7v0o | pdb_00007v0o
Cryo-EM structure of SINV/EEEV in complex with Fab fragment of a moderately/weakly neutralizing human antibody IgG-94
Structural highlights
FunctionPOLS_EEEVF Forms an icosahedral capsid with a T=4 symmetry composed of 240 copies of the capsid protein surrounded by a lipid membrane through which penetrate 80 spikes composed of trimers of E1-E2 heterodimers (By similarity). The capsid protein binds to the viral RNA genome at a site adjacent to a ribosome binding site for viral genome translation following genome release (By similarity). Possesses a protease activity that results in its autocatalytic cleavage from the nascent structural protein (By similarity). Following its self-cleavage, the capsid protein transiently associates with ribosomes, and within several minutes the protein binds to viral RNA and rapidly assembles into icosahedric core particles (By similarity). The resulting nucleocapsid eventually associates with the cytoplasmic domain of the spike glycoprotein E2 at the cell membrane, leading to budding and formation of mature virions (By similarity). In case of infection, new virions attach to target cells and after clathrin-mediated endocytosis their membrane fuses with the host endosomal membrane (By similarity). This leads to the release of the nucleocapsid into the cytoplasm, followed by an uncoating event necessary for the genomic RNA to become accessible (By similarity). The uncoating might be triggered by the interaction of capsid proteins with ribosomes (By similarity). Binding of ribosomes would release the genomic RNA since the same region is genomic RNA-binding and ribosome-binding (By similarity). Specifically inhibits interleukin-1 receptor-associated kinase 1/IRAK1-dependent signaling during viral entry, representing a means by which the alphaviruses may evade innate immune detection and activation prior to viral gene expression (By similarity). Inhibits host transcription (By similarity). Forms a tetrameric complex with XPO1/CRM1 and the nuclear import receptor importin (By similarity). This complex blocks the central channel of host nuclear pores thereby inhibiting the receptor-mediated nuclear transport and thus the host mRNA and rRNA transcription (By similarity). The inhibition of transcription is linked to a cytopathic effect on the host cell (By similarity).[UniProtKB:P03315][UniProtKB:P03316][UniProtKB:P09592][UniProtKB:P27284][UniProtKB:P36329] Provides the signal sequence for the translocation of the precursor of protein E3/E2 to the host endoplasmic reticulum. Furin-cleaved E3 remains associated with spike glycoprotein E1 and mediates pH protection of the latter during the transport via the secretory pathway. After virion release from the host cell, the assembly protein E3 is gradually released in the extracellular space.[UniProtKB:P03315] Plays a role in viral attachment to target host cell, by binding to the cell receptor. Synthesized as a p62 precursor which is processed by furin at the cell membrane just before virion budding, giving rise to E2-E1 heterodimer. The p62-E1 heterodimer is stable, whereas E2-E1 is unstable and dissociate at low pH. p62 is processed at the last step, presumably to avoid E1 fusion activation before its final export to cell surface. E2 C-terminus contains a transitory transmembrane that would be disrupted by palmitoylation, resulting in reorientation of the C-terminal tail from lumenal to cytoplasmic side. This step is critical since E2 C-terminus is involved in budding by interacting with capsid proteins. This release of E2 C-terminus in cytoplasm occurs lately in protein export, and precludes premature assembly of particles at the endoplasmic reticulum membrane.[UniProtKB:P03315] Constitutive membrane protein involved in virus glycoprotein processing, cell permeabilization, and the budding of viral particles. Disrupts the calcium homeostasis of the cell, probably at the endoplasmic reticulum level. This leads to cytoplasmic calcium elevation. Because of its lipophilic properties, the 6K protein is postulated to influence the selection of lipids that interact with the transmembrane domains of the glycoproteins, which, in turn, affects the deformability of the bilayer required for the extreme curvature that occurs as budding proceeds. Present in low amount in virions, about 3% compared to viral glycoproteins.[UniProtKB:P03315] Class II viral fusion protein. Fusion activity is inactive as long as E1 is bound to E2 in mature virion. After virus attachment to target cell and endocytosis, acidification of the endosome would induce dissociation of E1/E2 heterodimer and concomitant trimerization of the E1 subunits. This E1 trimer is fusion active, and promotes release of viral nucleocapsid in cytoplasm after endosome and viral membrane fusion. Efficient fusion requires the presence of cholesterol and sphingolipid in the target membrane. Fusion is optimal at levels of about 1 molecule of cholesterol per 2 molecules of phospholipids, and is specific for sterols containing a 3-beta-hydroxyl group.[UniProtKB:P03315] Publication Abstract from PubMedSelection and development of monoclonal antibody (mAb) therapeutics against pathogenic viruses depends on certain functional characteristics. Neutralization potency, or the half-maximal inhibitory concentration (IC(50)) values, is an important characteristic of candidate therapeutic antibodies. Structural insights into the bases of neutralization potency differences between antiviral neutralizing mAbs are lacking. In this report, we present cryo-electron microscopy (EM) reconstructions of three anti-Eastern equine encephalitis virus (EEEV) neutralizing human mAbs targeting overlapping epitopes on the E2 protein, with greater than 20-fold differences in their respective IC(50) values. From our structural and biophysical analyses, we identify several constraints that contribute to the observed differences in the neutralization potencies. Cryo-EM reconstructions of EEEV in complex with these Fab fragments reveal structural constraints that dictate intravirion or intervirion cross-linking of glycoprotein spikes by their IgG counterparts as a mechanism of neutralization. Additionally, we describe critical features for the recognition of EEEV by these mAbs including the epitope-paratope interaction surface, occupancy, and kinetic differences in on-rate for binding to the E2 protein. Each constraint contributes to the extent of EEEV inhibition for blockade of virus entry, fusion, and/or egress. These findings provide structural and biophysical insights into the differences in mechanism and neutralization potencies of these antibodies, which help inform rational design principles for candidate vaccines and therapeutic antibodies for all icosahedral viruses. Structural constraints link differences in neutralization potency of human anti-Eastern equine encephalitis virus monoclonal antibodies.,Williamson LE, Bandyopadhyay A, Bailey K, Sirohi D, Klose T, Julander JG, Kuhn RJ, Crowe JE Jr Proc Natl Acad Sci U S A. 2023 Mar 28;120(13):e2213690120. doi: , 10.1073/pnas.2213690120. Epub 2023 Mar 24. PMID:36961925[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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