Cryo-EM structures of alphavirus conformational intermediates in low pH-triggered prefusion states

Alphaviruses can cause severe human arthritis and encephalitis. During virus infection, structural changes of viral glycoproteins in the acidified endosome trigger virus-host membrane fusion for delivery of the capsid core and RNA genome into the cytosol to initiate virus translation and replication. However, mechanisms by which E1 and E2 glycoproteins rearrange in this process remain unknown. Here, we investigate prefusion cryoelectron microscopy (cryo-EM) structures of eastern equine encephalitis virus (EEEV) under acidic conditions. With models fitted into the low-pH cryo-EM maps, we suggest that E2 dissociates from E1, accompanied by a rotation (∼60°) of the E2-B domain (E2-B) to expose E1 fusion loops. Cryo-EM reconstructions of EEEV bound to a protective antibody at acidic and neutral pH suggest that stabilization of E2-B prevents dissociation of E2 from E1. These findings reveal conformational changes of the glycoprotein spikes in the acidified host endosome. Stabilization of E2-B may provide a strategy for antiviral agent development

Cryo-EM structures of alphavirus conformational intermediates in low pH-triggered prefusion states

Alphaviruses can cause severe human arthritis and encephalitis. During virus infection, structural changes of viral glycoproteins in the acidified endosome trigger virus-host membrane fusion for delivery of the capsid core and RNA genome into the cytosol to initiate virus translation and replication. However, mechanisms by which E1 and E2 glycoproteins rearrange in this process remain unknown. Here, we investigate prefusion cryoelectron microscopy (cryo-EM) structures of eastern equine encephalitis virus (EEEV) under acidic conditions. With models fitted into the low-pH cryo-EM maps, we suggest that E2 dissociates from E1, accompanied by a rotation (∼60°) of the E2-B domain (E2-B) to expose E1 fusion loops. Cryo-EM reconstructions of EEEV bound to a protective antibody at acidic and neutral pH suggest that stabilization of E2-B prevents dissociation of E2 from E1. These findings reveal conformational changes of the glycoprotein spikes in the acidified host endosome. Stabilization of E2-B may provide a strategy for antiviral agent development

Cryo-EM Structures of Eastern Equine Encephalitis Virus Reveal Mechanisms of Virus Disassembly and Antibody Neutralization

Alphaviruses are enveloped pathogens that cause arthritis and encephalitis. Here, we report a 4.4-Å cryoelectron microscopy (cryo-EM) structure of eastern equine encephalitis virus (EEEV), an alphavirus that causes fatal encephalitis in humans. Our analysis provides insights into viral entry into host cells. The envelope protein E2 showed a binding site for the cellular attachment factor heparan sulfate. The presence of a cryptic E2 glycan suggests how EEEV escapes surveillance by lectin-expressing myeloid lineage cells, which are sentinels of the immune system. A mechanism for nucleocapsid core release and disassembly upon viral entry was inferred based on pH changes and capsid dissociation from envelope proteins. The EEEV capsid structure showed a viral RNA genome binding site adjacent to a ribosome binding site for viral genome translation following genome release. Using five Fab-EEEV complexes derived from neutralizing antibodies, our investigation provides insights into EEEV host cell interactions and protective epitopes relevant to vaccine design

Genetic and Biochemical Characterization Reveals the Substrate Specificity of the Three-Member Azaguanine-like Transporter Family of Zea mays

Nucleobases, purines and pyrimidines, along with their precursors are central to nucleic acid biochemistry, a process essential for all forms of life. Not only do they play a role in metabolism of phospholipids, carbohydrates and glycoproteins, they have a pronounced role in synthesis of secondary metabolites in addition to serving as energy source. In maize (Zea mays), ZmAZG (azaguanine-like transporter family), one of the several nucleobase transporters, plays a major role in ensuring the movement of nucleobases between and within cells and between the organelles and cytoplasm. With an aim to characterize these transporters in Zea mays, this research was conducted. The study focused on identifying the substrate specificity and binding properties of the three putative members of the AZG-like family of Zea mays; ZmAZG1, ZmAZG2 and ZmAZG3. Transgenic yeast cells, deficient in their native nucleobase transporter, were assayed for their ability to uptake a panel of radiolabeled nucleobases to reveal the transport profiles of the heterologously-expressed ZmAZG transporters. The results showed that ZmAZG1 facilitates the transport of uracil, xanthine and hypoxanthine whereas ZmAZG2 and ZMAZG3 transport adenine, guanine, cytosine, hypoxanthine and xanthine. The kinetic properties of AZG-like transporters were further revealed using homologous competition between radiolabeled nucleobase and varying concentrations of same cold competitor, heterologous competition of radiolabeled nucleobase against a concentration series of various cold nucleobases, and heterologous competition between radiolabeled hypoxanthine as a substrate and an array of non-radioactive cold nucleobase competitors. Effects of protonophores and Na+ pump inhibitors on the function of the ZmAZG transporters revealed that they are proton-driven symporters. Moreover, all three ZmAZG transporters continually transported [3H]-hypoxanthine over a period of two hours without reaching a saturation point suggesting their pivotal role in hypoxanthine transport. Additionally, ZmAZG2 complemented the missing azaguanine-like transporter function in a transgenic homozygous double knock-out Arabidopsis mutant Atazg1-1/Atazg1-1, Atazg2-1/Atazg2-1 providing in planta confirmation of being a proton-driven symporter. These findings shed light on the significant role the AZG-like transporters play in maize growth and development

Molecular basis for the acid-initiated uncoating of human enterovirus D68

Enterovirus D68 (EV-D68) belongs to a group of enteroviruses that contain a single positive-sense RNA genome surrounded by an icosahedral capsid. Like common cold viruses, EV-D68 mainly causes respiratory infections and is acid-labile. The molecular mechanism by which the acid-sensitive EV-D68 virions uncoat and deliver their genome into a host cell is unknown. Using cryoelectron microscopy (cryo-EM), we have determined the structures of the full native virion and an uncoating intermediate [the A (altered) particle] of EV-D68 at 2.2- and 2.7-Å resolution, respectively. These structures showed that acid treatment of EV-D68 leads to particle expansion, externalization of the viral protein VP1 N termini from the capsid interior, and formation of pores around the icosahedral twofold axes through which the viral RNA can exit. Moreover, because of the low stability of EV-D68, cryo-EM analyses of a mixed population of particles at neutral pH and following acid treatment demonstrated the involvement of multiple structural intermediates during virus uncoating. Among these, a previously undescribed state, the expanded 1 ("E1") particle, shows a majority of internal regions (e.g., the VP1 N termini) to be ordered as in the full native virion. Thus, the E1 particle acts as an intermediate in the transition from full native virions to A particles. Together, the present work delineates the pathway of EV-D68 uncoating and provides the molecular basis for the acid lability of EV-D68 and of the related common cold viruses

Structural Basis of Zika Virus Specific Neutralization in Subsequent Flavivirus Infections

Zika virus (ZIKV), a mosquito-borne human flavivirus that causes microcephaly and other neurological disorders, has been a recent focus for the development of flavivirus vaccines and therapeutics. We report here a 4.0 Å resolution structure of the mature ZIKV in complex with ADI-30056, a ZIKV-specific human monoclonal antibody (hMAb) isolated from a ZIKV infected donor with a prior dengue virus infection. The structure shows that the hMAb interactions span across the E protein dimers on the virus surface, inhibiting conformational changes required for the formation of infectious fusogenic trimers similar to the hMAb, ZIKV-117. Structure-based functional analysis, and structure and sequence comparisons, identified ZIKV residues essential for neutralization and crucial for the evolution of highly potent E protein crosslinking Abs in ZIKV. Thus, this epitope, ZIKV’s “Achilles heel”, defined by the contacts between ZIKV and ADI-30056, could be a suitable target for the design of therapeutic antibodies