Viral membrane fusion proceeds through a sequence of steps that are driven by triggered conformational changes of viral envelope glycoproteins, so-called fusion proteins. Although high-resolution structural snapshots of viral fusion proteins in their prefusion and postfusion conformations are available, it has been difficult to define intermediate structures of the fusion pathway because of their transient nature. Flaviviruses possess a class II viral fusion protein (E) mediating fusion at acidic pH that is converted from a dimer to a trimer with a hairpin-like structure during the fusion process. Here we show for tick-borne encephalitis virus that exposure of virions to alkaline instead of acidic pH traps the particles in an intermediate conformation in which the E dimers dissociate and interact with target membranes via the fusion peptide without proceeding to the merger of the membranes. Further treatment to low pH, however, leads to fusion, suggesting that these monomers correspond to an as-yet-elusive intermediate required to convert the prefusion dimer into the postfusion trimer. Thus, the use of nonphysiological conditions allows a dissection of the flavivirus fusion process and the identification of two separate steps, in which membrane insertion of multiple copies of E monomers precedes the formation of hairpin-like trimers. This sequence of events provides important new insights for understanding the dynamic process of viral membrane fusion.
Key Points• BI 836858, an Fc-engineered anti-CD33 antibody, mediates autologous and allogeneic NK cell-mediated ADCC.• Decitabine increases ligands for activating NK receptors potentiating BI 836858 activity, providing a rationale for combination therapy.Acute myeloid leukemia (AML) is the most common type of acute leukemia, affecting older individuals at a median age of 67 years. Resistance to intensive induction chemotherapy is the major cause of death in elderly AML; hence, novel treatment strategies are warranted. CD33-directed antibody-drug conjugates (gemtuzumab ozogamicin) have been shown to improve overall survival, validating CD33 as a target for antibody-based therapy of AML.Here, we report the in vitro efficacy of BI 836858, a fully human, Fc-engineered, anti-CD33 antibody using AML cell lines and primary AML blasts as targets. BI 836858-opsonized AML cells significantly induced both autologous and allogeneic natural killer (NK)-cell degranulation and NK-cell-mediated antibody-dependent cellular cytotoxicity (ADCC). In vitro treatment of AML blasts with decitabine (DAC) or 5-azacytidine, 2 hypomethylating agents that show efficacy in older patients, did not compromise BI 836858-induced NK-cellmediated ADCC. Evaluation of BI 836858-mediated ADCC in serial marrow AML aspirates in patients who received a 10-day course of DAC (pre-DAC, days 4, 11, and 28 post-DAC) revealed significantly higher ADCC in samples at day 28 post-DAC when compared with pre-DAC treatment. Analysis of ligands to activating receptors (NKG2D) showed significantly increased NKG2D ligand [NKG2DL] expression in day 28 post-DAC samples compared with pre-DAC samples; when NKG2DL receptor was blocked using antibodies, BI 836858-mediated ADCC was significantly decreased, suggesting that DAC enhances AML blast susceptibility to BI 836858 by upregulating NKG2DL. These data provide a rationale for combination therapy of Fc-engineered antibodies such as BI 836858 with azanucleosides in elderly patients with AML.
In this study, we investigated in a flavivirus model (tick-borne encephalitis virus) the mechanisms of fusion inhibition by monoclonal antibodies directed to the different domains of the fusion protein (E) and to different sites within each of the domains by using in vitro fusion assays. Our data indicate that, depending on the location of their binding sites, the monoclonal antibodies impaired early or late stages of the fusion process, by blocking the initial interaction with the target membrane or by interfering with the proper formation of the postfusion structure of E, respectively. These data provide new insights into the mechanisms of flavivirus fusion inhibition by antibodies and their possible contribution to virus neutralization.
, EMBO J. 23: [728][729][730][731][732][733][734][735][736][737][738] 2004). In this study we compared the properties of this truncated form with the full-length trimer and found that the so-called stem-anchor region not only confers additional stability to the full-length molecule but also structurally modifies the protein domain carrying the fusion peptide loop. These data provide experimental evidence to support the model of a fusion process that leads to the interaction of the stem-anchor region with the fusion peptide loop in the postfusion trimer.The entry of enveloped viruses into cells requires the action of specific surface glycoproteins that are capable of undergoing triggered conformational changes for mediating the fusion of the viral membrane with a cellular membrane. Two classes of such viral fusion proteins have been identified so far that exhibit completely different structural organizations (reviewed in references 5 and 19). Class I fusion proteins form trimeric spikes and are present in orthomyxo-, paramyxo-, retro-, filo-, and coronaviruses. Class II fusion proteins on the other hand are oriented parallel to the viral membrane and form part of icosahedral protein networks in the envelopes of flavi-and alphaviruses (reviewed in references 8 and 13). These viruses enter cells by receptor-mediated endocytosis, and the acidic pH in endosomes triggers irreversible conformational changes in their fusion proteins resulting in fusion from within the endosomes.The atomic structures of C-terminally truncated forms of the fusion protein E1 from an alphavirus (Semliki Forest virus [SFV]) and the fusion protein E of two flaviviruses (tick-borne encephalitis virus [TBEV] and dengue virus) have been determined both in their pre-and postfusion conformations (4,6,14,(16)(17)(18)24). The corresponding structures of the flavivirus TBEV E protein are shown in Fig. 1. The C-terminally truncated flavivirus E proteins used for X-ray crystallography lack approximately 90 amino acids consisting of the double membrane anchor and the so-called stem region that extends from the C terminus of domain III and contains two ␣-helices (23) (Fig. 1C, D). In contrast to the class I fusion proteins, the fusion-associated structural rearrangements of the class II proteins do not involve a major refolding of the polypeptide chain but rather a reorganization of the three domains relative to each other (4,6,17). Most importantly, domain III changes its position from the end of the rod-like structure and moves to the side of domain II, resulting in a folded-back, hairpin-like structure in a trimeric complex which-in contrast to the native protein-is oriented perpendicular to the membrane (Fig. 1E).The folded-back orientation suggests that the fusion mechanism driven by class II fusion proteins is mechanistically similar to that proposed previously for viruses with class I fusion proteins (4,6,17). Although the stem-anchor region was not present in the truncated soluble E trimer (sE trimer) structure, the orientation of domain III sug...
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