Enveloped viruses employ a common mechanism to enter the host cell [1
]. The first steps, receptor binding and membrane fusion, are initiated by the envelope protein [2
]. While specific details vary among different viruses, the envelope proteins invariably go through a large conformational change [5
] before initiating membrane fusion. These large conformational changes allow the envelope protein to assume an extended fusion-initiation conformation: the envelope protein in the fusion-initiation state is able to bridge across the viral and the host membranes, subsequently bringing the two membranes into close proximity and starting the fusion process [7
]. Viral neutralization by antibodies may involve binding to the fusion-state structure or inhibiting its formation. Therefore, viral envelope proteins are important foci for the development of vaccines and therapeutics. Recent intense research focus on the Ebola and Zika viruses has provided new data for the structural modeling of these transitions.
Structural data for a number of viral envelope proteins are available in the Protein Data Bank (PDB) [6
]. Many of these known structures correspond to envelope proteins in the pre-fusion state, and some of the fusion-state structures only correspond to a partial molecule (usually in a low-pH environment). To date, there are no structures for complete viral envelope proteins in the fusion-initiation state; understanding the mechanics of the conformational change from the pre-fusion to the fusion-initiation state requires such a description.
Directly determining fusion-state structures for complete viral envelope proteins by experimental methods is difficult; molecular modeling offers a readily applicable alternative means to structural characterization. We describe the use of a knowledge-based methodology (homology modeling) to develop structures of viral envelope proteins in the fusion-initiation state. We further extend the basic idea of homology modeling to include a simple concept, “proteins and protein domains that fold similarly interact similarly”, as a result, developing structural models of envelope protein–antibody complexes. In this work we focus on envelope proteins from Ebola and Zika viruses. Ebola virus causes Ebola hemorrhagic fever, a severe and highly lethal infection: the 2013–2015 West African Ebola virus epidemic (December 2013–2015) resulted in approximately 11,000 confirmed deaths and 28,000 suspected cases [10
]. Zika virus is a member of the virus family Flaviviridae [11
] that includes the Dengue virus (DENV) and West Nile virus; in contrast to the above-mentioned Ebola virus, Zika virus causes a brief, relatively mild illness, but it has been linked to congenital microcephaly and Guillan–Barré Syndrome in humans [13
]. In mouse models, Zika virus causes microcephaly [15
], as well as damage to the male reproductive system [16
] and to adult neural stem cells [17
]. At the time of publication, 84 countries, territories, and subnational areas reported Zika transmission [18
]. The Ebola and Zika viruses represent persistent threats to public health; there are limited options available for the treatment or prevention of either virus. In this paper, we present the following models for Ebola virus glycoprotein (EBOV GP) and Zika virus envelope protein (ZIKV E):
A trimer model of EBOV GP in the fusion-initiation state with the Niemann–Pick C1 (NPC1) receptor and neutralizing antibodies.
A trimer model of ZIKV E in the fusion-initiation state with neutralizing antibodies and the surrounding 9-mer structure of ZIKV E in the pre-fusion state with neutralizing antibodies.
Our modeling approach is general and comprehensive and can be used for developing structures of other pathogen proteins in their functional states for understanding their functions; the developed structure-based knowledge can further add to sequence-based information and improve vaccine design for viruses and other pathogens. Importantly, all of the developed models are testable experimentally, potentially leading to the discovery of new targets for drugs and vaccines and the optimization of known vaccine and drug targets.
Ebola virus has a small (∼18–19 kb) negative-stranded RNA genome [19
] that encodes eight viral proteins. Envelope glycoprotein (GP) is the viral surface protein responsible for host cell entry [20
], and it has a sequence of 676 residues in all known Ebola strains. The N-terminus (residues 1–32) forms a signal peptide, and the remaining protein residues (33–676) are collectively referred to as the GP portion. The host endoprotease furin cleaves GP into two segments: GP1 (residues 33–501) and GP2 (residues 502–676) [22
]. GP1 is responsible for receptor binding to the host cell [23
], while GP2 acts as a class I viral fusion protein [25
]. Several functional domains of EBOV GP are denoted in Figure 1
. Like influenza and HIV envelope proteins, EBOV GP is a homotrimer in its functional state [26
Since the 2015 epidemic, significantly more Ebola virus sequence information has become available, including sequences of GP (more than 1000 unique EBOV GP sequences can be found in the current NCBI Ebola virus database) [27
]. Significant efforts have been devoted towards solving the GP structure using various experimental approaches, including X-ray crystallography [26
], NMR [31
], and electron microscopy [33
]. Through this work, domain structures have been solved in different functional states, and as has the trimer structure of the EBOV GP mucin-like region deletion mutant (GPmuc) in the pre-fusion state [26
]. All of these pieces of information contribute to a basis for developing a structural model of the GP in the fusion-initiation state. Zika virus possesses a non-segmented, single-stranded, positive-sense RNA genome (10 kb) [12
]. The urgency in finding ways to combat the virus has increased significantly since a causal relation between Zika virus and the apoptosis of human neurons was discovered [13
]. For EBOV, the envelope protein (GP) forms homotrimers that are distributed sparsely on the surface of the virus, as in influenza and HIV [26
]. By contrast, ZIKV E’s form homodimers and cover the entire viral surface (the capsid comprises 90 homodimers, arranged as 30 rhombic faces with 3 dimers each) [12
]. However, the partial structure of ZIKV E in the fusion-initiation state (at low pH) shows a trimer arrangement [36
]. Determining how ZIKV E transforms from a pre-fusion dimer to a fusion-state trimer presents a considerable challenge to the structural modeling community. Although ZIKV E and EBOV GP are phylogenetically distinct viruses, their fusion subunits both adopt a trimer structure; in this study, we investigate some of the similarities, differences, and consequences of these fusion-state structures.
Viral cell entry is a complex process involving multiple molecular participants and conformational changes. We provide a plausible mechanism in atomic detail for the pre-fusion-to-fusion-state transition for both the Ebola and Zika viruses. While these two viruses are very different, their fusion proteins share common features in the initiation of cell entry. The most apparent common feature is a trimer arrangement of the proteins in the fusion-initiation state. This can be seen in other viruses, such as influenza and HIV. Secondly, these fusion proteins undergo large conformational changes to transition from the pre-fusion- to fusion-initiation-state structures. To prevent viral cell entry, antibodies can act on the fusion protein using two different mechanisms: (1) preventing the structural transition, and (2) blocking fusion peptide access to the cell membrane.
With the known structures of these proteins in the pre-fusion state and partial structures of the proteins in the fusion-initiation state (Figure A1
), we developed near-complete fusion-state structural models of EBOV GP and ZIKV E. Using a spring-loaded mechanism proposed for influenza hemagglutinin (HA) structural transition, the fusion-initiation-state trimer structure of EBOV GP was developed. The fusion-initiation-state trimer structure of ZIKV E was developed from three dimers on the surface of the virus by utilizing the 3-fold symmetry.
Combining the information of EBOV GP/ZIKV E structures in both the pre-fusion and the fusion-initiation states, as well as the structures of these proteins in complexes with different neutralizing antibodies, we investigated the mechanisms used by the antibodies to prevent the process of viral cell entry. We found that KZ52 (Ebola) and EDE1-8 (Zika) prevented cell entry by blocking the pre-fusion-to-fusion structural transition. We propose that the connecting bridge in the GP1 trimer (S90–P93 and P126–R130) serves as a gate for the pre-fusion-to-fusion structural transition: if this gate is locked, the structural transition step is inhibited, and the viral life cycle is halted. We also demonstrated that the antibody 2A0G6 binds to ZIKV E in the fusion-initiation state and possibly prevents the fusion peptide from reaching the cell membrane. The antibody mAb100 is able to bind EBOV GP in both the pre-fusion and the fusion-initiation states, preventing viral cell entry by both inhibiting the structural transition and blocking fusion peptide access to the cell membrane.
With the collective efforts to expand the information base in both biological sequences and structures, GenBank and PDB provide invaluable information. This information enables the expansion of structural modeling to a new realm that includes proteins in higher-order structures and/or transitions. Using EBOV GP and ZIKV E as examples, we have demonstrated that a conceptually straightforward, knowledge-based approach allows us to develop models of the fusion-initiation-state structures for both proteins in their functional units. With this information, we were able to study the binding of neutralizing antibodies to these proteins and propose the exact mechanisms for how these antibodies block cell entry to stop viral replication.