Proteolysis of the Ebola Virus Glycoproteins Enhances Virus Binding and Infectivity

Ebola virus infection requires the surface viral glycoprotein to initiate entry into the target cells. The trimeric glycoprotein is a highly glycosylated viral protein which has been shown to interact with host C-type lectin receptors and the soluble complement recognition protein mannose-binding lectin, thereby enhancing viral infection. Similarly to mannose-binding lectin, ficolins are soluble effectors of the innate immune system that recognize particular glycans at the pathogen surface. In this study, we demonstrate that ficolin-1 interacts with the Zaire Ebola virus (EBOV) glycoprotein, and we characterized this interaction by surface plasmon resonance spectroscopy. Ficolin-1 was shown to bind to the viral glycoprotein with a high affinity. This interaction was mediated by the fibrinogen-like recognition domain of ficolin-1 and the mucin-like domain of the viral glycoprotein. Using a ficolin-1 control mutant devoid of sialic acid-binding capacity, we identified sialylated moieties of the mucin domain to be potential ligands on the glycoprotein. In cell culture, using both pseudotyped viruses and EBOV, ficolin-1 was shown to enhance EBOV infection independently of the serum complement. We also observed that ficolin-1 enhanced EBOV infection on human monocyte-derived macrophages, described to be major viral target cells,. Competition experiments suggested that although ficolin-1 and mannose-binding lectin recognized different carbohydrate moieties on the EBOV glycoprotein, the observed enhancement of the infection likely depended on a common cellular receptor/partner. In conclusion, ficolin-1 could provide an alternative receptor-mediated mechanism for enhancing EBOV infection, thereby contributing to viral subversion of the host innate immune system. E bola virus (EBOV), a member of the Filoviridae family, can cause a severe, often fatal, hemorrhagic fever (HF) in humans and nonhuman primates (1). The first Ebolavirus species was discovered in 1976 in the Democratic Republic of Congo (previously called Zaire), near the Ebola River (2). Subsequently, 28 outbreaks appeared sporadically until March 2014, when the most devastating outbreak occurred in western Africa, including the countries of Guinea, Liberia, and Sierra Leone (3-6). Most outbreaks are caused by the Zaire Ebola virus subspecies (7), which is the most pathogenic (case fatality rate, up to 90%). EBOV is classified in the category A agents of the Centers for Disease Control and Prevention (CDC) because it represents a substantial threat to public health, and consequently, its handling requires a biosafety level 4 (BSL-4) laboratory. The development of effective therapies against EBOV became an urgent priority during the spread of the most recent epidemics throughout Africa, which, in addition to causing the loss of thousands of human lives, caused economic and social instability (8).The trimeric transmembrane glycoprotein (GP) of EBOV plays a crucial role in EBOV infection by mediating its cellular attachment and entry into host cells (9, 1...

Section: Gp Interaction With Ficolinssupporting

confidence: 77%

Enhancement of Ebola Virus Infection via Ficolin-1 Interaction with the Mucin Domain of GP Glycoprotein

Favier¹,

Gout

Reynard

et al. 2016

“…EBOV GP 1,2 is primed for fusion in a low-pH endosomal compartment by cathepsins B and L. These proteases cleave GP 1 (130 kDa) to a 20-kDa species and a key, 19-kDa species (4,11,21) that confer enhanced virus binding to and infection of the host cell (11,21). In addition to cathepsins B and L, which function at pH ϳ5, ZEBOV GP 1,2 can be primed more quantitatively to 19-kDa GP 1 with thermolysin, a protease that functions at neutral pH (21).…”

Section: Resultsmentioning

confidence: 99%

“…Although the mucin-like domain at the C-terminal end of GP 1 can facilitate initial virus attachment to cells (1,16,18,23,25), this domain is fully dispensable for entry. Moreover, 19-kDa GP 1 clearly contains receptor binding activity, since virus binding and infection are actually enhanced after the mucin-like domain is removed (GP 1,2 ⌬) (11,21). Additionally, 19-kDa GP 1 most likely includes residues from the amino-terminal portion of GP 1 , since mutations within the first 150 residues (residues 33 to 183) impair virus infection (3,13,15,17) and since a recombinant protein including residues 54 to 201 binds specifically to permissive cells and inhibits EBOV GP 1,2 -mediated infection (13).…”

mentioning

confidence: 99%

The Primed Ebolavirus Glycoprotein (19-Kilodalton GP _1,2 ): Sequence and Residues Critical for Host Cell Binding

Dube¹,

Brecher²,

Delos

et al. 2009

150

214

Entry of ebolavirus (EBOV) into cells is mediated by its glycoprotein (GP 1,2 ), a class I fusion protein whose structure was recently determined (J. E. Lee et al., Nature 454:177-182, 2008). Here we confirmed two major predictions of the structural analysis, namely, the residues in GP 1 and GP 2 that remain after GP 1,2 is proteolytically primed by endosomal cathepsins for fusion and residues in GP 1 that are critical for binding to host cells. Mass spectroscopic analysis indicated that primed GP 1,2 contains residues 33 to 190 of GP 1 and all residues of GP 2 . The location of the receptor binding site was determined by a two-pronged approach. We identified a small receptor binding region (RBR), residues 90 to 149 of GP 1 , by comparing the cell binding abilities of four RBR proteins produced in high yield. We characterized the binding properties of the optimal RBR (containing GP 1 residues 57 to 149) and then conducted a mutational analysis to identify critical binding residues. Substitutions at four lysines (K95, K114, K115, and K140) decreased binding and the ability of RBR proteins to inhibit GP 1,2 -mediated infection. K114, K115, and K140 lie in a small region modeled to be located on the top surface of the chalice following proteolytic priming; K95 lies deeper in the chalice bowl. Combined with those of Lee et al., our findings provide structural insight into how GP 1,2 is primed for fusion and define the core of the EBOV RBR (residues 90 to 149 of GP 1 ) as a highly conserved region containing a two-stranded ␤-sheet, the two intra-GP 1 disulfide bonds, and four critical Lys residues.

“…As a result, antibodies against these sites would likely be most functional outside the cell or on the cell surface. Third, the required receptor-binding event appears to occur only after proteolytic remodeling in the endosome (5,8,18). Hence, antibodies that target the receptor-binding sites may not recognize viral-surface GP and thus may not be present in the endosome for neutralization.…”

Section: Discussionmentioning

confidence: 99%

Structure of an Antibody in Complex with Its Mucin Domain Linear Epitope That Is Protective against Ebola Virus

Olal

Kuehne

Bale

et al. 2012

Antibody 14G7 is protective against lethal Ebola virus challenge and recognizes a distinct linear epitope in the prominent mucinlike domain of the Ebola virus glycoprotein GP. The structure of 14G7 in complex with its linear peptide epitope has now been determined to 2.8 Å. The structure shows that this GP sequence forms a tandem ␤-hairpin structure that binds deeply into a cleft in the antibody-combining site. A key threonine at the apex of one turn is critical for antibody interaction and is conserved among all Ebola viruses. This work provides further insight into the mechanism of protection by antibodies that target the protruding, highly accessible mucin-like domain of Ebola virus and the structural framework for understanding and characterizing candidate immunotherapeutics.