Structure and pathogenesis of Ebola Virus

Ebola virus is a non-segmented negative-strand RNA virus with a genome size of about
18-19 kb. The genome of Ebola virus is made up from 7 important gene which encodes viral
proteins that is essential to the life cycle of Ebola virus. The 7 important genes are nucleoprotein
(NP) gene, viral protein (VP) 35 gene, VP40 gene, glycoprotein (GP) gene, VP30 gene, VP24
gene and polymerase (L) gene (Baseler et al. 2017). In between each gene, 3’ and 5’ untranslated
regions (UTR) can be found. This is exceptionally important for miRNA as RNA interfering
pathway of miRNA involves complementary binding of the seed sequences of miRNA to the
UTR of the mRNA and this results in gene silencing. Therefore, miRNA may be a potential
candidate as a therapeutic agent in impeding the transcription of viral gene, which in turn will
directly affect the replication of Ebola virus. One recent study had elucidated the possible human
miRNAs that can bind to UTR of Ebola viral genes through aligning the miRNA to viral gene
sequences. Intriguingly, all the Ebola viral genes are specifically targeted by one or more human
miRNA (Golkar et al. 2016). This study had shown that human miRNA can be potentially used
in therapeutic purposes and further research has to be done to determine the effect of each
miRNA that targets the specific viral genes.

Ebola virus has a diameter of 90 nm but the length of virus may vary and it usually
appears in filamentous forms (Bharat et al. 2012). The core structure of Ebola virus is made up
from RNA genome, NP, VP35, VP30 and polymerase (L) and these four viral proteins which are
essential in transcription and replication of Ebola virus forms the ribonucleoprotein complex
(RNP). NP encapsidates the Ebola viral genome and this will promote the formation of
nucleocapsid (Boehmann et al. 2005). Ebola virus infection is known to have detrimental effects
on the innate immune response by blocking the interferon (IFN) induction and the main culprit is
VP35. VP35 not only affect the host immune system, it also acts as a polymerase cofactor in
RNA polymerase transcription and replication complex. VP35 is capable of inhibiting the
activation of interferon regulatory factor 3 (IRF-3) which ultimately leads to the impairment of
IFN-β production (Cardenas et al. 2006). In addition, recent findings had discovered the ability
of VP35 in suppressing the miRNA-directed silencing in mammalian cells. Based on the finding,
VP35 can effectively suppress the establishment of silencing by miR-30 (Zhu et al. 2012). This
result poses a challenge in developing an effective miRNA in targeting Ebola virus as the virus is
able to identified the innate antiviral effect of miRNA in preventing viral replication and develop
viral proteins that will disrupt the gene silencing of miRNA. Furthermore, VP30 plays a role as a
transcriptional activator and can also interacts with NP to promote RNA synthesis of Ebola virus
(Xu et al. 2017). VP24 is a viral protein needed in the assembly of capsid, together with VP35
and NP and these three proteins must be present (Huang et al. 2002). These close associations of
proteins indicate the importance of a group of viral proteins working together to ensure viral
replication is successful. Any changes or disruption affecting either one viral protein will affect
the capsid production and this association may be a potential therapeutic target that could be
targeted to impede viral replication. VP40 is the primary matrix protein and it involves in
budding of Ebola virus from the plasma membrane. VP40 can also interact with host factor
Endosomal Sorting Complex Required for Transport (ESCRT) pathway and this interaction is
involved in the efficiency of formation of virion (Madara et al. 2015). The role of RNA-
dependent RNA polymerase L of Ebola virus is relatively straightforward as it mainly transcribes
and replicates viral genomes.

The pathogenesis of Ebola virus is relatively complex as the clinical manifestations are
due to direct cytopathogenic effect and indirect host-mediated effectors. Ebola virus not only can
directly affect the function of different organ, it can also evade or even impair the host immune
system. Ebola virus can affect a wide range of cells and organs, which includes adrenal cells,
hepatocytes, epithelial cells, kidney, spleen and lymph node and this accounts for the complexity
of the pathogenic effect of Ebola virus (Moghadam et al. 2015). Before Ebola virus is widely
disseminated in the body, Ebola virus will first infect macrophages and dendritic cells at the site
of entry. Macrophages and dendritic cells are the early replication site for Ebola virus and then
traveled to lymph node and spleen (Leroy et al. 2011). Once Ebola virus reaches lymph node or
spleen, more viral replication takes places and there is no doubt that Ebola virus are able to
disseminate to different parts of the body.

Ebola virus was known to be a deadly virus because it can alter the host innate and
adaptive immune response to favor viral replication. During Ebola virus infection, immune
suppression and overactivation in different aspect of immune response and blood coagulation
disorder will occur. These complications will then ultimately lead to Ebola hemorrhagic fever
where it is characterized by shock, fever and coagulation defect. Immune overactivation is
particularly one of the main concern of Ebola virus infection as it is associated with cytokine
storm (Marcinkiewicz et al. 2014). Previous study had shown that within the first hour of Ebola
virion binding and entering the macrophage, the level of gene products, such as cytokines, had
been elevated, even before the viral genes are expressed. The activation of cytokine production
begins with the interaction of shed GP of Ebola virus and toll-like receptor 4 (TLR4). This
interaction is able to induce the release of pro-inflammatory and anti-inflammatory cytokine,
such as tumour necrosis factor α (TNFα), Interleukin (IL) 6, IL10, IL10, IL12, IL8, IL1β and
IL1RA (Escudero-Perez et al. 2014). One recent study also shows that Ebola virus can bind to T-
cell immunoglobulin and mucin domain-containing protein 1 (Tim-1) to induce a cytokine storm.
Ebola virus bind directly CD4+ T cells and trigger the release of inflammatory mediators
(Younan et al. 2017). Therefore, CD4+ T cells, macrophage and dendritic cells are responsible
for the elevated levels of inflammatory cytokines and chemokines in a short period of time. The
lethality of cytokine storm was also known after a report indicating that patients that do not
survive develops high levels of certain inflammatory cytokines and chemokines in a short while
after Ebola viral infection and the level still remain high until death occurs (Baize et al. 2002). In
conclusion, cytokine storm plays a very important role in contributing high mortality rate of
Ebola viral infection and CD4+ T cells, macrophage and dendritic cells may be the crucial
therapeutic targets. Ebola virus had not only upregulated many inflammatory mediators,
coagulation factors is also upregulated. Tissue factor is involved in the production of thrombin
and eventually leads to deposition of fibrin. On the other hand, Protein C is markedly decrease in
Ebola virus infection and it is important in anticoagulation (Geisbert et al. 2003). As the
anticoagulation effect is impaired, consumption of coagulation factor to form fibrin thrombi and
platelets consumption also increased and these combination of effects results in coagulopathy
(Bray & Geisbert 2003).

The ability of Ebola virus to evade immune response had allowed Ebola virus to replicate
and disseminate throughout the body. Ebola virus had evolved in such a way that, it can inhibit
type-1 interferon (IFN) response. Type-1 IFN involves in innate antiviral activity and in the
context of Ebola virus, it plays a role in inhibiting the release of viral particles (Neil et al. 2007).
However, the structural protein of Ebola virus, which is VP24 and VP35 are develop to disrupt
the IFN response. VP35 inhibits the retinoic acid-inducible gene-I (RIG-I) and prevents the
phosphorylation of IRF-3 and IRF-7 to become activate state. As a result, there are insufficient
IRF-3 to activate the expression of IFN-α (Cardenas et al. 2006). Other studies also reveal that
VP35 can bind to the kinase domains of inhibitor of κB kinase epsilon (IKKε) and TANK-
binding kinase 1 (TBK-1). These kinase interact directly with IRF-3 and IRF-7 to convert them
into phosphorylated form and the binding of VP35 disrupts this interaction (Prins et al. 2009).
Besides VP35, VP24 also plays an important role in inhibiting IFN signaling and render the cells
to be insensitive towards exogenous IFN. One of the study had shown that VP24 is able to bind
to unique non-classical nuclear localization signal (NLS) on karyopherin alpha5 (KPNA5) that is
important for the efficient translocation of tyrosine phosphorylated signal transducer and
activator of transcription 1 (PY-STAT1) into the nucleus (Basler 2015). VP24 competes with
PY-STAT1 and results in less PY-STAT1 translocated into nucleus. Since PY-STAT1 binds to
IFN stimulated response elements (IRSE) to induce IFN stimulated gene (ISG) expression, ISG
expression will decrease as there are less PY-STAT1 in binding IRSE (Xu et al. 2015). This
study is important because it shows that introducing exogenous IFN will not be the best way in
inducing antiviral effect but instead, the molecular mechanism that cause this effect was
elucidated, which is the competitive nature of VP24. As previously mention, Ebola virus can
alter the host adaptive immune system and this was done through suppressing dendritic cells
(DC) maturation (Mahanty et al. 2003). Dendritic cell are antigen-presenting cells that process
the antigens and present the processed antigen to T cells. For dendritic cells infected with Ebola
virus, retinoic acid-inducible gene I (RIG-I) signaling pathway is affected and this results in
prevention of upregulation of major histocompatibility complex class I (MHC-I), MHC-II,
CD40, CD80, and CD83. These changes inhibit maturation of dendritic cells and surprisingly,
VP35 is also responsible for this (Yen et al. 2014). VP35 interact with VP24 through several
innate response antagonist domains (IRAD) locations and resulted in a cooperative effect that
caused lack of maturation in dendritic cells (Lubaki et al. 2013). As the co-stimulatory molecule
and MHC are decrease, less antigen was presented to the CD4+ and CD8+ T cells to activate
these cells, which are important in stimulating the adaptive immune response. Again, the
interaction of viral proteins of Ebola virus not only is necessary in viral replication, it is also
important in altering normal innate and adaptive immune response.

miRNA as a novel therapeutic approach

 To date, there is still no drug or vaccine that is effective and used widely in treating Ebola
virus. Most of the treatment depends on a good supportive care and relies on the patient’s own
immune system (Duraffour et al. 2017). However, not everyone will have a good immune
system in combating Ebola virus infection and Ebola virus itself involves in impeding normal
function of immune system, which further worsen the problem. Therefore, intense research was
done to develop and discover potential cure for Ebola virus. Favipiravir is one of the new
antiviral drug which act as a RNA polymerase inhibitor. One animal studies even show that all
mice treated with Favipiravir after Ebola virus challenge survived (Smither et al. 2014).
Nevertheless, further research is still needed as the results from Phase II clinical trial was still not
clear and one study had also found that Favipiravir benefits patients with medium to high
viremia, but not very high viremia (Sissoko et al. 2016). Recent research revealed that siRNA
targets Ebola viral proteins, such as L, VP35 and VP24 (Bixler et al. 2017). In this case, miRNA
may also have the potential to be one of the therapeutic approach in treating Ebola virus
infection.
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