Voltage-gated sodium (Nav) channels are essential for the rapid depolarization of nerve and muscle1, and are important drug targets2. A family of bacterial Nav channels, exemplified by NaChBac (Na+-selective Channel of Bacteria)3, provides a good model system for structure-function analysis. Here we report the crystal structure of NavAP, a NaChBac orthologue from marine bacteria alpha proteobacterium HIMB114, at 3.05 Å resolution. The channel comprises an asymmetric tetramer. The carbonyl oxygen atoms of Thr178 and Leu179 constitute an inner site within the selectivity filter (178TLSSWE183) where a Ca2+ can bind and resides in the crystal structure. The outer mouth of the Na+ selectivity filter, defined by Ser181 and Glu183, is closed, as is the activation gate at the intracellular side of the pore. The voltage sensors adopt a depolarized conformation with all the gating charges exposing to the extracellular side. We hypothesize that NavAP is captured in an inactivated conformation. Comparison of NavAP with NavAb4 reveals significant conformational rearrangements that may underlie the electromechanical coupling mechanism of voltage-gated channels.

The major facilitator superfamily glucose transporters, exemplified by human GLUT1-4, have been central to the study of solute transport. Using lipidic cubic phase crystallization and microfocus X-ray diffraction, we determined the structure of human GLUT3 in complex with D-glucose at 1.5 Å resolution in an outward-occluded conformation. The high-resolution structure allows discrimination of both α- and β-anomers of D-glucose. Two additional structures of GLUT3 bound to the exofacial inhibitor maltose were obtained at 2.6 Å in the outward-open and 2.4 Å in the outward-occluded states. In all three structures, the ligands are predominantly coordinated by polar residues from the carboxy terminal domain. Conformational transition from outward-open to outward-occluded entails a prominent local rearrangement of the extracellular part of transmembrane segment TM7. Comparison of the outward-facing GLUT3 structures with the inward-open GLUT1 provides insights into the alternating access cycle for GLUTs, whereby the C-terminal domain provides the primary substrate-binding site and the amino-terminal domain undergoes rigid-body rotation with respect to the C-terminal domain. Our studies provide an important framework for the mechanistic and kinetic understanding of GLUTs and shed light on structure-guided ligand design.

SARS-CoV-2 variants continue to emerge during the global pandemic and may facilitate escape from current antibody therapies and vaccine protection. Here, we showed that the South African variant B.1.351 was the most resistant to current monoclonal antibodies and convalescent plasma from COVID-19-infected individuals, followed by the Brazilian variant P.1 and the UK variant B.1.1.7. This resistance hierarchy corresponded with Y144del and 242-244del mutations in the N-terminal domain and K417N/T, E484K and N501Y mutations in the receptor binding domain (RBD) of SARS-CoV-2. Crystal structural analysis of B.1.351 triple mutant (417N-484K-501Y) RBD complexed with monoclonal antibody P2C-1F11 revealed the molecular basis for antibody neutralization and escape. B.1.351 and P.1 also acquired the ability to use mouse and mink ACE2 receptor for entry. Our results demonstrate major antigenic shifts and potential broadening of the host range for B.1.351 and P.1 variants, which pose serious challenges to our current antibody therapies and vaccine protection.

The pandemic of COVID-19, caused by SARS-CoV-2, is a major global health threat. Epidemiological studies suggest that bats (Rhinolophus affinis) are the natural zoonotic reservoir for SARS-CoV-2. However, the host range of SARS-CoV-2 and intermediate hosts that facilitate its transmission to humans remain unknown. The interaction of coronavirus with its host receptor is a key genetic determinant of host range and cross-species transmission. SARS-CoV-2 uses angiotensin-converting enzyme 2 (ACE2) as the receptor to enter host cells in a species-dependent manner. In this study, we characterized the ability of ACE2 from diverse species to support viral entry. By analyzing the conservation of five residues in two virus-binding hotspots of ACE2 (hotspot 31Lys and hotspot 353Lys), we predicted 80 ACE2 proteins from mammals that could potentially mediate SARS-CoV-2 entry. We chose 48 ACE2 orthologs among them for functional analysis, and showed that 44 of these orthologs—including domestic animals, pets, livestock, and animals commonly found in zoos and aquaria—could bind the SARS-CoV-2 spike protein and support viral entry. In contrast, New World monkey ACE2 orthologs could not bind the SARS-CoV-2 spike protein and support viral entry. We further identified the genetic determinant of New World monkey ACE2 that restricts viral entry using genetic and functional analyses. These findings highlight a potentially broad host tropism of SARS-CoV-2 and suggest that SARS-CoV-2 might be distributed much more widely than previously recognized, underscoring the necessity to monitor susceptible hosts to prevent future outbreaks.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes the global pandemic of COVID-19. SARS-CoV-2 is classified as a biosafety level-3 (BSL-3) agent, impeding the basic research into its biology and the development of effective antivirals. Here, we developed a biosafety level-2 (BSL-2) cell culture system for production of transcription and replication-competent SARS-CoV-2 virus-like-particles (trVLP). This trVLP expresses a reporter gene (GFP) replacing viral nucleocapsid gene (N), which is required for viral genome packaging and virion assembly (SARS-CoV-2 GFP/ΔN trVLP). The complete viral life cycle can be achieved and exclusively confined in the cells ectopically expressing SARS-CoV or SARS-CoV-2 N proteins, but not MERS-CoV N. Genetic recombination of N supplied in trans into viral genome was not detected, as evidenced by sequence analysis after one-month serial passages in the N-expressing cells. Moreover, intein-mediated protein trans-splicing approach was utilized to split the viral N gene into two independent vectors, and the ligated viral N protein could function in trans to recapitulate entire viral life cycle, further securing the biosafety of this cell culture model. Based on this BSL-2 SARS-CoV-2 cell culture model, we developed a 96-well format high throughput screening for antivirals discovery. We identified salinomycin, tubeimoside I, monensin sodium, lycorine chloride and nigericin sodium as potent antivirals against SARS-CoV-2 infection. Collectively, we developed a convenient and efficient SARS-CoV-2 reverse genetics tool to dissect the virus life cycle under a BSL-2 condition. This powerful tool should accelerate our understanding of SARS-CoV-2 biology and its antiviral development.

The pandemic of a newly emerging coronavirus (SARS-CoV-2), the causative agent of severe pneumonia disease , is a major global health threat. Epidemiological studies suggest that bats are the natural zoonotic reservoir for SARS-CoV-2, however, the host range of SARS-CoV-2 and the identity of intermediate hosts that may facilitate the transmission to humans remains unknown. Coronavirus-receptor interaction is a key genetic determinant of the host range, cross-species transmission, and tissue tropism. SARS-CoV-2 uses Angiotensin-converting enzyme II (ACE2) as the receptor to enter its host cells in a species-dependent manner. It has been shown that human, palm civet, pig and bat ACE2 can support virus entry, while the murine ortholog cannot. In this study, we aimed to characterize ACE2 from diverse species for its ability to support viral entry. We found that ACE2 is expressed in a wide range of host species, with high conservation especially in mammals. By analyzing critical amino acid residues in ACE2 for virus entry, based on the well-characterized SARS-CoV spike protein interaction with ACE2 (human, bat, palm civet, pig and ferret ACE2), we identified approximately eighty ACE2 proteins from mammals could potentially function as the receptor to mediate SARS-CoV-2 entry. Functional assays showed that 44 of these mammalian ACE2 orthologs, including domestic animals, pet animals, livestock animals and even animals in the zoos or aquaria, could bind viral spike protein and support SARS-CoV-2 entry. In summary, our study demonstrates that ACE2 from a remarkably broad range of species support SARS-CoV-2 entry. These findings highlight a potentially broad host tropism and suggest that SARS-CoV-2 might be distributed much more widely than previously recognized, emphasizing the necessity to monitor the susceptible hosts, especially their potential of cross-species, which could prevent the future outbreaks.

A highly specific and very sensitive serological SARS-CoV-2 antibody assay with overall accuracy at 97.3% was developed using CHO-expressed SARS-CoV-2 S1 protein for screening medical staff and others for SARS-CoV-2 infection. AbstractBackground: Thousands of medical staff had been infected with SARS-CoV-2 virus with hundreds of deaths reported. Such loss could be prevented if there is a serologic assay for SARS-CoV-2-specific antibodies for serological surveillance of its infection at the early stage of disease.Methods: Using CHO cell expressed full length SARS-CoV-2 S1 protein as capturing antigen, a COVID-19/SARS-CoV-2 S1 serology ELISA kit was developed and validated with negative samples collected prior to the outbreaks or during the outbreak, and positive samples from patients confirmed with COVID-19. Results:The specificity of the ELISA kit was 97.5%, as examined against total 412 normal human samples. The sensitivity was 97.1% by testing against 69 samples from hospitalized and/or recovered COVID-19 patients. The overall accuracy rate reached 97.3%. The assay was able to detect SARS-CoV-2 antibody on day one after the onset of COVID-19 disease. The average antibody levels increased during the hospitalization and after been discharged for two weeks. SARS-CoV-2 antibodies were detected in 28 out of 276 asymptomatic medical staff and one out of five nucleic acid test-negative "Close contacts" of COVID-19 patient. Conclusion:With the assays developed here, we can screen medical staff, in-coming patients, passengers and people who are in close contact with the confirmed patients to identify the "innocent viral spreaders", protect the medical staff and stop the further spreading of the virus.

NaChBac is a bacterial voltage-gated sodium (Na v ) channel that shows sequence similarity to voltage-gated calcium channels. To understand the ion-permeation mechanism of Na v channels, we combined molecular dynamics simulation, structural biology and electrophysiological approaches to investigate the recently determined structure of Na v Rh, a marine bacterial NaChBac ortholog. Two Na + binding sites are identified in the selectivity filter (SF) in our simulations: The extracellular Na + ion first approaches site 1 constituted by the side groups of Ser181 and Glu183, and then spontaneously arrives at the energetically more favorable site 2 formed by the carbonyl oxygens of Leu179 and Thr178. In contrast, Ca 2+ ions are prone to being trapped by Glu183 at site 1, which then blocks the entrance of both Na + and Ca 2+ to the vestibule of the SF. In addition, Na + permeates through the selective filter in an asymmetrical manner, a feature that resembles that of the mammalian Na v orthologs. The study reported here provides insights into the mechanism of ion selectivity on Na + over Ca 2+ in mammalian Na v channels.