 12**).

**Figure 12.** In silico‐derived small molecules, it binds the filovirus *VP35* protein and inhibit its polymerase cofactor ac‐ tivity. (a) {4‐[(2R)‐3‐(2‐chlorobenzoyl)‐2‐(2‐chlorophenyl)‐4‐hydroxy‐5‐oxo‐2,5‐dihydro‐1H‐pyrrol‐1‐yl]phenyl}acetic acid. (b) *3D with ligand.* (c) The *pharmacophore map of ligand in the active site of VP35* [49].

#### **8. Conclusions**

The light of the above discussion results, the Filoviridae genome coding seven proteins, with the exception of Ebola virus that coded for the eight proteins, including the GP gene which coded for secretory glycoproteins, membrane glycoproteins, and thus it is subdivided into subunits called as GP1 and GP2. However, the sGP has not the membrane region. The Ebola genome contains six interagency regions, having functions in regulation of transcription of genes and the CAP‐polyA to protect the mRNA. Those regions contain a RNA 2D confirmation boots. The immune evasion processes in Filoviridae generally, and essentially for Ebola virus based on two complementary process; one intercellular by GP and second intracellular where the roles remarkable of VP35 by inhibition of RIG‐I and INF‐3, therefore, the roles of VP40 and VP24 inhibition of INF‐β signal.

#### **Acknowledgements**

**7. Inhibition strategies of Ebola virus**

the virus more accessible (**Figure 12**).

112 Ebola

The involvement of VP35 in diverse parts of the infection (replication, inhibition of RIG1, TAK, and inhibition of interferon) made it a principal target of several medicines to inhibit it. Thus, all the Ebola proteins are crystallized and available in databases as RCSB, addition to the full‐ length genome sequenced is available in NCBI databases make research and information about

**Figure 12.** In silico‐derived small molecules, it binds the filovirus *VP35* protein and inhibit its polymerase cofactor ac‐ tivity. (a) {4‐[(2R)‐3‐(2‐chlorobenzoyl)‐2‐(2‐chlorophenyl)‐4‐hydroxy‐5‐oxo‐2,5‐dihydro‐1H‐pyrrol‐1‐yl]phenyl}acetic

The light of the above discussion results, the Filoviridae genome coding seven proteins, with the exception of Ebola virus that coded for the eight proteins, including the GP gene which

acid. (b) *3D with ligand.* (c) The *pharmacophore map of ligand in the active site of VP35* [49].

**8. Conclusions**

The authors would like to thank the ministry of higher education, University Hassan II of Casablanca, Faculty of Sciences and Technics and the Laboratory of Virology, Microbiology, Quality and Biotechnologies/Ecotoxicology and Biodiversity.

#### **Author details**

Rahma Ait Hammou, Yassine Kasmi, Khadija Khataby, Fatima Ezzahra Laasri, Said Boughribil and My Mustapha Ennaji\*

\*Address all correspondence to: m.ennaji@yahoo.fr

Laboratory of Virology, Microbiology, Quality and Biotechnologies/Eco-toxicology and Biodiversity, Team of Virology, Oncology and Medical Biotechnologies, Faculty of Sciences and Techniques, University Hassan II of Casablanca, Mohammedia, Morocco

Rahma Ait Hammou and Yassine Kasmi contributed equally to this work.

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116 Ebola


## **Ebola Virus's Glycoproteins and Entry Mechanism**

Khadija Khataby, Yassine Kasmi, Rahma Ait Hammou, Fatima Ezzahra Laasri, Said Boughribi and My Mustapha Ennaji

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/64032

#### **Abstract**

Ebola virus glycoprotein (GP) is the only protein that is expressed on the surface of the virus. The GP proteins play critical roles in the entry of virus into cell and in the evasion of the immune system. The GP gene transcript to membrane GP is constituted of two subunits GP1 and GP2,and the secretory GP (sGP). The main function of GP1/2 is to attach virus to target cell's membrane, whereas sGP has multiple functions on Ebola pathogen‐ esis, such as inactivate neutrophils through CD16b causing lymphocyte apoptosis and vascular dysregulation. There are many studies that focused on better understanding the GP mechanism and aim at developing new antibodies and drugs such as VSV-EBOV, cAd3-EBO Z, rVSVN4CT1 VesiculoVax, 'C-peptide' based on the GP2 C-heptad repeat region (CHR) targeted to endosomes (Tat-Ebo) and MBX2270. In this chapter, we discuss the Ebola viral glycoproteins, genomic organization, synthesis, and their roles and functions. On the other hand, we treat the mechanisms of pathogenicity associated with Ebola GPs.

**Keywords:** EBOLA, virus, glycoprotein (GP), entry, mechanism, pathogenesis, struc‐ ture

#### **1. Introduction**

Since the beginning of the year 2012, cases of Ebola virus have been reported in four African countries: Guinea, Liberia, Sierra Leone and Nigeria. WHO announced the end of Ebola outbreak in January 2016 [1]; despite this, according to the WHO, new cases are declared later in Sierra Leone, Liberia and Guinea [2, 3]. What this highlights is that the risk of the Ebola

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

epidemic is still standing. The Ebola haemorrhagic fever weans and is often fatal in humans. It is caused by *Filoviridae*, citing the species Zaire Ebola virus (ZEBOV).

Ebola virus is a pathogenic agent of Ebola haemorrhagic fever; it is a single-stranded RNA with negative sense and with a genome length of approximately 18,920 nucleotides. Since it belongs to *Filoviridae*, its diameter is about 80 nm with a twisted filamentous form. Generally, the virus length is up to 1.1 µm [4], but particles of 14 µm were detected in the culture of liver tissue [5]. The viral RNA contains information about eight proteins, VP24, VP30, VP35, VP40, L, NP, sGP and GP1/2. Each of the protein expressed by EBOV is known for its multi-func‐ tionality that is why it is nominated as Swiss Army Knife, essentially VP35 and GP that present multi-functionality in the pathogenesis process and in the inhibition of immune responses in the host. EBOV is classified as a Category A priority pathogen by the National Institute of Allergy and Infectious Diseases (NIAID) and a Category A agent of bioterrorism by the Centers of Disease Control and Prevention (CDC) [6].

The principal characteristics of the Ebola virus is the presence of a heavily glycosylated GP on the external surface of viral membrane. Crystallographic studies revealed that GP on the viral surface exists in the trimeric form [7].

This chapter aims to provide a current overview on the treatment of GP of the Ebola virus, genomic organization, synthesis, their roles and functions, and the mechanisms of viral entry associated with GP and replication.

#### **2. Phylogenetic information of Ebola virus**

A total of 132 sequences are collected from the NCBI gene database. The samples were collected from the Makona river (80 sequences from Sierra Leon and 52 from Guinea), and they were collected in the period between 1 June 2014 and 30 August 2015. They underwent a global alignment and phylogenetic analysis by MEGA 6.4 and BEAST [8, 9].

Phylogenetic analysis reveals a greater genetic diversity with the presence of three distinct lines. The first line represents a set of sequences found only in Guinea, and that is most closely

**Figure 1.** Phylogram of Ebola virus obtained with BEAST, the phylogenic software based on Bayesian evolutionary analysis. The first cluster (GP1) contains sequences from Guinea alone. In second cluster (GP2), we may find sequences from Sierra Leone and Guinea. In third cluster (GP3), the sequences from Fore'cariah, Dalaba are collected.

related to viruses taken early in March 2014. The cluster found in the large Conakry region is relative to two other lines. The second cluster contains sequences from Sierra Leone and Guinea; it could be a reintroduction of Sierra Leone or Guinea streaming in related strains to those initially introduced in Sierra Leone. Finally, a third group of viruses is located in Conakry, Fore'cariah, Dalaba, and to a limited extent in Coyah. Several sequences from Sierra Leone are grouped within the third cluster. Such phylogenetic structure suggests that there have been multiple migrations of EBOV into Guinea from Sierra Leone [10, 11]. However, following the phylodynamic studies, the virus had accumulated a significant number of mutations, 7 × 10–4 substitutions per site per year [12]. **Figure 1** represents a phylogram of the Ebola virus.

#### **3. Structural roles and genomic information on gylcoprotein**

epidemic is still standing. The Ebola haemorrhagic fever weans and is often fatal in humans.

Ebola virus is a pathogenic agent of Ebola haemorrhagic fever; it is a single-stranded RNA with negative sense and with a genome length of approximately 18,920 nucleotides. Since it belongs to *Filoviridae*, its diameter is about 80 nm with a twisted filamentous form. Generally, the virus length is up to 1.1 µm [4], but particles of 14 µm were detected in the culture of liver tissue [5]. The viral RNA contains information about eight proteins, VP24, VP30, VP35, VP40, L, NP, sGP and GP1/2. Each of the protein expressed by EBOV is known for its multi-func‐ tionality that is why it is nominated as Swiss Army Knife, essentially VP35 and GP that present multi-functionality in the pathogenesis process and in the inhibition of immune responses in the host. EBOV is classified as a Category A priority pathogen by the National Institute of Allergy and Infectious Diseases (NIAID) and a Category A agent of bioterrorism by the Centers

The principal characteristics of the Ebola virus is the presence of a heavily glycosylated GP on the external surface of viral membrane. Crystallographic studies revealed that GP on the viral

This chapter aims to provide a current overview on the treatment of GP of the Ebola virus, genomic organization, synthesis, their roles and functions, and the mechanisms of viral entry

A total of 132 sequences are collected from the NCBI gene database. The samples were collected from the Makona river (80 sequences from Sierra Leon and 52 from Guinea), and they were collected in the period between 1 June 2014 and 30 August 2015. They underwent a global

Phylogenetic analysis reveals a greater genetic diversity with the presence of three distinct lines. The first line represents a set of sequences found only in Guinea, and that is most closely

**Figure 1.** Phylogram of Ebola virus obtained with BEAST, the phylogenic software based on Bayesian evolutionary analysis. The first cluster (GP1) contains sequences from Guinea alone. In second cluster (GP2), we may find sequences

from Sierra Leone and Guinea. In third cluster (GP3), the sequences from Fore'cariah, Dalaba are collected.

It is caused by *Filoviridae*, citing the species Zaire Ebola virus (ZEBOV).

of Disease Control and Prevention (CDC) [6].

**2. Phylogenetic information of Ebola virus**

alignment and phylogenetic analysis by MEGA 6.4 and BEAST [8, 9].

surface exists in the trimeric form [7].

120 Ebola

associated with GP and replication.

The Ebola genome mainly consists of seven genes, which encode eight proteins. Genes are delimited by conserved transcriptional signals; each gene starts with an initiation site at 3′ and ends with a (polyadenylation) stopover site. Zaire Ebola virus (EBOV) is a member of *Filoviridae* order of *Mononegavirales*. According to the ICTV (International Committee on Taxonomy of Viruses) classification, *Mononegavirales* order contains four families in addition to *Filoviridae*, which are *Bornaviridae, Nyamiviridae, Paramyxoviridae* and *Rhabdoviridae*.

Families of the *Mononegavirales* can be distinguished by the size of the genome and coding capacity, virion's morphology (filamentous, pleomorphic, or ball-shaped and bacilliform), viral pathogenesis and by its hosting organism. The nomenclature *Filoviridae* was changed several times for various reasons and also due to the nature of information discovered through the study of these viruses. The aim was to prepare a classification that reflects the knowledge and correct the international code of nomenclature [13]. Unfortunately, in the *Filoviridae* case, approaching ICTV has not been useful because both species and virus names had already been implemented in non-Latinized binomial form [14, 15]. The new nomenclature writes "*Zaire ebolavirus*" means the species "**Ebola virus,"** which is a member of Zaire Ebola virus—and is the most common member. However, the abbreviation is "EBOV." The new classification also contains a new genre called "**Cuevavirus**," which is newly discovered and contains the species "*Lloviu cuevavirus*" [14].

The common characteristics of this family are as follows: a negative genome RNA, which is linear, single-stranded, mono-segmented and filamentous; the genes in genome are ordered in specific ways, always starting with gene for envelope protein (3′UTR) and finishing with gene for the RNA polymerase (5'UTR); the viral replication occurs after synthesizing antigenome; RNA is stored within the helical nucleocapsid; and RNA polymerase gene is the largest gene of *Filoviridae* [16].

Besides the common characteristics, there are others that allow the distinctions between the genomes of *Filoviruses* from *Mononegavirales* order, such as the location of overlapping genes in *Filoviridae*. Overlaps are of the length 18–20 bases and are limited to the conserved sequences determined for transcription signals [17]. The bioinformatics analysis of the amino acids' sequences of the protein from *Filoviridae* has shown different degrees of identities. The analysis has shown that the nucleoprotein (NP) is the most conserved protein with a significant identity with the exception of the C-terminal portion. The rates of conservation for other proteins are as follows: 33% for VP35, 27% for VP40, 34% for GP, 33% for VP30 and 37% for VP24 [17–19].

In **Figure 2**, the genome of EBOV is shown schematically. From this scheme it is evident that the fourth gene from the 3′ of the viral genome encodes for glycoprotein (GP). This gene contains two reading frames: GP is one of the genes that encode for two mRNA—ORF I of the GP gene encodes for an sGP of about 50–70 kDa (it is non-structural glycoprotein that is efficiently secreted by infected cells) and ORF II encodes for a transmembrane glycoprotein of 120–150 kDa. The length of this gene is about 2406 nucleotides and is located between nucleotide 5900 and 8308. It is located before the VP40 gene and after the VP30 gene.

**Figure 2.** The full length genome of Ebola is about 19,000 base, where L is the length of the gene and is more conserved then the VP40, which is the more polymorphisized gene; however, the GP is within an average polymorphism. The sequence 6.039–6.923 coding for the GP1 is followed by polyadenine before the GP2 sequence in position 6.924–8.068.

The GP gene is localized between the nucleotides 5883 and 8288, where the mRNA of small non-structural secreted glycoprotein is encoded between the regions 6022 and 7116; however, the membrane GP is encoded by the regions 6022 and 8051 of nucleotides, where the region 6022–6906 is responsible for the subunit GP1 and the nucleotide sequence form 6906 to 8051 in the EBOV's genome coding is responsible for GP2; here we observed that for sGP and GP1/2 the coding starts separately.

The GP gene advances by conserved transcription start and stop signals: "CUACUUC UAAUU" as the start transcription signal for nucleotides 5883 and 5894, and "UAAUUCU" as the stop transcription signal. The end of the gene of GP containing a region encoding (UUUUUU) or including that of the GP gene (UAAUUCUUUUU) is typically sited in the region 8278–8288. It is plausible to think that the purpose of these poly U's into the genome, and these sites, is to form a poly-adenine during the transcription for retaining the mRNA and forming a premature protein [19].

A major difference between the Ebola and Marburg species has been shown with regards to the GP gene. In contrast to Ebola in which the genome contains information about two different forms of GP (sGP and GP), the GP gene of Marburg only codes a single GP.

The work of Sullivan et al. [20] has dismissed that a single mutation in the position 77 or 121 in the sequence of GP2 is able to influence the cytotoxicity and the immunogenicity of the virus. A post-transcriptional modification is also able to have the same effect [20]. The post-tran‐ scriptional change may be due to the effects of prophylactic drugs or antibodies that are specifically reactive with the GP2 [20]. The structure of EBOV GP and its interaction with the human antibody KZ52 is shown in **Figure 3**. Therefore, the GP1 subunit of GP binds with the GP2 subunit by a non-covalent bond; therefore, the residues of GP at the positions 266 and 476 do not significantly affect viral entry [21].

sequences of the protein from *Filoviridae* has shown different degrees of identities. The analysis has shown that the nucleoprotein (NP) is the most conserved protein with a significant identity with the exception of the C-terminal portion. The rates of conservation for other proteins are as follows: 33% for VP35, 27% for VP40, 34% for GP, 33% for VP30 and 37% for VP24 [17–19]. In **Figure 2**, the genome of EBOV is shown schematically. From this scheme it is evident that the fourth gene from the 3′ of the viral genome encodes for glycoprotein (GP). This gene contains two reading frames: GP is one of the genes that encode for two mRNA—ORF I of the GP gene encodes for an sGP of about 50–70 kDa (it is non-structural glycoprotein that is efficiently secreted by infected cells) and ORF II encodes for a transmembrane glycoprotein of 120–150 kDa. The length of this gene is about 2406 nucleotides and is located between

nucleotide 5900 and 8308. It is located before the VP40 gene and after the VP30 gene.

**Figure 2.** The full length genome of Ebola is about 19,000 base, where L is the length of the gene and is more conserved then the VP40, which is the more polymorphisized gene; however, the GP is within an average polymorphism. The sequence 6.039–6.923 coding for the GP1 is followed by polyadenine before the GP2 sequence in position 6.924–8.068.

The GP gene is localized between the nucleotides 5883 and 8288, where the mRNA of small non-structural secreted glycoprotein is encoded between the regions 6022 and 7116; however, the membrane GP is encoded by the regions 6022 and 8051 of nucleotides, where the region 6022–6906 is responsible for the subunit GP1 and the nucleotide sequence form 6906 to 8051 in the EBOV's genome coding is responsible for GP2; here we observed that for sGP and

The GP gene advances by conserved transcription start and stop signals: "CUACUUC UAAUU" as the start transcription signal for nucleotides 5883 and 5894, and "UAAUUCU" as the stop transcription signal. The end of the gene of GP containing a region encoding (UUUUUU) or including that of the GP gene (UAAUUCUUUUU) is typically sited in the region 8278–8288. It is plausible to think that the purpose of these poly U's into the genome, and these sites, is to form a poly-adenine during the transcription for retaining the mRNA and

A major difference between the Ebola and Marburg species has been shown with regards to the GP gene. In contrast to Ebola in which the genome contains information about two different

The work of Sullivan et al. [20] has dismissed that a single mutation in the position 77 or 121 in the sequence of GP2 is able to influence the cytotoxicity and the immunogenicity of the virus. A post-transcriptional modification is also able to have the same effect [20]. The post-tran‐ scriptional change may be due to the effects of prophylactic drugs or antibodies that are specifically reactive with the GP2 [20]. The structure of EBOV GP and its interaction with the

forms of GP (sGP and GP), the GP gene of Marburg only codes a single GP.

GP1/2 the coding starts separately.

122 Ebola

forming a premature protein [19].

**Figure 3.** The Ebola virus glycoprotein has a calyx structure-like tribal as it is demonstrated by the crystal structure. According to the crystal structure of the protein, it is shown that three subunits of GP1 (blue) are bound to three GP2's subunits (green). In yellow it is shown that the human antibody KZ52 interacted with the GP at the base of the chalice [7, 14].

Multiple studies have shown that the GP gene products have the following multiple roles in the process of pathogenesis:




#### **4. Synthesis of GP and maturation**

#### **4.1. The transmembrane glycoprotein GP1/2**

EBOV GP has 676 amino acids in length with an apparent molecular weight of 150 kDa. The glycoprotein is synthesized as a precursor of pre-GP (or GP0) considering the length of the gene. A series of post-translational events leads to the maturation of the viral glycoprotein, *N*-glycosylation of the protein in the endoplasmic reticulum and the *O*-glycosylation in the Golgi. The pre-GP precursor is finally cleaved in the trans-Golgi compartment by a protease, furin, into two subunits: extracellular GP1(501 amino acids) and the GP2 transmembrane subunit (175 amino acids), which are interconnected together by disulphide bridges [24]. GP plays a role in the pathogenesis through regulation of the adaptive response. It is at the origin of [24]


#### **4.2. The secretory glycoprotein (sGP)**

We hypothesize that the alteration of the homeostasis system and vascular system observed during Ebola virus disease could be, at least in part, caused by these soluble glycoproteins [25].

Recent works show that the source of the expression of GP1/2 or sGP is the ribosomal slippage process, where eight adenines nucleotides play an important role in the inversion of the expression of sGP and GP1/2,.If the transcription complex reads only seven adenines, it encodes for the secretory GP, while the reading of the eight adenines leads to the transcription of the pre-GP1/2 by dint of two disulphide-linked subunits GP1 and GP2. **Figure 4** illustrates this translational frameshifting or ribosomal frameshifting shown in the Ebola virus GP gene [26].

**Figure 4.** Illustration of the translational frameshifting of the GP gene due to the open reading frame.

#### **5. Viral cycle and pathogenicity**


**4. Synthesis of GP and maturation**

**4.1. The transmembrane glycoprotein GP1/2**


**4.2. The secretory glycoprotein (sGP)**



of [24]

124 Ebola

[26].


EBOV GP has 676 amino acids in length with an apparent molecular weight of 150 kDa. The glycoprotein is synthesized as a precursor of pre-GP (or GP0) considering the length of the gene. A series of post-translational events leads to the maturation of the viral glycoprotein, *N*-glycosylation of the protein in the endoplasmic reticulum and the *O*-glycosylation in the Golgi. The pre-GP precursor is finally cleaved in the trans-Golgi compartment by a protease, furin, into two subunits: extracellular GP1(501 amino acids) and the GP2 transmembrane subunit (175 amino acids), which are interconnected together by disulphide bridges [24]. GP plays a role in the pathogenesis through regulation of the adaptive response. It is at the origin

We hypothesize that the alteration of the homeostasis system and vascular system observed during Ebola virus disease could be, at least in part, caused by these soluble glycoproteins [25].

Recent works show that the source of the expression of GP1/2 or sGP is the ribosomal slippage process, where eight adenines nucleotides play an important role in the inversion of the expression of sGP and GP1/2,.If the transcription complex reads only seven adenines, it encodes for the secretory GP, while the reading of the eight adenines leads to the transcription of the pre-GP1/2 by dint of two disulphide-linked subunits GP1 and GP2. **Figure 4** illustrates this translational frameshifting or ribosomal frameshifting shown in the Ebola virus GP gene

**Figure 4.** Illustration of the translational frameshifting of the GP gene due to the open reading frame.

The pathogenesis mechanism of the Ebola virus begins with the infection of the immune system's cells such as macrophages, dendritic cells (DCs) and monocytes, which are the first that come to contact with the viral particles. An alteration of interferon's production is observed in the infected cells, and the level of alteration is different from one cell to another. DCs and monocytes are the cells that show important level of alterations and, consequently, the INF production level decreases significantly. Furthermore, lymphocyte apoptosis and a global dysfunction of specific immune system's cells are observed with the increase of viral burden. In addition, the virus is responsible for other dysfunctions such as neutrophils inactivation, induction of apoptosis, inhibitor of immune response, and involvement in the process of viral entry in epithelial cells, vascular dysregulation and evasion [24, 27, 28]. The spread of virus throughout the body including the vital organs with immune system dysfunc‐ tion leads to death.

The Ebola virus attacks the whole body causing increasing disseminated intravascular coagulation that degrades quickly the haemostasis and the functioning of vital organs. Infection destroys the endothelial cells, mononuclear phagocytes (monocytes, macrophages, dendritic cells, mast cells) and hepatocyte [29]. The mechanism of pathogenicity and the viral cycle can be divided into three phases:



#### **5.1. Target cells and receptors**

The tropism of the Ebola virus depends on the expression of the receptor at the entry of the virus by the target cell. Several receptors of *Filoviridae* were determined.

The Ebola GP is bound to the C-type lectins as DC-SIGN, L-SIGN and hMGL expressed by monocytic, dendritic and macrophage cells [30–33]. The virus uses other ubiquitous molecules expressed by non-monocytic cells to internalize the target cells too [34]. The Ebola virus also uses a process called antibody-dependent enhancement (ADE) to attach the host cells and facilitate the entry [35]. It is shown that the GP binds with the IgG Fc receptor IIIb and forms a cross-linking virus-antibody-complement complex to Fcγ III, which explains the rapid spread of virus throughout the body (liver, brain, heath and endothelial cell) [35]; other study demonstrated that T-cell immunoglobulin and mucin domain 1 are receptors for the Ebola virus [35].

The Ebola virus infects most types of cells. However, macrophages and dendritic cells allow a strong replication and spread of the virus through the lymph and blood circulatory system. Thus, the virus reaches lymph nodes, liver and spleen, and spreads to other tissues.

#### **5.2. Extracellular role of GP**

The infective dose of EBOV is about 1–10 virion by aerosol in non-human primates. Despite this small amount of the virus, the formation and composition of the virion allows it to cause problems for the infected bodies.

However, after infection, it tries to prevent and interfere with the immune response via glycoproteins EBOV (GP), which is one of the reasons why the Ebola virus is fatal. The EBOV glycoprotein is the only viral protein expressed on the surface of the virion and is essential for binding to host cells and the catalysis of membrane fusion in addition to other roles of pathogenicity. The GP is combined with carbohydrates that help in the prevention of the immune system; also, the coating of the protein in a sweet layer makes it more difficult for the immune system to identify that a virus is present. On the other hand, the GP released into the intracellular medium inhibited host antibodies. It is also accompanied by the rapid neutrali‐ zation of certain populations of T lymphocytes by a super-antigen effect. The GP is the essential protein in the mechanism of penetration. The GP-secreted/transmembrane GP inhibit the effect of neutralization of the natural antibodies (Ab), thanks to the carbohydrates combined with GP. The GP related to Ab easily gets attached on the cell membrane by C1q (thanks to the complement immune). This attachment facilitates and promotes deposition on host cell and this is followed by the penetration of the virus via the macropinosome pathway (this is the same capture solute of intercellular lipid lane) to the cell. Moreover, citing the possibility of using the protein G and calthrin, it indicates the role of the actin in the penetration of virus and suggests that the virus promotes, locates and retakes a large part of it action by interactions (**Figure 5**).

**Figure 5.** The mechanism of recognition and virus entry. GP binds to the antibodies of unknown site in the Fab re‐ gions. C1q complement allows the binding of the antibody bound to the virus with the target cells. The internalization of virus is the objective of multiple receptors known to the GP, they are of relatively non-specific type.

The GP binds with neutrophils and endothelial cells by the DC‑SIGN (dendritic-cell-specific ICAM3‑grabbing non-integrin) and L‑SIGN (liver and lymph node SIGN), which provide links to cell-GP via carbohydrate determinants. These bonds formed with the neutrophil receptor CD16 cause a significant reduction in the signal CR3 and Fcγ receptor II B, avoiding virus clearance. It has been shown that the strong pro-inflammatory responses were induced by the commitment of the EBOV GP with the TLR-4 and by the activation of the NF-κB transcription factor. The GP is responsible for cytotoxicity on endothelial cells by secretion of enzymes, proteolytic endosomes (such as cathepsin), that cause the destruction of the vascular endo‐ thelium and increase in vascular permeability and haemorrhagic signs.

In addition, the GP also binds with multiple nearby IgG, which allows the binding of C1 to the Fc region of antibodies that is thermo labile, and interacts with the cell surface molecules. This complex consists of C1q and two pro-enzymes of serine protease, C1r and C1s [26], which allow the virus to bind to cell membranes. At that time the virus enters and internalizes the cell via the macropinocytosis [36] (**Figure 6**).

The amount of BST2 into cell does not change but the surface of BST2 decreases in the presence of the GP. This explains that the GP hides the BST2 receptors in its absence, and VP40 that commune-precipitates and co-localizes binds with BST2. This reveals that GP plays a role in the inhibition of this interaction [37].

#### **5.3. Intracellular action of GP**

**5.2. Extracellular role of GP**

126 Ebola

(**Figure 5**).

problems for the infected bodies.

The infective dose of EBOV is about 1–10 virion by aerosol in non-human primates. Despite this small amount of the virus, the formation and composition of the virion allows it to cause

However, after infection, it tries to prevent and interfere with the immune response via glycoproteins EBOV (GP), which is one of the reasons why the Ebola virus is fatal. The EBOV glycoprotein is the only viral protein expressed on the surface of the virion and is essential for binding to host cells and the catalysis of membrane fusion in addition to other roles of pathogenicity. The GP is combined with carbohydrates that help in the prevention of the immune system; also, the coating of the protein in a sweet layer makes it more difficult for the immune system to identify that a virus is present. On the other hand, the GP released into the intracellular medium inhibited host antibodies. It is also accompanied by the rapid neutrali‐ zation of certain populations of T lymphocytes by a super-antigen effect. The GP is the essential protein in the mechanism of penetration. The GP-secreted/transmembrane GP inhibit the effect of neutralization of the natural antibodies (Ab), thanks to the carbohydrates combined with GP. The GP related to Ab easily gets attached on the cell membrane by C1q (thanks to the complement immune). This attachment facilitates and promotes deposition on host cell and this is followed by the penetration of the virus via the macropinosome pathway (this is the same capture solute of intercellular lipid lane) to the cell. Moreover, citing the possibility of using the protein G and calthrin, it indicates the role of the actin in the penetration of virus and suggests that the virus promotes, locates and retakes a large part of it action by interactions

**Figure 5.** The mechanism of recognition and virus entry. GP binds to the antibodies of unknown site in the Fab re‐ gions. C1q complement allows the binding of the antibody bound to the virus with the target cells. The internalization

The GP binds with neutrophils and endothelial cells by the DC‑SIGN (dendritic-cell-specific ICAM3‑grabbing non-integrin) and L‑SIGN (liver and lymph node SIGN), which provide links

of virus is the objective of multiple receptors known to the GP, they are of relatively non-specific type.

Macrophages and dendritic cells are the first to be infected, but the viruses can infect most cell types with the notable exception of lymphocytes and other non-adherent cells [38]. Several researches have shown that the EBOV's binding with the receptors is relatively non-specific. For example, EBOV may also attach to C-type lectins, which interact with glycans on EBOV GP as well as on phosphatidylserine (PtdSer) receptors which interacts with the viral envelope, which leads to a better EBOV entry [39, 40]. PtdSer receptors include Gas6 or protein S and TAM family receptors (TYRO3, AXL and MER).

It is suggested that EBOV enters cells through endocytosis clathrin [41]. The typical architec‐ ture of Ebola virions (length 1–2 µm, diameter 80–100 nm) is larger than the diameter of the clathrin-coated pits (85–110 nm).

It was found that their internalization was independent of clathrin- or caveolae-mediated endocytosis, but they co-localized with sorting nexin (SNX) 5 [42]. Once it is internalized, the virus must be carried in an intracellular compartment containing the factors essential for the activation of GP. The virus is initially inside the cell and in macropinosomes. The proteases cathepsin B and cathepsin L cysteine cleave GP and remove over 60% of the peptide mass, while interacting with NPC1 GP1 they intended to promote fusion of the viral membrane with the membrane of the bladder, accompanied with a pH drop in macropinosomes announcing the end of this step, which causes the fusion of the membrane of the host to the virus. Then, the complex transcription is released first, followed by the release of the viral genome [37, 43] (**Figure 6**).

**Figure 6.** The diagram summarizes the virus entry mechanism and the various stages of internalization and replica‐ tion. After attachment of the virus to the cell membrane, it activates the formation of macropinosomes via intracellular signals including the role of HAVCR1 (TM1), which recently has demonstrated the roles of the TIM-1 as a receptor or a cofactor for entry of Ebola virus. Moreover, the expression of endogenous TIM-1 reduced in very permissive cell lines leads to a reduction of the infectivity of Ebola virus [44]. Cleavage of the GP via Cathepsin B and L allows the fusion of endosomal membrane with virus causing the release of virus into the cytoplasm. By order, the VP30, VP35 and L are the first that are released into the cytoplasm and the viral genome negative sb RNA (Image from ViralZone2014 [45]).

Transcription and replication complex, VP35, N and L ensure the transfer of RNA– to RNA+ for transcription and translation of viral genome. The first step is the activation of the tran‐ scription complex by the fixation of zinc in the active site (70–90) of co-activator VP30. The VP30 binds with VP35, L and N to start the transcription and translation. The mRNAs are translated using host ribosomes. At this point, we can say that *filoviridae* are independent in their replication machinery and they need only a transcriptional signal (zinc and ribosomes) from the host cell.

The maturation of the GP track in the Golgi apparel, where the GP sequence is cleaved into GP 1 and GP2, is expressed on the surface of the cytoplasmic membrane and sGP.

The VP35, VP24 and VP40 play roles in the inhibition of immune responses by inhibition of the translation and signalization of antiviral genes by the succession of kinase-phosphorylation reactions.


**Table 1.** The collection of crystallographic structures related to Ebola's GP.

**Figure 6.** The diagram summarizes the virus entry mechanism and the various stages of internalization and replica‐ tion. After attachment of the virus to the cell membrane, it activates the formation of macropinosomes via intracellular signals including the role of HAVCR1 (TM1), which recently has demonstrated the roles of the TIM-1 as a receptor or a cofactor for entry of Ebola virus. Moreover, the expression of endogenous TIM-1 reduced in very permissive cell lines leads to a reduction of the infectivity of Ebola virus [44]. Cleavage of the GP via Cathepsin B and L allows the fusion of endosomal membrane with virus causing the release of virus into the cytoplasm. By order, the VP30, VP35 and L are the first that are released into the cytoplasm and the viral genome negative sb RNA (Image from ViralZone2014 [45]).

Transcription and replication complex, VP35, N and L ensure the transfer of RNA– to RNA+ for transcription and translation of viral genome. The first step is the activation of the tran‐ scription complex by the fixation of zinc in the active site (70–90) of co-activator VP30. The VP30 binds with VP35, L and N to start the transcription and translation. The mRNAs are translated using host ribosomes. At this point, we can say that *filoviridae* are independent in their replication machinery and they need only a transcriptional signal (zinc and ribosomes)

The maturation of the GP track in the Golgi apparel, where the GP sequence is cleaved into

The VP35, VP24 and VP40 play roles in the inhibition of immune responses by inhibition of the translation and signalization of antiviral genes by the succession of kinase-phosphorylation

GP 1 and GP2, is expressed on the surface of the cytoplasmic membrane and sGP.

from the host cell.

reactions.

128 Ebola

When the complex polymerase binds along the RNA template, the polymerase complex stops and is re-introduced at each junction of genes and transcription, thus individual genes appear sequentially in their 3′–5′ order. The region 3′ in the genome and anti-genome viral contains promoter's sites of replication for positive and negative sense RNA synthesis; they are approximately 176b [46]. The virus acts on microtubules and immunosuppressive genes to inhibit cell division. As the number of virions increases, it causes a burst of the host cell and then death or apoptosis due to the speed of the replication of virions, which are approximately 109 plaque-forming units (PFUs) in tissue during 7–10 days [26].

The spread of the virus in the body and vital organs causes haemorrhage and fever due to unskilled hyper activation of cytokines via transmembrane GP, and the activity of the NK causes diarrhoea because of the infection of digestive cells of the system. In addition, the pneumocystis, hepatocytes and cardiovascular cell infection accelerates the death of the patient.

#### **5.4. Strategies for the inhibition of the** *Ebola virus*

Several methods can be used to inhibit *filoviridae* and more particularly the glycoprotein. The inhibition of the GP induces entry cell inhibition and then limited viral infection. **Table 1** shows different crystalline forms of GP with cellular proteins, which develop the pathology and suggest a site to inhibit. Inhibitors must beagle to inhibit the alone active site of GP1 or both active sites of GP2. The inhibitors may be antibodies or small molecules that interact with EBOV proteins in the way to limit its action. The EBOV has the ability to use multiple ways as immune pathway, therefore it is important to design inhibitor or cocktail of inhibitors that inhibit multiple targets at the same time. In our opinion, the best way to reduce devastating action of EBOV is the collective inhibition of GP and VP30. The inhibition of GP is to reduce the side effects caused by the GP. Many antibodies and inhibitors were developed, and some of them during clinical trials [47]. However, the inhibition of VP30 is also the best way to inhibit the replication process and then remove the virus via mRNA degradation by RNAase.

#### **6. Biosecurity, biosafety and Ebola virus**

A good understanding of the mechanisms of the virus' action allows us to manipulate the viruses in the level of biosafety, which is lower than BSL-4. The virus is composed of two complementary and essential units for the infectious act, genome and VP30, VP35, VP40 and L proteins; merging of genome with these four proteins is capable of inducing infection. As Ebola is a negative sense single-stranded RNA virus, the isolation of their genome from its microenvironment composed of four proteins cannot trigger a viral reaction. As such Ebola cannot synthesize DNA genome, therefore, its cDNA copy is synthesized only in a laboratory, in which all manipulation conditions are easily manageable.

The best way to master and control the transcription and translation processes in the laboratory is by not allowing a transcription/translation of the total genome. Moreover, by isolating the proteins from their genome and other proteins, the pathogenic effects can be stopped. The pathogenic effect is caused by the cooperation and integration of different proteins that make up the genome of the viruses; however, the loss of a single protein causes the inhibition of virus by losing their genetic information via the degradation of cellular RNA as in the case of VP30.

It is possible to clone cDNA in *E. coli* in the BSL-2 laboratory [52, 53], which is consistent with the approach outlined in the BMBL and is responsible for developing and implementing an appropriate biosecurity measure. By cloning the cDNA of a gene, it is possible that it will accelerate the scientific research process and help in discovering new drugs. The study of individually cloned altered proteins is also possible in animals' models.

#### **7. Conclusions**

When the complex polymerase binds along the RNA template, the polymerase complex stops and is re-introduced at each junction of genes and transcription, thus individual genes appear sequentially in their 3′–5′ order. The region 3′ in the genome and anti-genome viral contains promoter's sites of replication for positive and negative sense RNA synthesis; they are approximately 176b [46]. The virus acts on microtubules and immunosuppressive genes to inhibit cell division. As the number of virions increases, it causes a burst of the host cell and then death or apoptosis due to the speed of the replication of virions, which are approximately

The spread of the virus in the body and vital organs causes haemorrhage and fever due to unskilled hyper activation of cytokines via transmembrane GP, and the activity of the NK causes diarrhoea because of the infection of digestive cells of the system. In addition, the pneumocystis, hepatocytes and cardiovascular cell infection accelerates the death of the

Several methods can be used to inhibit *filoviridae* and more particularly the glycoprotein. The inhibition of the GP induces entry cell inhibition and then limited viral infection. **Table 1** shows different crystalline forms of GP with cellular proteins, which develop the pathology and suggest a site to inhibit. Inhibitors must beagle to inhibit the alone active site of GP1 or both active sites of GP2. The inhibitors may be antibodies or small molecules that interact with EBOV proteins in the way to limit its action. The EBOV has the ability to use multiple ways as immune pathway, therefore it is important to design inhibitor or cocktail of inhibitors that inhibit multiple targets at the same time. In our opinion, the best way to reduce devastating action of EBOV is the collective inhibition of GP and VP30. The inhibition of GP is to reduce the side effects caused by the GP. Many antibodies and inhibitors were developed, and some of them during clinical trials [47]. However, the inhibition of VP30 is also the best way to inhibit the

replication process and then remove the virus via mRNA degradation by RNAase.

A good understanding of the mechanisms of the virus' action allows us to manipulate the viruses in the level of biosafety, which is lower than BSL-4. The virus is composed of two complementary and essential units for the infectious act, genome and VP30, VP35, VP40 and L proteins; merging of genome with these four proteins is capable of inducing infection. As Ebola is a negative sense single-stranded RNA virus, the isolation of their genome from its microenvironment composed of four proteins cannot trigger a viral reaction. As such Ebola cannot synthesize DNA genome, therefore, its cDNA copy is synthesized only in a laboratory,

The best way to master and control the transcription and translation processes in the laboratory is by not allowing a transcription/translation of the total genome. Moreover, by isolating the proteins from their genome and other proteins, the pathogenic effects can be stopped. The

109 plaque-forming units (PFUs) in tissue during 7–10 days [26].

**5.4. Strategies for the inhibition of the** *Ebola virus*

**6. Biosecurity, biosafety and Ebola virus**

in which all manipulation conditions are easily manageable.

patient.

130 Ebola

Based on the data presented in this chapter, Ebola has developed multiple pathways and modes for the evasion of the immune system and internalization in target cells. Further studies are necessary for a good understanding of the entry mechanism. However, the specific proteins of virus or even the cDNA genome disassociated of proteins can be studied in BSL-2 because the effects of virus depends on the presence of genome associated with structural and func‐ tional proteins, which allows to study the virus in laboratories at biosafety level 3 or 2. They may even accelerate the process of finding new inhibitors by pharmaceutical and vaccination companies.

#### **Acknowledgements**

The authors thank the Ministry of Higher Education, University Hassan II of Casablanca, Faculty of Sciences and Technics and the Laboratory of Virology, Microbiology, Quality and Biotechnologies/Ecotoxicology & Biodiversity-Team of Virology, oncology and medical Biotechnologies.

### **Author details**

Khadija Khataby, Yassine Kasmi, Rahma Ait Hammou, Fatima Ezzahra Laasri, Said Boughribi and My Mustapha Ennaji\*

\*Address all correspondence to: m.ennaji@yahoo.fr

Laboratory of Virology, Microbiology, Quality and Biotechnologies/Ecotoxicology & Biodiversity, Faculty of Sciences and Techniques, University Hassan II of Casablanca, Casablanca, Morocco

Khadija Khataby and Yassine Kasmi contributed equally to this work.

#### **References**


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