MicrobiologyBytes

Ebolavirus (EBOV) and Marburgvirus (MARV) that compose the filovirus family of negative strand RNA viruses infect a broad range of mammalian cells. Recent studies indicate that cellular entry of this family of viruses requires a series of cellular protein interactions and molecular mechanisms, some of which are unique to filoviruses and others are commonly used by all viral glycoproteins. Details of their cell entry pathway are highlighted in a new paper.

Filovirus Entry: A Novelty in the Viral Fusion World. (2012) Viruses 4(2): 258-275; doi:10.3390/v4020258
Fliovirus entry into cells is initiated by the interaction of the viral glycoprotein1 subunit (GP1) with both adherence factors and one or more receptors on the surface of host cells. On epithelial cells, we recently demonstrated that TIM-1 serves as a receptor for this family of viruses, but the cell surface receptors in other cell types remain unidentified. Upon receptor binding, the virus is internalized into endosomes primarily via macropinocytosis, but perhaps by other mechanisms as well. Within the acidified endosome, the heavily glycosylated GP1 is cleaved to a smaller form by the low pH-dependent cellular proteases Cathepsin L and B, exposing residues in the receptor binding site (RBS). Details of the molecular events following cathepsin-dependent trimming of GP1 are currently incomplete; however, the processed GP1 specifically interacts with endosomal/lysosomal membranes that contain the Niemann Pick C1 (NPC1) protein and expression of NPC1 is required for productive infection, suggesting that GP/NPC1 interactions may be an important late step in the entry process. Additional events such as further GP1 processing and/or reducing events may also be required to generate a fusion-ready form of the glycoprotein. Once this has been achieved, sequences in the filovirus GP2 subunit mediate viral/cellular membrane fusion via mechanisms similar to those previously described for other enveloped viruses. This multi-step entry pathway highlights the complex and highly orchestrated path of internalization and fusion that appears unique for filoviruses.

MicrobiologyBytes: Yes folks, it’s that naughty Niemann Pick C1 (NPC1) protein again!
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When it comes to nasty pathogens, Marburg virus is among the nastiest. Cousin to Ebola virus, Marburg causes fever, rash, delirium, and severe hemorrhaging, often ending in organ failure and death. It is rare in the wild, but was a central focus of weaponization by the Soviet Union, and remains a concern for terrorism experts who fear its lethal potential and resistance to treatment.

One reason that treatments have proved so elusive is because the virus is so hard to work with – hazmat suits, self-contained breathing gear, and electronically secured airlocks are all required for even the simplest of studies with live virus. But another reason is that the virion (the virus particle) is heterogeneous in shape, and that heterogeneity has confounded standard imaging techniques (X-ray crystallography, cryo-electron microscopy), which require purified, identical particles to obtain their highest resolution. Researchers have now got around that problem by using a sophisticated combination of imaging techniques that provide the first clear three-dimensional picture of the intact Marburg virion structure.

Marburg Virus Structure Revealed in Detail. (2011) PLoS Biol 9(11): e1001198. doi:10.1371/journal.pbio.1001198
and:
Cryo-Electron Tomography of Marburg Virus Particles and Their Morphogenesis within Infected Cells. (2011) PLoS Biol 9(11): e1001196. doi:10.1371/journal.pbio.1001196
Several major human pathogens, including the filoviruses, paramyxoviruses, and rhabdoviruses, package their single-stranded RNA genomes within helical nucleocapsids, which bud through the plasma membrane of the infected cell to release enveloped virions. The virions are often heterogeneous in shape, which makes it difficult to study their structure and assembly mechanisms. We have applied cryo-electron tomography and sub-tomogram averaging methods to derive structures of Marburg virus, a highly pathogenic filovirus, both after release and during assembly within infected cells. The data demonstrate the potential of cryo-electron tomography methods to derive detailed structural information for intermediate steps in biological pathways within intact cells. We describe the location and arrangement of the viral proteins within the virion. We show that the N-terminal domain of the nucleoprotein contains the minimal assembly determinants for a helical nucleocapsid with variable number of proteins per turn. Lobes protruding from alternate interfaces between each nucleoprotein are formed by the C-terminal domain of the nucleoprotein, together with viral proteins VP24 and VP35. Each nucleoprotein packages six RNA bases. The nucleocapsid interacts in an unusual, flexible “Velcro-like” manner with the viral matrix protein VP40. Determination of the structures of assembly intermediates showed that the nucleocapsid has a defined orientation during transport and budding. Together the data show striking architectural homology between the nucleocapsid helix of rhabdoviruses and filoviruses, but unexpected, fundamental differences in the mechanisms by which the nucleocapsids are then assembled together with matrix proteins and initiate membrane envelopment to release infectious virions, suggesting that the viruses have evolved different solutions to these conserved assembly steps.

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Filoviruses cause lethal hemorrhagic fever in humans and nonhuman primates. The family Filoviridae includes two genera: Marburgvirus (MARV) and Ebolavirus (EBOVs). MARV was discovered in 1967 in Marburg, Germany during an outbreak in laboratory staff exposed to tissues from monkeys imported from Uganda. The Zaire virus was discovered in 1976 in Yambuku, Zaire during a 312-person outbreak associated with 90% mortality. With the exception of Reston Ebolavirus that appears to be pathogenic in nonhuman primates but not in humans and is endemic in the Philippines, all known filoviruses are pathogenic in primates including humans and are endemic in Africa. Bats are implicated as reservoirs and vectors for transmission of filoviruses in Africa. Ebolavirus sequences have been found in various bats. Bats naturally or experimentally infected with Ebolaviruses are healthy and shed virus in their feces for up to 3 weeks.

In 2002, colonies of Schreiber’s bats (Miniopterus schreibersii), sustained massive die-offs in caves in France, Spain and Portugal. This paper report the first discovery of an ebolavirus-like filovirus in bats from Europe.

Discovery of an Ebolavirus-Like Filovirus in Europe. (2011) PLoS Pathog 7(10): e1002304. doi:10.1371/journal.ppat.1002304
Filoviruses, amongst the most lethal of primate pathogens, have only been reported as natural infections in sub-Saharan Africa and the Philippines. Infections of bats with the ebolaviruses and marburgviruses do not appear to be associated with disease. Here we report identification in dead insectivorous bats of a genetically distinct filovirus, provisionally named Lloviu virus, after the site of detection, Cueva del Lloviu, in Spain.

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Infections by the Ebola and Marburg filoviruses cause a rapidly fatal haemorrhagic fever in humans for which no approved antivirals are available. Filovirus entry is mediated by the viral spike glycoprotein (GP), which attaches viral particles to the cell surface, delivers them to endosomes and catalyses fusion between viral and endosomal membranes. Additional host factors in the endosomal compartment are probably required for viral membrane fusion; however, despite considerable efforts, these critical host factors have defied molecular identification.

A new paper describes a genome-wide screen of human cells to identify host factors required for Ebola virus entry. Cells defective for the homotypic fusion and vacuole protein-sorting (HOPS) complex or cholesterol transporter protein Niemann–Pick C1 (NPC1) are resistant to infection by Ebola virus and Marburg virus, but remain fully susceptible to a suite of unrelated viruses. Membrane fusion mediated by filovirus glycoproteins and virus escape from the vesicular compartment require the NPC1 protein, independent of its known function in cholesterol transport. These findings uncover unique features of the entry pathway used by filoviruses and indicate potential antiviral strategies to combat these deadly agents.

Ebola virus entry requires the cholesterol transporter Niemann–Pick C1. (2011) Nature 477: 7364 doi:10.1038/nature10348
Infections by the Ebola and Marburg filoviruses cause a rapidly fatal haemorrhagic fever in humans for which no approved antivirals are available. Filovirus entry is mediated by the viral spike glycoprotein (GP), which attaches viral particles to the cell surface, delivers them to endosomes and catalyses fusion between viral and endosomal membranes. Additional host factors in the endosomal compartment are probably required for viral membrane fusion; however, despite considerable efforts, these critical host factors have defied molecular identification. Here we describe a genome-wide haploid genetic screen in human cells to identify host factors required for Ebola virus entry. Our screen uncovered 67 mutations disrupting all six members of the homotypic fusion and vacuole protein-sorting (HOPS) multisubunit tethering complex, which is involved in the fusion of endosomes to lysosomes, and 39 independent mutations that disrupt the endo/lysosomal cholesterol transporter protein Niemann–Pick C1 (NPC1). Cells defective for the HOPS complex or NPC1 function, including primary fibroblasts derived from human Niemann–Pick type C1 disease patients, are resistant to infection by Ebola virus and Marburg virus, but remain fully susceptible to a suite of unrelated viruses. We show that membrane fusion mediated by filovirus glycoproteins and viral escape from the vesicular compartment require the NPC1 protein, independent of its known function in cholesterol transport. Our findings uncover unique features of the entry pathway used by filoviruses and indicate potential antiviral strategies to combat these deadly agents.

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Ebola glycoprotein (GP) is the only virus protein expressed on the surface of Ebola virus and mediates entry into target cells. However, several studies report that GP expression also causes cytotoxicity, although the underlying mechanism remains unknown. GP is also believed to be a key determinant of Ebola pathogenesis and virus-like particles (VLPs) containing GP are shown to activate human endothelial cells and macrophages. The other virus proteins tested were not cytotoxic. Collectively, these reports indicate that Ebola GP imparts cell rounding and cytotoxicity in addition to facilitating virus entry. As full-length GP but not the secreted form (sGP) is shown to cause cytotoxicity, this suggests that the release of sGP during Ebola virus infection could be a mechanism used by the virus to prevent cytotoxicity and replicate and spread throughout the body.

Ebola glycoprotein accumulates in the endoplasmic reticulum. Virology Journal 2011, 8:11 doi:10.1186/1743-422X-8-11
The Filoviridae family comprises of Ebola and Marburg viruses, which are known to cause lethal hemorrhagic fever. However, there is no effective anti-viral therapy or licensed vaccines currently available for these human pathogens. The envelope glycoprotein (GP) of Ebola virus, which mediates entry into target cells, is cytotoxic and this effect maps to a highly glycosylated mucin-like region in the surface subunit of GP (GP1). However, the mechanism underlying this cytotoxic property of GP is unknown. To gain insight into the basis of this GP-induced cytotoxicity, HEK293T cells were transiently transfected with full-length and mucin-deleted (Δmucin) Ebola GP plasmids and GP localization was examined relative to the nucleus, endoplasmic reticulum (ER), Golgi, early and late endosomes using deconvolution fluorescent microscopy. Full-length Ebola GP was observed to accumulate in the ER. In contrast, GPΔmucin was uniformly expressed throughout the cell and did not localize in the ER. The Ebola major matrix protein VP40 was also co-expressed with GP to investigate its influence on GP localization. GP and VP40 co- expression did not alter GP localization to the ER. Also, when VP40 was co-expressed with the nucleoprotein (NP), it localized to the plasma membrane while NP accumulated in distinct cytoplasmic structures lined with vimentin. These latter structures are consistent with aggresomes and may serve as assembly sites for filoviral nucleocapsids. Collectively, these data suggest that full-length GP, but not GPΔmucin, accumulates in the ER in close proximity to the nuclear membrane, which may underscore its cytotoxic property.

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The filoviruses, Marburg and Ebola, cause lethal hemorrhagic fever and are highest-priority bioterrorism agents. Filovirus particles contain a rod-like nucleocapsid and are normally filamentous, though other shapes are seen. It is poorly understood how such large filamentous particles are assembled and released from infected cells. Researchers studied Marburg virus production in infected cells using electron tomography. This technique allows virus particles to be visualized in three dimensions at different stages during assembly. They found that in early stages of virus production, highly infectious filamentous viruses are produced, whereas after prolonged infection poorly infectious spherical viruses are released. We also define the sequence of steps in filamentous virus release. The intracellular nucleocapsid first travels to the plasma membrane of the cell, where it binds laterally along its whole length. One end is then wrapped by the plasma membrane and wrapping proceeds rapidly until the virus protrudes vertically from the cell surface. The rear end of the virus particle then pinches off from the cell. They propose that other important filamentous and rod-shaped viruses also follow this series of steps of assembly and budding.

Electron Tomography Reveals the Steps in Filovirus Budding. 2010 PLoS Pathog 6(4): e1000875. doi:10.1371/journal.ppat.1000875

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• How do you make a vaccine against Ebola virus?
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Ebola and Marburg virus are filoviruses that cause outbreaks of highly lethal haemorrhagic fever. Mortality rates in these diseases average more than 50%, with the highest recorded rates seen for Ebola Zaire virus (88%) and Marburg Angola virus (90%). Infection with these filoviruses produces a very high fever followed by interference with blood coagulation and vascular permeability, causing internal bleeding, bruising and skin rashes. After an asymptomatic incubation period, which can last days to weeks, symptoms of a typical filovirus infection emerge; headache, nausea, fever and malaise followed by more serious haemorrhagic symptoms and, in fatal cases, death results from multi-organ failure owing to shock.

Present treatments for filovirus infection are palliative, and consist primarily of supportive care, including hydration and pain management. There is no effective treatment or cure for these diseases. Therefore, vaccine development is crucially important as a strategy for fighting filovirus outbreaks. However, vaccine efficacy testing for Ebola virus is very difficult. There is no readily identifiable high-risk human population that can be targeted for a placebo-controlled clinical trials because disease outbreaks are unpredictable and sporadic, both geographically and temporally. Normally, clinical trials of medicines and vaccines intended for human use follow a lengthy but predictable sequence of safety and efficacy testing.

Because of its sporadic nature, the incidence of Ebola virus infection in human populations is not predictable and does not allow for adequate testing. Moreover, the immune correlates of protection from filovirus disease in humans remain unknown and therefore cannot be used to assess candidate vaccine efficacy. To facilitate the licensing of medicines when efficacy cannot be evaluated in the setting of natural infection, the U.S. Food and Drug Administration (FDA) introduced a new regulation in 2002 as an alternative licensing pathway for pharmaceutical products that target highly lethal pathogens. The FDA’s “animal rule” allows approval based on animal efficacy data. The animal rule is intended to be used as a pathway for regulatory approval only when there is no other way to licence a vaccine (Correlates of protective immunity for Ebola vaccines: implications for regulatory approval by the animal rule. 2009 Nature Reviews Microbiology 7: 393-400).

In the case of Ebola virus, the relevant animal models are non-human primates and mice. The immune correlates of Ebola virus infection consist of immunoglobulin G responses, although other factors, such as T cells, are also likely to be important in a successful immune response. Current vaccine candidates against Ebola virus include the virus glycoprotein and nucleocapsid proteins. Initial animal testing of Ebola vaccines has shown a protective effect in non-human primates and positive antibody titres in humans.

To date, no vaccines have received regulatory approval and been licensed using the FDA animal rule. This pathway does not diminish the level of regulatory contol required for vaccine approval; extensive human testing is still required to demonstrate safety and immunogenicity. The predictive relationship between animals and humans for protective efficacy is unknown, and therefore an immune correlate is used to bridge the gap between animal efficacy studies and human immunogenicity trials. It has not yet been determined what level of efficacy in animals will be required for vaccine approval, but other vaccines currently administered to the U.S. population have shown efficacies in human trials that are as low as 18%. Even this level of efficacy will provide a benefit against pathogens such as filoviruses with high mortality rates, and therefore may be acceptable against emerging natural infections or bioterrorism threats.

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