A Brief Passage of Time in the Deadly Ebola Virus

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As if from a science fiction movie, the Ebola virus is one of the most lethal viruses known to man. The haste with which it dispatches its victims is truly gruesome! Ebola was first identified in Africa in1976, and can be caused by any four of the five Ebola viruses: Bundibugyo virus, Ebola virus, Sudan virus, and Taï forest virus. The fifth virus, Reston virus is not thought to be disease causing for humans. Ebola is a member of the family Filoviridae in the order Mononegavirales. EVD has a case fatality rate of 90%. Infections of the virus cause a rapidly fatal haemorrhagic fever. Humans become infected by contact with bodily fluids from an infected person or objects that have been contaminated. The virus can also be contracted from infected animals. The reservoir of Ebola is still unknown, but scientists believe fruit bats are the most likely hosts.

 

Incubation spans two to twenty-one days and symptoms include: weakness, fever, aches, diarrhoea, vomiting and stomach pain, throat soreness, difficulty breathing, or swallowing and bleeding. Within days the virus causes a condition known as disseminated intravascular coagulation, which is marked by both blood clots and haemorrhaging. Patients exhibit symptoms of spontaneous bleeding from body orifices and any breaks in the skin, including injection sites. The virus attacks the gastrointestinal tract, skin, and internal organs. Death is brought on by haemorrhaging, shock, or renal failure and occurs within 8 to 17 days. No FDA approved therapy for Ebola virus exists, though the FDA is currently fast tracking a RNA interference therapy specific to the Ebola virus being developed by Tekmira Pharmaceuticals.

 

 

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So what does the Ebola virus look like? In fact, it has a quite menacing look. To start with, it has a threadlike structure, which is characteristic of all filoviruses. The virons are tubular and have a diameter around 80 nm. To increase its menacing appearance, lipid bilayer anchors called glycoproteins, project 10 nm spikes from the viral envelope. They’re generally somewhere between 800 nm and 1000nm long. The nucleocapsid in the center of the viron is made of a RNA wound helically with the proteins NP, VP35, VP30, and last but not least L. The viral proteins VP40 and VP24 are found in the viral tegument or the area between the envelope and nucleocapsid.

 

 

Ebola_Virion

 

 

Each viron of Ebola virus has a single stranded, negative sense RNA linear genome. It’s between 18,959 and 18,961 nucleotides in length. One frightening aspect of the virus is that replication of the virus can occur with only 427 nucleotides from the 3’ end and 731 nucleotides from the 5’ end. The virus codes for seven structural proteins and one non-structural protein. Having a negative sense polarity, the virus’ RNA structure begins in the 3’ position and ends in the 5’ position. Following the 3’ position is a non-transcribed region known as the leader. Next, a nucleoprotein of 739 amino acids that plays a central role in the virus’ replication. Then comes a protein known as VP35, which is responsible for binding a double stranded RNA and inhibiting the host cells alpha/beta interferon. After this comes the VP40 protein, which uses the COPII transport system for intracellular transport. Then comes the sGP that serves as a structural protein in the virus and then the VP30 protein, which is a RNA binding protein. Next comes the VP24 protein that the virus uses to prevent Heterogeneous ribonucleoprotien particles C1/C2 binding to Karyopherin Alpha-1 and partially alter its nuclear import. This is followed by an L protein which caps and Polyadenylates mRNA’s. Next comes a non-transcribed trailer, and finally 3’. What’s interesting about the leader and trailer is that they carry signals which control transcription, replication, and package the viral genomes into new virons.

 

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As I mentioned earlier the virus’s lipid bilayer anchors glycoproteins, which project 10 nm spikes. What I didn’t mention was that these spikes mediate the entry of the virus into the cell. The spikes attach viral particles to the cell surface. The viral particles adhere to lectin proteins and are taken up and transported to endosomes which contain host proteins cathepsin B and Niemann Pick C1 (NPC1) and catalyze fusion between viral and the endosomes membranes. In a study published in Nature by the MIT’s Whitehead Institute for Biomedical Research, it was found that for the Ebola virus to enter it required the cholesterol transporter Niemann-Pick C1 (NPC1). In the study, cells with mutated NPC1 proteins were exposed to the Ebola virus and something very, very interesting happened: The cells survived and appeared to be immune to the virus. This is interesting for that fact that this particular mutation causes a naturally occurring genetic disease (unfortunately terminal). So technically these people with Niemann-Pick disease type C, would be immune to some of the earth’s most deadly viruses. Also significant, it shows that the Ebola uses the NPC1 protein to enter the cell. The same immunity was found with Ebola’s filovirus cousin the Marburg virus. So the NPC1 protein is essential for the entry of filoviruses and acts as a receptor for the virus that mediates the infection by binding directly to the viral envelope glycoprotein.

 

 

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An interesting study found that scientists could inhibit the infection of the Ebola virus through identification and targeting with a chemical probe. The study revealed that the Ebola virus glycoprotein is cleaved by the earlier mentioned cathepsin protease and that the glycoprotein is a ligand for NPC1 protein. They used a small molecule called adamantine dipeptide to inhibit the Ebola virus’ infection. They then used a structure activity relationship to identify a more potent inhibitor for the virus and a photoaffinity-labeling agent. They employed these agents to identify the target as NPC1. It’s one of very few promising viral therapies.

 

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The Ebola virus, like all viruses, must reproduce its components and assemble itself within cells, by hijacking cellular machinery to reproduce its infectious virons. So let’s go over the replication process and then look further into the proteins that affect this. The virus begins by attaching itself to the host’s receptor through its glycoprotein spike (peplomer) Then, it goes through the process of macropinocytososis forming a pocket around the virus when it enters the host. The viral membrane then fuses with the vesicle membrane, and the nucleocapsid is released into the host cells cytoplasm. Then the negative-sense genomic ssRNA, which is enclosed in a protein shell, is used as a template for synthesis (3’-5’) of the polyadenylated messenger RNA. The cellular machinery begins translating mRNA into viral proteins. Viral proteins are processed, and the glycoprotein precursor is cleaved to heavily glycosylated GP1 and GP2. The GP1 and GP2 molecules assemble first into heterodimers, and then into trimmers to give the surface glycoprotein spikes. The secreted glycoprotein precursor is cleaved to sGP and delta peptide both of which are released from the cell. As the number of viral proteins in the cell rise, a switch happens. Things go from translation to replication… The negative sense genomic RNA is used as a template and a extra single stranded RNA is synthesized then used as a template for the synthesis of new genomic ssRNA which is rapidly encapsidated. Then things start to get really bad for the cell: Newly formed nucleocapsids and envelope proteins associate at the host cell’s plasma membrane. Budding occurs, and the cell is destroyed.

 

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The four proteins NP, VP35, VP30 and, L are what compose the nucleocapsid complex. This one thing is what sets the Ebola virus apart from most viruses of the order of Mononegavirales, which only have 3 nucleocapsids. The virus only needs NP, VP35, and L to facilitate replication. So when Ebola gets into the cell it first needs to transcribe itself.  To accomplish this, it uses its nucleocapsid protein VP30 which activates transcription of the virus. The protein works by binding to RNA through a zinc finger Cys-His motif. The zinc finger can actually be used to interact directly with RNA. Interestingly, the virus can’t actually transcribe itself without an intact zinc binding. Once the virus binds it will initiate transcription. The virus begins transcribing itself by binding a RNA polymerase to one binding site located within the leader region of the genome. The RNA polymerase then moves across the RNA template and begins to transcribe individual genes from the 3’ to 5’ order. In the transcription process it must transcribe its genome. It does this through transcribing itself into seven messenger RNAs. The RNAs length is determined by extremely protected start stop signals. It’s predicted that stable stem loop intramolecular base pairing structures are formed from these start and stop signals. These stem-loops might actually get in the way of the progression of the virus, so the zinc finger can be used to resolve or cover the structure through interacting with the RNA. Therefore, transcription can continue as planned. Due to the nature of polymerase being released from the RNA template after the formation mRNA, the proteins like NP (closest to the 3’ end) have the highest levels of the enzyme whereas proteins like L (closest to the 5’ end) are transcribed at the lowest level. The cell may want to stop the virus from transcribing itself but the VP30 is also able to stop the termination of RNA synthesis right after transcription has been initiated.

 

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The most conserved protein of Ebola is VP24. VP24 can bind the Karyopherin Alpha 1 protein and actually inhibit the transcription and replication of the virus’ genome by blocking nuclear accumulation signal transducers and activators of transcription (STAT1). This protein is also identified as ‘Ebola Virus Inhibitor’.  But, if you haven’t produced RNAi, things are going to start to get really, really nasty. The virus will have used up all of your cellular machinery and at this point is readying itself for departure. Though consistent with everything Ebola does, it will leave in the most destructive way possible! Not content to leave through exocytosis or even apoptosis, instead it uses viral budding or self-exocytosis. To accomplish this, the VP40 matrix is necessary for the virus to say farewell. The VP40 can mediate its own release from cells. An integral component of VP40 is the Proline Rich (PY) motif on its N-terminus. The PY motif can mediate interactions with the WW domain. The WW domain mediates interactions with protein ligands and both binds proline-rich with specific proline motifs. One can picture how the virus takes advantage of this system. The specific ligands that the VP40 takes advantage of are in the type I WW-domain. An example would be the neutral precursor cell expressed developmentally down-regulated protein 4 (NEDD4). VP40 is able to mediate its own release through the PY domain. VP40 can also interact physically and functionally with ligase of the WW-domain. VP40 must be transported to the plasma membrane before it can induce budding. So the VP40 interacts with the vesicle trafficking protein SEC24C and uses it to hijack the COPII transport system, to transport it from the rough endoplasmic reticulum. Sar1p is a protein of the GTPase family, that hydrolyzes Guanosine-5’-triphosphate (GTP). Sar1p acts as a sort of molecular switch that switches between an activated membrane-embedded GTP-bound form, and an inactive soluble GTP-bound form. Ebola uses Sar1p to its advantage. The inactive GTP-bound form is attracted to Sar1p, so the two spontaneously bind. Sar1p rapidly changes and forms into a hydrophobic tail, which can be inserted into the lipid bilayer. Then other protein complexes like Sec23p bind to the membrane sequentially. The proteins of the virus continue assembling themselves to form a large complex, which distorts the membrane until it buds a vesicle.  This causes the new virons to escape from the cell and carry on to infect other cells.

 

Since Ebola was first discovered, efforts to find a cure or vaccine have been futile. Perhaps the greatest weapon we have in our arsenal to fight the Ebola Virus is RNA interference. This may seem confusing given the earlier stated fact that RNAi can be suppressed by the virus. It turns out that the virus is able to suppress the natural production of RNAi however when siRNAs are introduced or the production is switched back on we have quite a powerful tool.  Many eukaryotic organisms use RNA interference as a mechanism of gene regulation. RNAi works to inhibit gene expression through small RNA molecules. This generally leads to the destruction of targeted mRNA. The molecules are one of two different types of small RNA molecules: A small non-coding RNA molecule that functions as transcriptional and post-transcriptional regulation of gene expression known as micro RNA (miRNA). Most micro RNA’s come from RNA’s that are transcribed in the cell nucleus, and a double stranded RNA molecule that interferes with the expression of specific genes complimentary to nucleotide sequences known as small interfering RNA (siRNA). siRNA are either produced in the cell or are delivered into cells experimentally.

 

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These therapies are extremely precise. The pathway that activates RNA interference is known as the enzyme endoribonuclease dicer. Dicer begins by cleaving double-stranded RNA and per-microRNA into double stranded fragments of siRNA approximately 20 nucleotides in length. The Ebola RNAi therapy works by using modified siRNAs. The combination of siRNAs targets the virus’s polymerase L protein, VP24, and VP35 protein. They deliver the siRNA through stable nucleic acid lipid particles (SNALPs). SNALPs are microscopic particles approximately 120 nanometers in length. They are made up of a lipid bilayer, composed of a mixture of cationic and fusogenic lipids, coated with a diffusible polyethylene. Once the host cells notice Ebola they will attempt to release Alpha/Beta interferon induced by RIG-I signalling to warn the cells around. But, the Ebola virus has a few more tricks up its sleeve: The virus’s VP35 protein binds double stranded RNA to inhibit interferon. But the Ebola virus isn’t quite finished yet: The virus supresses RNA silencing. During the virus replication one strand of siRNA (the guide strand), is loaded into an RNA-induced silence complex (RISC). This targets the Viral RNA for destruction. The previously mentioned VP35 encodes the recombination signal sequences (RSS), which block the RNAi’s pathway. Eventually though, enough of our dsRNA and siRNA accumulate and RSS can no longer mask itself. So the cytoplasmic dsRNA sensors RIG-I/MDA5, PKR, and 2’5’ OAS/RNAseL are all activated. Then, ranges of antiviral responses are activated, including our old friend interferon. The virus’s translation is inhibited and the virus RNA is degraded.

 

In a proof of concept study by Tekmira, a Vancouver based Biotechnology Company, rhesus monkeys and marques monkeys where exposed to the deadly Ebola virus and then given the siRNA therapy. Two out of the three rhesus monkeys were protected from the virus and all of the marques monkeys were protected. This is a remarkable achievement! It proves that RNAi is an effective therapy and cure for the deadly Ebola virus in non-human primates. Human trials are currently underway and the US FDA has fast-tracked the process. If Human trials succeed the implications of this ingenious therapy are enormous.

 

What makes Ebola so different than other viruses is it’s ability to attack and overcome its host so efficiently. It quickly migrates throughout the lymph nodes and the blood stream to infect the parenchyma of most organs. The only cells it will not infect are skeletal/cardiac muscle and bone.

 

Although this is a truly morbid subject, Ebola serves as one of the most efficient viruses in nature. So efficient and so deadly, that frequently contamination wipes out the host population before the virus can spread to a wider demographic. One of natures most deadly killing machines limits its exposure by the very nature of being so virulent. 

 

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Comments
  1. Aglagoh Godsway says:

    02/07/2014

    I became very frightened when i took my time to read about the Ebola Virus and if i understood what i read very well, the disease is very lethal than HIV AIDS and i recommend every individual protect himself/herself from this deadly disease. I think we must be very careful about the meats that eat since the virus is carried by animals too. May God have mercy on us and cure his land.

  2. Robin Jesme, RN says:

    28/07/2014

    After reading the blog, one of your statements stood out;—his, it uses its nucleocapsid protein VP30 which activates transcription of the virus. The protein works by binding to RNA through a zinc finger Cys-His motif. The zinc finger can actually be used to interact directly with RNA. Interestingly, the virus can’t actually transcribe itself without an intact zinc binding— Maybe way too simplistic, but what if the necessary zinc were not available for binding? Could this possibly stop the spread?

    1. conor.hearn@gmail.com author says:

      29/07/2014

      Thanks for your comment. Inhibiting a zinc finger is an excellent idea and is used for other viral treatments such as HIV. The zinc finger is essential for transcription, so in theory you may inhibit the transcription of the Ebola virus through a zinc ejector. Zinc ejectors interact with the zinc finger through one of several compounds such as Azodicarbonamide (which upsets the bonds by attacking the sulphur atoms) and ultimately releases the zinc from the zinc finger. This in turn disrupts the polypeptide chain. The process of disrupting the bond is known as chelation. This disruption of the zinc finger will likely limit the replication of the virus. However as treatments based on zinc inhibitors are used for the treatment of HIV, there is a growing body of information about the challenges associated with this approach. Like Ebola, HIV uses a similar zinc finger for transcription. One of the main concerns like so many viral treatments is resistant strains. In the mid to late 2000’s, researchers from Germany found that an antiviral protein previously shown to inhibit other viruses also protected against Ebola and Marburg virus infection. If the treatment is adaptive enough to keep pace with viral resistance, then this may yield a viable treatment.

      Some good articles for reference:

      http://en.wikipedia.org/wiki/Zinc_finger_inhibitor
      http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0039159

        1. conor.hearn@gmail.com author says:

          10/10/2014

          Thanks for your comment. You make an excellent point. As with HIV, transcription can be blocked by using another metal such as copper to displace zinc. Metal ions may be a very promising way to address a number of viruses. Please refer to table 2 in the following article:

          http://www.researchgate.net/publication/8051263_Interaction_of_viral_proteins_with_metal_ions_role_in_maintaining_the_structure_and_functions_of_viruses/links/00b4951c00b76339af000000

          Also, here are some excellent articles:

          http://www.ncbi.nlm.nih.gov/pubmed/10459834

          http://www.ncbi.nlm.nih.gov/pmc/articles/PMC149768/

          https://www.landesbioscience.com/pdf/Iuchi08Hanas.pdf

  3. Bev says:

    30/07/2014

    What is the potential rate of mutation of Ebola? Is it possible that the virus might be airborn infective in mutant strains?

    1. conor.hearn@gmail.com author says:

      30/07/2014

      In a study entitled, The Origin and Evolution of Ebola and Marburg Viruses from The Center for Information Biology, National Institute of Genetics, Mishima, Japan it was found that the Ebola virus is evolving at a rate around one hundred times slower than those of retroviruses and human influenza. This is promising for the development of a vaccine to treat Ebola. When viruses mutate through replication, the mutations occur within base pairs of RNA or DNA. This causes substitutions in nucleotide base pairs in the new copy. Sometimes these changes cause the virus to infect or replicate better and sometimes these changes have the opposite effect. However, the changes have more of an effect on our ability to target the destruction of the virus then allowing it to change its form of transmission.

      Ebola is a zoonotic virus that jumps between species. It is found in bats, non-human primates, and pigs, with several other species under investigation. At the National Center for Foreign Animal Disease, at the University of Manitoba in Canada a study was done where EBOV was transmitted from infected piglets to a species of macaque monkey. These animals were kept in separate cages and did not have direct physical contact with one another. After analysis the transmission was theorized to be airborne droplets. These airborne droplets can travel a short distance and do not present the same type of threat that a truly airborne virus presents.

      So it is unlikely that at the current rate of mutation the Ebola virus could spawn an airborne variant.

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  5. Choko says:

    08/09/2014

    Nice research! How do you explain the CFR in my country Sierra Leone. It stands at 35% average! And also why is it spreading so rapidly!

    1. conor.hearn@gmail.com author says:

      09/09/2014

      Thanks for your comment. According to research compiled on the following site: https://gmggranger.wordpress.com/tag/disease/ it appears the CFR is in the 40% range in Sierra Leone. The author points out that there seems to be quite a lot of variance in the numbers in different geographic locations. It’s also important that the CFR compared to previous outbreaks is much lower. This may be a result of better protocols for the treatment of the sick. As far as spread of the virus, I believe the following Wikipedia article provides good information: http://en.wikipedia.org/wiki/Ebola_virus_epidemic_in_West_Africa. Extreme Poverty, lack of water and sanitation, as well as rumours and denial all seem to contribute to the spread of the virus. I would be very interested in your insights if you could share your thoughts.

  6. Carolynn says:

    13/09/2014

    I congratulate you for your effort in research. I am a fellow Canadian who has been doing health care work in Liberia. You are on tract with the contributing factors to the fast spread of Ebola. Other very real ones are ‘fear and cultural behaviour patterns’. People are used to knowing where to go for help – village herbalists or health care facilities. Most health care facilities have been closed – so even if your child is serious with Malaria, there has not been help. Fear that their sick family member taken to an isolation center would die. People are used to being with the sick loved ones – not isolated from them- and want them to die at home. They go to stay in another community with friends rather than hear of the possibility of a terminal disease, the stigma that goes with it, and the inability to burial their own. Visitation and care of grave sites later are important in the culture.

    1. conor.hearn@gmail.com author says:

      13/09/2014

      Thank you for your comment. Your explanation of fear and cultural behaviour patterns makes a lot of sense and speaks to the real human emotions that underly this tragedy. I can only assume that with many health care facilities closed, there is an enormous impact on other illnesses. Can you tell me if there is adequate basic medical equipment to protect health care workers? I’ve read that there are shortages in many care facilities, which makes caring for any illness far more difficult. Thanks again for your contribution.

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  8. Larry says:

    16/10/2014

    Thanks so much for this article. What I see here is a catch-22 situation. I just read an article about the use of zinc as an inhibitor of rhinovirus and flu virus replication and was just wondering if this could be effective against the Ebola virus too and if zinc supplements could be used to disrupt viral replication. The zinc finger argument however blows this idea out of the water and so I am beginning to think that administering the zinc ejector alongside copper supplements or copper nano-particles may be a likely treatment.

  9. Kato Solomon says:

    05/11/2014

    Reseacrh can be targeted on the urest transcription of the viron making it inactive to the human host. Good luck

    1. conor.hearn@gmail.com author says:

      12/11/2014

      Thanks for your comment. I’m familiar with RE1-Silencing Transcription factor (REST) and Neuron-Restrictive Silencer Factor (NRSF), but not with UREST. I assume you mean inhibiting gene expression using an RNA binding protein thus blocking the attachment of RNA polymerase to the promoter and preventing transcription of the genes into the messenger RNA?

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