The Relationship Between Ebola Virus and Host Factors

By: Amanda He (currently being edited)

Introduction to Ebolavirus

Ebolaviruses (EBOV) are enveloped viruses that belong to the Filoviridae family. EBOV has received a significant amount of international attention because it causes severe hemorrhagic fevers in humans with a fatality rate of approximately 60% [1]. A minority of cases with infection results in flu-like symptoms with the addition of mild blood coagulopathy, blood loss, and increase in white blood cells, but these patients have a full recovery. The majority of cases develop severe illness with excessive hemorrhaging and blood clotting, which results in shock and death. However, it is important to note that a major factor of the fatality rate of Ebolavirus disease is the access to proper healthcare. EBOV is extremely infectious and can be transmitted through exposure to bodily fluids such as feces, saliva, urine, vomit, and semen from an infected individual and can enter the body through broken skin or unprotected mucous membrane. For a healthcare worker who may be interacting with a number of different patients, it is important to wear appropriate protective equipment and practice appropriate infection control and sterilization measures [2]. Potentially infected individuals must be isolated from other patients to prevent further outbreaks. Although an individual may be infected, the symptoms are not automatic as the virus has an incubation period ranging from 2 to 21 days [3]. The average incubation period is 7-10 days. Through isolation of a potentially infected individual, it can prevent the spread to others.

Several studies have characterized EBOV infection to disable the immune system and the vascular system. The disabling of the vascular system is what leads to the symptoms such as hemorrhage, hypotension, and blood pressure drop [Ansari]. Autopsy of EBOV infected patients found the viruses located primarily in the endothelial cells, mononuclear phagocytic cells, and several fibroblasts and hepatic sinusoids [3]. As a result, it is likely that the initial infection of EBOV targets macrophages and monocytes [4]. The infection of macrophages and monocytes leads to increased synthesis of tumor necrosis factor-α (TNF-α), which induces fever and lymphoid cell apoptosis [3]. Additionally, infection of macrophages and monocytes can induce the release of various pro-inflammatory proteins.

As of 2014, there are no approved therapeutic strategies to treat infection. As a result, there has been a significant emphasis on studying the host factors EBOV recruits [5]. Host factors are traits found in an individual that could have an effect on their susceptibility to disease. In recent studies, certain host factors such as cathepsin B, heat shock 70 kDa protein 5, and STAT1 have been identified to play a role in EBOV infection. Further investigation of host factors can help grasp a better understanding about EBOV and potentially lead to a treatment.

Ebola Genome and Replication Cycle

Figure 2: The negative-strand RNA genome of Ebolavirus.
Image source: http://viralzone.expasy.org/all_by_species/207.html

EBOV has a filamentous shaped virus with a lipid envelope, lipid bilayer coat, and genome. The lipid bilayer coats serves as a mechanism to protect the genome and assists with entry into the host cell [6]. The genome of EBOV consists of single stranded, nonsegmented, negative-sense RNA virus with approximately 19,000 bases [7]. The EBOV genome encodes for seven proteins: glycoprotein (GP), matrix protein viral protein 40 (VP40), nucleoprotein (NP), viral protein 24 (VP24), viral protein 30 (VP30), viral protein 35 (VP35), and large protein, an enzyme subunit (L) [1].

Each of these proteins play a significant role in EBOV infection. GP is found on the viral envelope and plays a role in viral attachment and entry into the host cell [7]. VP40 is found in the viral lipid coat and plays a role in virus structure and stability [1]. Researchers found that with the expression of only one EBOV protein, VP40, the virus is still able to form virus like particles in human cells. All the remaining proteins are used to compose the nucleocapsid. The nucleocapsid is the capsid surrounding the RNA, which plays an important role in viral transcription and replication.

HOPS and NPC1

Figure 3: Comparison of mutated NPC1 and NPC2 on susceptibility to EBOV infection.
Image source: http://www.nature.com/nature/journal/v477/n7364/full/nature10348.html [8]

In a genome-wide haploid genetic screen to identify host factors required for EBOV entry, the most mutations found were in homotypic fusion and vacuole protein-sorting (HOPS) multisubunit tethering complex and Niemann-Pick C1 (NPC1). HOPS is involved in fusion of endosomes to lysosomes and plays a role in endosome maturation, while NPC1 is an endo/lysosomal cholesterol transporter protein that can affect endosome/lysosome fusion and fussion and calcium homeostasis [8].

Carette et al took skin fibroblast samples from healthy individuals and tested the susceptibility to Filviridae infection, which typically utilizes the GP protein to enter the host cell [8]. Since both NPC1 and NPC2 are cholesterol transporter proteins, both were used and mutated to not fully express. Although both NPC1 and NPC2 had identical cholesterol levels as shown through filipin staining, only the NPC1 mutant was less susceptible to infection to EBOV [Fig 3A,B]. NPC2 had similar infection percentages as the wild-type [Fig 3B]. The researchers wanted to confirm that the NPC1 is required for EBOV GP-mediated infection, so they used U18666A, a small molecule that causes NPC1 deficiency by targeting NPC1 [8]. When Vero and HAP1 cells were exposed to increasing concentrations of U18666A, it was able to inhibit viral infection of EBOV-GP, but not the rabies virus [Fig 3D]. From this result, there is a likely chance that NPC1 may have an interaction with EBOV GP-protein that allows it to infect the host cell.

Figure 4: Identification of EBOV virus entry and exit in HAP1 cells vs HOPS and NPC1 knockout strains.
Image source: http://www.nature.com/nature/journal/v477/n7364/full/nature10348.html [8]

Once it was confirmed that NPC1 and HOPS complex were needed for virus infection, a series of experiments ranging from virus binding to VSV M protein-release assay to electron microscopy were run to identify how virus infection would be inhibited in cells deficient for HOPS complex and NPC1. The HAP1 cells were mutated to generate, VPS33AGT and VPS11GT – the HOPS knockout strains, and NPC1GT – the NPC1 knockout strain. Quantifying the amount of virions that were able to bypass the infection block that the knockout strains established, they found that both knockout strains had a significantly lower total amount of cellular infection from EBOV-GP in comparison to the control, HAP1 cell [Fig 4C]. However, NPC1 knockout strains had the lowest amount of infection as they had roughly 62% less infection than the HAP1 cells, while the HOPS knockout strains had roughly 37% less [Fig 4C].

In Fig 4D, viral escape into the cytoplasm was measured. The presence of red surrounding the blue of the viral particles indicates membrane fusion [8]. In the wild-type cells that were not treated anything, there is a significantly high amount of red throughout when exposed to EBOV, which means that there is more viruses escaping the cell [Fig 4D]. However, the wild-type cells that are treated with U18666A have reduced the number of viruses escaping. The U18666A treated cells had identical results to the HOPS knockout strain and the NPC1 knockout strain. Overall, the results consistently confirm that HOPS and NPC1 are somehow used by EBOV to enter and exit host cells. Through knocking out the complexes or using inhibitory molecules, there was a prevalent decrease in the number of infected cells. It could be of potentially interest to look into the NPC1 gene for EBOV therapeutics as it was roughly twice as effective as HOPS.

HSPA5

Figure 4: Pre-treatment with EGCG on HeLa cells and its effect on EBOV infection.
Image source: http://www.sciencedirect.com/science/article/pii/S0166354214002034 [5]

An endoplasmic reticulum (ER) chaperone called heat shock 70 kDa protein 5 (HSPA5) has been identified as a EBOV-associated host factor [9]. HSPA5 is a highly conserved ER protein that assists with protein folding and assembly. It has additional roles in regulation of ER stress responses. However, various viruses are able to utilize HSPA5 for their own purposes such as assistance in development of mature viral envelope proteins and viral entry [5]. It is likely that HSPA5 may be utilized by EBOV assist in infection.

In Reid, et al, 2014, the laboratory members investigated how inhibition of HSPA5 affects EBOV infection. First, they determined whether or not HSPA5 had an essential role in EBOV infection by inhibiting HSPA5 activity at the ATP-binding site. Epigallocatechin gallate (EGCG) is a small molecule that is able to bind to the ATP-binding site of HSPA5 and hinder its functions. In the experiment, EGCG was introduced into HeLa cells with at varying concentrations and incubated for two hours prior to infection with EBOV. Two days after EBOV infection, the cells were fixed and stained for with Hoechst 33342 [Fig 4]. Using immunofluroscence imaging, they identified the identified the significant decrease in viral infection. Infected cell nuclei were fluoresced a green color, while uninfected cell nuclei were stained blue [Fig 4B]. Quantifying the amount of cells that fluoresced green in proportion to the blue uninfected cells, there was a direct correlation between the levels of EGCG to the percentage of EBOV inhibition. There was a consistent pattern of increased EBOV inhibition with increased concentration of EGCG [Fig 4A].

Figure 5: Effect on EBOV expression and protein level when HSPA5 is knocked out.
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As there are known viruses that utilizes HSPA5 for viral protein maturation and viral entry, further experiments were ran to identify the protein production when HSPA5 is inhibited. 293T cells were treated with HSPA5 target siRNA and then infected with EBOV for 24 or 48 hours. RNA was isolated and quantified for presence of viral and HSPA5 mRNA [Fig 5A,C]. Only the 293T cells that had HSPA5 knocked out had significantly lower relative expression of EBOV mRNA, while the untransfected control and non-target siRNA control both had similar high levels of expressed EBOV mRNA [Fig 5A]. When used in a Western blot, they were able to identify that without the expression of HSPA5, there was VP24 formation and reduced formation of VP40 [Fig 5B,D]. The lack of HSPA5 preventing the formation of VP24, a protein involved in suppressing type I interferon production, and VP40, a protein involved in viral budding, shows that it is utilized by EBOV in a similar fashion to other viruses [5].

A takeaway from the Reid, et al study is the possibility of targeting HSPA5 to inhibit viral infection of EBOV instead of the EBOV genome. In both in vitro and in vivo experiments targeting HSPA5 resulted in significant reduction in virus replication and higher survival of mice against the lethal dosage of EBOV. By targeting a host factor instead of the viral genome, there would be greater difficulty for the virus to make escape mutants [5].

STAT1 and VP24

VP24 is one of the eight proteins that EBOV encodes in its genome. It has a number of different roles ranging from virus replication and assembly to binding to assisting in immune suppression [10]. Through X-ray crystallography, VP24 has been shown to bind directly to signal transducer and activator of transcription 1-α/β (STAT1). STAT1 is a transcription factor that up-regulates genes when signaled by interferons [10]. STAT proteins often undergo tyrosine-phosphorylation to form homodimers and heterodimers. The dimer formations can interact with interferon α/β expression [11]. STAT1 is able to form homodimers by itself (STAT1:STAT1), which interact with karyopherin α1, a nuclear localization signal receptor [11]. The reason why EBOV are fairly resistant to type I and II interferon treatments is because of VP24 and its ability to bind directly to STAT1’s karyopherin α1 site. Regardless of whether or not interferon-β was added, VP24 would bind to karyopherin α1 [11]. When EBOV VP24 binds to STAT1’s karyopherin α1 site, the virus hinders the STAT1’s ability to establish cell-intrinsic antiviral signaling [12]. As a direct result, EBOV is able to facilitate viral replication.

Figure 6: Ribbon structure of VP24 protein in EBOV with emphasis on virulence sites.
Image source: http://www.plospathogens.org/article/info%3Adoi%2F10.1371%2Fjournal.ppat.1002550 [10]

Further investigation of the binding between STAT1 and VP24 must be conducted in order to establish a potential drug that would prevent VP24 from binding. Once VP24 is no longer binding, STAT1 will be free to amplify interferon signals and establish a stronger defense against EBOV.