Ebolavviruksen musiininkaltainen domeeni (jatkoa)

Oxford University Press Impact of Ebola Mucin-Like Domain on Antiglycoprotein Antibody Responses Induced by Ebola Virus-Like Particles

Osvaldo Martinez, Lee Tantral, [...], and Christopher F. Basler

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Ebola viruses (EBOVs) are enveloped, single-stranded, negative-sense RNA viruses belonging to the family Filoviridae that cause a hemorrhagic fever in humans, with a mortality rate of up to 90%. EBOV attachment and entry into host cells is mediated by the viral envelope glycoprotein (GP) [1–3]. The membrane-anchored GP is a trimer. GP is first produced as a 676-residue–long precursor, GP0, which is cleaved by furin into disulfide-bonded GP1 and GP2. 

These GP1/GP2 heterodimers assemble into a chalice-shaped trimer that is expressed on the virion surface [4]. 

GP1 is distal to the membrane surface and contains the receptor-binding domain (N-terminal residues 54–201) and a heavily glycosylated mucin-like domain. 

 GP2 possesses a transmembrane domain, the fusion peptide, and heptad repeats required for virus–cell membrane fusion [5, 6].

When expressed from 293T cells, the EBOV matrix protein, virion protein 40 (VP40), induces the production of virus-like particles (VLPs) that are biochemically and morphologically similar to EBOV. If coexpressed with VP40, GP becomes incorporated into the VLPs and can mediate entry into target cells [7, 8]. GP-mediated entry likely occurs via macropinocytosis [9, 10], although other endocytic pathways have also been implicated in entry [9–14]. 

For productive entry, GP is cleaved by cellular cathepsins B and L in acid endosomes such that a substantial portion of the protein is removed and the remaining approximately 19 kDa cleavage product is sufficient to mediate membrane fusion reactions [15–19].

Among the regions of GP removed by cathepsin cleavage is the highly glycosylated mucin-like domain. The mucin-like domain is not required for viral entry, because mucin-domain–deleted GP is able to mediate viral attachment and entry in pseudotyped virus systems.

 EBOV GP interacts with lectin-binding receptors present on some cell types, including antigen-presenting cells (APCs) , thereby promoting virus attachment and entry [20–24]. The mucin-like domain has specifically been shown to be important for interaction of GP with the human macrophage galactose-specific and N-acetylgalactosamine-specific C-type lectin (hMGL), promoting EBOV infection [25]. 

 In addition, it may serve immune-modulating functions. 

For example, its presence is correlated with an ability to alter cellular signaling, including mitogen-activated protein kinase (MAPK-)  signaling [26, 27] and has been shown to enhance Ebola VLP-mediated cytokine secretion from stimulated dendritic cells [27].

 Furthermore, VLPs with wild-type but not mucin domain–deleted GP can activate toll-like receptor 4–dependent responses [28].

The GP mucin-like domain obstructs access to GP epitopes and epitopes expressed on other surface proteins [29].

 For example, expression of high levels of GP [30] blocks access to surface major histocompatibility complex class 1 molecules (MHC1) and β-integrins [29], resulting in loss of specific anti-MHC1 or β-integrin antibody binding, decreased CD8 T cell access to MHC1 [31] and induction of cell rounding as a consequence of anchorage loss [32]. 

Furthermore, removal of the mucin-like domain with cathepsin L uncovers epitopes for neutralizing antibodies [16, 33]. However, a mouse model of EBOV infection has shown that immunization of mice with a GP-expressing vaccine elicited mucin domain–specific monoclonal antibodies, some of which were protective [34].

The contribution of antibody responses to EBOV infection and its role in protective immunization remain unclear [35]. 

Studies have demonstrated the presence of EBOV-neutralizing antibodies in the sera of human survivors of infection [33, 36]. 

However, passive transfer of neutralizing anti-EBOV antibodies has shown mixed efficacy, failing to be protective in the more relevant nonhuman primate models of EBOV infection but exhibiting some efficacy in rodent models.

 Furthermore, there is also evidence of enhanced EBOV binding to target cells in the presence of anti-EBOV antibodies [37–39].

EBOV VLPs are immunogenic and have been used to vaccinate and protect mice and nonhuman primates in experimental animal models of EBOV infection [40, 41]. This immunogenicity is due, at least in part, to the mucin domain of the GP, yet studies have shown that the mucin-like domain itself may block access to the GP and adjacent immune molecules potentially inhibiting immune responses, including neutralizing antibody, raising the question of whether vaccines would be more efficacious with or without the GP mucin-like domain. Therefore, to test the hypothesis that the mucin-like domain modulates antibody responses to GP, including neutralization, mice were immunized with VLPs expressing EBOV wild-type GP, GP lacking the mucin-like domain, and a GP expressing a heterologous mucin-like domain; the ensuing antibody response was assessed.