Cathepsins B and L activate Ebola but not Marburg virus glycoproteins for efficient entry into cell lines and macrophages independent of TMPRSS2 expression

Cathepsins B and L activate Ebola but not Marburg virus glycoproteins for efficient entry into cell lines and macrophages independent of TMPRSS2 expression. 2014; Towner et al., 2006). Similarly to EBOV, virions of MARV are covered by the homotrimeric spikes of GP, the sole envelope viral protein responsible for cell entry. GP is a heavily glycosylated type I transmembrane protein, with O-linked glycans and many of the N-linked oligosaccharides clustered in the mucin-like domain (MLD) (Bukreyev et al., 1993; Feldmann et al., 1991; Will et al., 1993). The GP gene of ebolaviruses codes for the shorter secreted glycoprotein (sGP), while expression of the transmembrane full-length GP requires transcriptional editing of mRNA (Sanchez et al., 1996; Volchkov et al., 1995). In contrast, the GP gene of marburgviruses codes for transmembrane full-length GP; unlike Palmitic acid ebolaviruses, marburgviruses do not produce sGP. GP undergoes trimerization within the endoplasmic reticulum; homotrimerization is essential for fusion activity of GP during viral entry into target cells (Mittler et al., 2013). In the trans-Golgi network, MARV GP is cleaved at the Arg-435 residue into GP1 (~160 KDa) and GP2 (~38 KDa) subunits. GP2 carries the transmembrane domain, is incorporated into the viral or cellular membrane, and is linked to GP1 via an intra-molecular disulfide bridge formed by the Cys-37 and Cys-610 residues (Mittler et al., 2013; Volchkov et al., 2000). During the lifecycle, all Palmitic acid filoviruses share the requirement of proteolytic GP processing in endosomal compartments, which exposes the receptor-binding site (RBS) for the interaction with intracellular filovirus receptor, cholesterol transporter protein Niemann-Pick C1 protein (Carette et al., 2011; Cote et al., 2011), as a necessary step in cell entry. The cysteine endoproteases cathepsin B and cathepsin L were identified as key host enzymes that remove the MLD and glycan cap from filovirus GP (Chandran et al., 2005). It was shown for EBOV that these two domains shield RBS from access to the receptor in uncleaved GP (Lee et al., Palmitic acid 2008). Although the use of endosomal cysteine proteases as host Palmitic acid factors for entry is a general property of members of the family they can be generated (Flyak et al., 2015; King et al., 2018), raising a hypothetical concern about its use as a monotherapy. Here, we present a comprehensive study of biological properties of a panel of MARV mAbs from a human survivor (Flyak et al., 2015). We found that the non-neutralizing mAbs MR228 and MR235 strongly mediated neutrophil and monocyte phagocytosis and natural killer (NK) cell activation, with MR228 conferring protection through the induction of Fc domain-mediated mechanisms. We also show that MR228 and MR235 possess overlapping epitopes in the GP2 wing region. Next, we show that interaction of MR235 mAb with GP exposes neutralizing epitope(s) and dramatically increases binding of RBS-specific mAbs to membrane-anchored GP, which results in enhanced virus neutralization. These data suggest a cooperative mechanism for MARV neutralization by antibodies that target different GP Palmitic acid epitopes and an important role of non-neutralizing mAbs in virus clearance during infection. RESULTS Most of the tested neutralizing mAbs and a single non-neutralizing MR228 mAb protect against MARV challenge VAV3 protective, non-neutralizing MR228 binds a linear epitope within the GP2 wing region, which partially overlaps with that of the GP2 wing murine mAb 30G4 (Fig. 3E). The MR235 epitope also partially overlaps with the epitope of MR228 but shifts slightly toward the furin cleavage site and does not overlap with that of 30G4. Taken together, these data suggest that the non-neutralizing, but protective mAb MR228 and mAb MR235 bind to epitopes located in the GP2 wing region, and possibly weakly interact with GP1. Protection by the non-neutralizing antibody MR228 is mediated by Fc fragment effector functions We next asked if the single non-neutralizing mAb identified in the study that protects protection by MR228 mAb in the mouse model of MARV infection. Open in a separate window Fig. 4. MR228 protects against MARV infection (Baize et al., 2000; Geisbert et al., 2003), we performed confocal microscopic analysis of lymph nodes from infected mice treated with mAbs to better understand the mechanisms of.