Structural basis for VLDLR recognition by eastern equine encephalitis virus.

Eastern equine encephalitis virus (EEEV) is the most virulent alphavirus that infects humans, and many survivors develop neurological sequelae, including paralysis and intellectual disability. Alphavirus spike proteins comprise trimers of heterodimers of glycoproteins E2 and E1 that mediate binding to cellular receptors and fusion of virus and host cell membranes during entry. We recently identified very-low density lipoprotein receptor (VLDLR) and apolipoprotein E receptor 2 (ApoER2) as cellular receptors for EEEV and a distantly related alphavirus, Semliki Forest virus (SFV). Here, we use single-particle cryo-electron microscopy (cryo-EM) to determine structures of the EEEV and SFV spike glycoproteins bound to the VLDLR ligand-binding domain and found that EEEV and SFV interact with the same cellular receptor through divergent binding modes. Our studies suggest that the ability of LDLR-related proteins to interact with viral spike proteins through very small footprints with flexible binding modes results in a low evolutionary barrier to the acquisition of LDLR-related proteins as cellular receptors for diverse sets of viruses.

Shifts in receptors during submergence of an encephalitic arbovirus.

Western equine encephalitis virus (WEEV) is an arthropod-borne virus (arbovirus) that frequently caused major outbreaks of encephalitis in humans and horses in the early twentieth century, but the frequency of outbreaks has since decreased markedly, and strains of this alphavirus isolated in the past two decades are less virulent in mammals than strains isolated in the 1930s and 1940s1-3. The basis for this phenotypic change in WEEV strains and coincident decrease in epizootic activity (known as viral submergence3) is unclear, as is the possibility of re-emergence of highly virulent strains. Here we identify protocadherin 10 (PCDH10) as a cellular receptor for WEEV. We show that multiple highly virulent ancestral WEEV strains isolated in the 1930s and 1940s, in addition to binding human PCDH10, could also bind very low-density lipoprotein receptor (VLDLR) and apolipoprotein E receptor 2 (ApoER2), which are recognized by another encephalitic alphavirus as receptors4. However, whereas most of the WEEV strains that we examined bind to PCDH10, a contemporary strain has lost the ability to recognize mammalian PCDH10 while retaining the ability to bind avian receptors, suggesting WEEV adaptation to a main reservoir host during enzootic circulation. PCDH10 supports WEEV E2-E1 glycoprotein-mediated infection of primary mouse cortical neurons, and administration of a soluble form of PCDH10 protects mice from lethal WEEV challenge. Our results have implications for the development of medical countermeasures and for risk assessment for re-emerging WEEV strains.

Structural basis for VLDLR recognition by eastern equine encephalitis virus.

Alphaviruses are arthropod-borne enveloped RNA viruses that include several important human pathogens with outbreak potential. Among them, eastern equine encephalitis virus (EEEV) is the most virulent, and many survivors develop neurological sequelae, including paralysis and intellectual disability. The spike proteins of alphaviruses comprise trimers of heterodimers of their envelope glycoproteins E2 and E1 that mediate binding to cellular receptors and fusion of virus and host cell membranes during entry. We recently identified very-low density lipoprotein receptor (VLDLR) and apolipoprotein E receptor 2 (ApoER2), two closely related proteins that are expressed in the brain, as cellular receptors for EEEV and a distantly related alphavirus, Semliki forest virus (SFV) 1 . The EEEV and SFV spike glycoproteins have low sequence homology, and how they have evolved to bind the same cellular receptors is unknown. Here, we used single-particle cryo-electron microscopy (cryo-EM) to determine structures of the EEEV and SFV spike glycoproteins bound to the VLDLR ligand-binding domain. The structures reveal that EEEV and SFV use distinct surfaces to bind VLDLR; EEEV uses a cluster of basic residues on the E2 subunit of its spike glycoprotein, while SFV uses two basic residues at a remote site on its E1 glycoprotein. Our studies reveal that different alphaviruses interact with the same cellular receptor through divergent binding modes. They further suggest that the ability of LDLR-related proteins to interact with viral spike proteins through very small footprints with flexible binding modes results in a low evolutionary barrier to the acquisition of LDLR-related proteins as cellular receptors for diverse sets of viruses.

Protonation and Non-Innocent Ligand Behavior in Pyranopterin Dithiolene Molybdenum Complexes.

The complex [TEA][Tp*MoIV(O)(S2BMOPP)] (1) [TEA = tetraethylammonium, Tp* = tris(3,5-dimethylpyrazolyl)hydroborate, and BMOPP = 6-(3-butynyl-2-methyl-2-ol)-2-pivaloyl pterin] is a structural analogue of the molybdenum cofactor common to all pyranopterin molybdenum enzymes because it possesses a pyranopterin-ene-1,2-dithiolate ligand (S2BMOPP) that exists primarily in the ring-closed pyrano structure as a resonance hybrid of ene-dithiolate and thione-thiolate forms. Compound 1, the protonated [Tp*MoIV(O)(S2BMOPP-H)] (1-H) and one-electron-oxidized [Tp*MoV(O)(S2BMOPP)] [1-Mo(5+)] species have been studied using a combination of electrochemistry, electronic absorption, and electron paramagnetic resonance (EPR) spectroscopy. Additional insight into the nature of these molecules has been derived from electronic structure computations. Differences in dithiolene C-S bond lengths correlate with relative contributions from both ene-dithiolate and thione-thiolate resonance structures. Upon protonation of 1 to form 1-H, large spectroscopic changes are observed with transitions assigned as Mo(xy) → pyranopterin metal-to-ligand charge transfer and dithiolene → pyranopterin intraligand charge transfer, respectively, and this underscores a dramatic change in electronic structure between 1 and 1-H. The changes in electronic structure that occur upon protonation of 1 are also reflected in a large >300 mV increase in the Mo(V/IV) redox potential for 1-H, resulting from the greater thione-thiolate resonance contribution and decreased charge donation that stabilize the Mo(IV) state in 1-H with respect to one-electron oxidation. EPR spin Hamiltonian parameters for one-electron-oxidized 1-Mo(5+) and uncyclized [Tp*MoV(O)(S2BDMPP)] [3-Mo(5+)] [BDMPP = 6-(3-butynyl-2,2-dimethyl)-2-pivaloyl pterin] are very similar to each other and to those of [Tp*MoVO(bdt)] (bdt = 1,2-ene-dithiolate). This indicates that the dithiolate form of the ligand dominates at the Mo(V) level, consistent with the demand for greater S → Mo charge donation and a corresponding increase in Mo-S covalency as the oxidation state of the metal is increased. Protonation of 1 represents a simple reaction that models how the transfer of a proton from neighboring acidic amino acid residues to the Mo cofactor at a nitrogen atom within the pyranopterin dithiolene (PDT) ligand in pyranopterin molybdenum enzymes can impact the electronic structure of the Mo-PDT unit. This work also illustrates how pyran ring-chain tautomerization drives changes in resonance contributions to the dithiolene chelate and may adjust the reduction potential of the Mo ion.

Structural basis for continued antibody evasion by the SARS-CoV-2 receptor binding domain.

Many studies have examined the impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants on neutralizing antibody activity after they have become dominant strains. Here, we evaluate the consequences of further viral evolution. We demonstrate mechanisms through which the SARS-CoV-2 receptor binding domain (RBD) can tolerate large numbers of simultaneous antibody escape mutations and show that pseudotypes containing up to seven mutations, as opposed to the one to three found in previously studied variants of concern, are more resistant to neutralization by therapeutic antibodies and serum from vaccine recipients. We identify an antibody that binds the RBD core to neutralize pseudotypes for all tested variants but show that the RBD can acquire an N-linked glycan to escape neutralization. Our findings portend continued emergence of escape variants as SARS-CoV-2 adapts to humans.