Structural basis for the assembly of the DNA polymerase holoenzyme from a monkeypox virus variant (preprint)

The ongoing pandemic caused by a monkeypox virus (MPXV) variant has spread all over the world and raised great public health concerns. The DNA polymerase F8 of MPXV, associated with its processivity factors A22 and E4, is responsible for viral genome replication in the perinuclear sites of the infected cells as well as a critical target for developing antiviral drugs. However, the assembly and working mechanism for the DNA polymerase holoenzyme of MPXV remains elusive. Here, we present the cryo-EM structure of the DNA polymerase holoenzyme F8/A22/E4 from the 2022 West African strain at an overall resolution of 3.5 angstrom and revealed the precise spatial arrangement. Surprisingly, unlike any other previously reported B-family DNA polymerase, the holoenzyme complex is assembled as a dimer of heterotrimers, of which the extra interface between the thumb domain of F8 and A22 shows a clash between A22 and substrate DNA , suggesting an auto-inhibition state. Supplying an exogenous double-stranded DNA could notably shift the hexameric form into a trimeric form, which exposes the DNA binding site of thumb domain and might represent a more active state. The structures provide a molecular basis for the design of new antiviral therapeutics that target the MPXV DNA polymerase holoenzyme.

Structural basis for the enhanced infectivity and immune evasion of Omicron subvariants (preprint)

The Omicron variants of SARS-CoV-2 have recently become the globally dominant variants of concern in the COVID-19 pandemic. At least five major Omicron sub-lineages have been characterized: BA.1, BA.2, BA.3, BA.4 and BA.5. They all possess over 30 mutations on the Spike (S) protein. Here we report the cryo-EM structures of the trimeric S proteins from the five subvariants, of which BA.4 and BA.5 share the same mutations of S protein, each in complex with the surface receptor ACE2. All three receptor binding domains of S protein from BA.2 and BA.4/BA.5 are up, while the BA.1 S protein has two up and one down. The BA.3 S protein displays increased heterogeneity, with the majority in the all up RBD state. The differentially preferred conformations of the S protein are consistent with their varied transmissibilities. Analysis of the well defined S309 and S2K146 epitopes reveals the underlie immune evasion mechanism of Omicron subvariants.

Novel neutralization mechanism of a human antibody with pan-coronavirus reactivity (preprint)

The recurrent outbreak of coronaviruses and variants underscores the need for broadly reactive antivirals and vaccines. Here, a novel broad-spectrum human antibody named 76E1 was isolated from a COVID-19 convalescent patient and showed broad neutralization activity against multiple α- and β-coronaviruses, including the SARS-CoV-2 variants and also exhibited the binding breath to peptides containing the epitope from γ- and δ- coronaviruses. 76E1 cross-protects mice from SARS-CoV-2 and HCoV-OC43 infection in both prophylactic and treatment models. The epitope including the fusion peptide and S2’ cleavage site recognized by 76E1 was significantly conserved among α-, β-, γ- and δ- coronaviruses. We uncovered a novel mechanism of antibody neutralization that the epitope of 76E1 was proportionally less exposed in the prefusion trimeric structure of spike protein but could be unmasked by binding to the receptor ACE2. Once the epitope exposed, 76E1 inhibited S2’ cleavage, thus blocked the membrane fusion process. Our data demonstrate a key epitope targeted by broadly-neutralizing antibodies and will guide next-generation epitope-based pan-coronavirus vaccine design.

ACE2-Targeting Monoclonal Antibody As A "Pan" Coronavirus Blocker In Vitro and In A Mouse Model (preprint)

Coronaviruses have caused three major outbreaks of infectious disease since the beginning of 21st century. Broad-spectrum strategies that can be utilized in both current and future coronavirus outbreaks and mutation-tolerant are sought after. Here we report a monoclonal antibody 3E8 targeting human angiotensin-converting enzyme 2 (ACE2) neutralized pseudo-typed coronaviruse SARS-CoV-2, SARS-CoV-2-D614G, SARS-CoV and HCoV-NL63, without affecting physiological activities of ACE2 or causing toxicity in mouse model. 3E8 also blocked live SARS-CoV-2 infection in vitro and in a mouse model of COVID-19. Cryo-EM studies revealed the binding site of 3E8 on ACE2 and identified Histone 34 of ACE2 as a critical site of anti-viral epitope. Overall, our work has provided a potential pan coronavirus management strategy and disclosed a pan anti-coronavirus epitope on human ACE2 for the first time.

Antibody-dependent enhancement (ADE) of SARS-CoV-2 infection in recovered COVID-19 patients: studies based on cellular and structural biology analysis (preprint)

Antibody-dependent enhancement (ADE) has been reported in several virus infections including dengue fever virus, severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) coronavirus infection. To study whether ADE is involved in COVID-19 infections, in vitro pseudotyped SARS-CoV-2 entry into Raji cells, K562 cells, and primary B cells mediated by plasma from recovered COVID-19 patients were employed as models. The enhancement of SARS-CoV-2 entry into cells was more commonly detected in plasma from severely-affected elderly patients with high titers of SARS-CoV-2 spike protein-specific antibodies. Cellular entry was mediated via the engagement of Fc{gamma}RII receptor through virus-cell membrane fusion, but not by endocytosis. Peptide array scanning analyses showed that antibodies which promote SARS-CoV-2 infection targeted the variable regions of the RBD domain. To further characterize the association between the spike-specific antibody and ADE, an RBD-specific monoclonal antibody (7F3) was isolated from a recovered patient, which potently inhibited SARS-Cov-2 infection of ACE-2 expressing cells and also mediated ADE in Raji cells. Site-directed mutagenesis the spike RBD domain reduced the neutralization activity of 7F3, but did not abolish its binding to the RBD domain. Structural analysis using cryo-electron microscopy (Cryo-EM) revealed that 7F3 binds to spike proteins at a shift-angled pattern with one up and two down RBDs, resulting in partial overlapping with the receptor binding motif (RBM), while a neutralizing monoclonal antibody that lacked ADE activity binds to spike proteins with three up RBDs, resulting in complete overlapping with RBM. Our results revealed that ADE mediated by SARS-CoV-2 spike-specific antibodies could result from binding to the receptor in slightly different pattern from antibodies mediating neutralizations. Studies on ADE using antibodies from recovered patients via cell biology and structural biology technology could be of use for developing novel therapeutic and preventive measures for control of COVID-19 infection.

Structural basis for bivalent binding and inhibition of SARS-CoV-2 infection by human potent neutralizing antibodies (preprint)

Neutralizing monoclonal antibodies (nAbs) to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) represent promising candidates for clinical intervention against coronavirus virus diseases 2019 (COVID-19). We isolated a large number of nAbs from SARS-CoV-2 infected individuals capable of disrupting proper interaction between the receptor binding domain (RBD) of the viral spike (S) protein and the receptor angiotensin converting enzyme 2 (ACE2). In order to understand the mechanism of these nAbs on neutralizing SARS-CoV-2 virus infections, we have performed cryo-EM analysis and here report cryo-EM structures of the ten most potent nAbs in their native full-length IgG or Fab forms bound to the trimeric S protein of SARS-CoV-2. The bivalent binding of the full-length IgG is found to associate with more RBD in the "up" conformation than the monovalent binding of Fab, perhaps contributing to the enhanced neutralizing activity of IgG and triggering more shedding of the S1 subunit from the S protein. Comparison of large number of nAbs identified common and unique structural features associated with their potent neutralizing activities. This work provides structural basis for further understanding the mechanism of nAbs, especially through revealing the bivalent binding and their correlation with more potent neutralization and the shedding of S1 subunit.

Engineered Trimeric ACE2 Binds and Locks "Three-up" Spike Protein to Potently Inhibit SARS-CoVs and Mutants (preprint)

SARS-CoV-2 enters cells via ACE-2, which binds the spike protein with moderate affinity. Despite a constant background mutational rate, the virus must retain binding with ACE2 for infectivity, providing a conserved constraint for SARS-CoV-2 inhibitors. To prevent mutational escape of SARS-CoV-2 and to prepare for future related coronavirus outbreaks, we engineered a de novo trimeric ACE2 (T-ACE2) protein scaffold that binds the trimeric spike protein with extremely high affinity (KD < 1 pM), while retaining ACE2 native sequence. T-ACE2 potently inhibits all tested pseudotyped viruses including SARS-CoV-2, SARS-CoV, eight naturally occurring SARS-CoV-2 mutants, two SARSr-CoVs as well as authentic SARS-CoV-2. The cryo-EM structure reveals that T-ACE2 can induce the transit of spike protein to "three-up" RBD conformation upon binding. T-ACE2 thus represents a promising class of broadly neutralizing proteins against SARS-CoVs and mutants.

AXL Promotes SARS-CoV-2 Infection of Pulmonary and Bronchial Epithelial Cells (preprint)

The current coronavirus disease 2019 (COVID-19) pandemic presents a global public health challenge. The viral pathogen responsible, Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), binds to a host receptor ACE2 through its spike (S) glycoprotein, which mediates membrane fusion and virus entry. Although the role of ACE2 as a receptor for SARS-CoV-2 is clear, studies have shown that ACE2 expression across different human tissues is extremely low, especially in pulmonary and bronchial cells. Thus, other host receptors and/or co-receptors that promote the entry of SARS-CoV-2 into cells of the respiratory system might exist. In this study, we have identified tyrosine-protein kinase receptor UFO (AXL), specifically interacts with SARS-CoV-2 S on the host cell membrane. When overexpressed in cells that do not highly express either AXL or ACE2, AXL promotes virus entry as efficiently as ACE2. Strikingly, deleting AXL, but not ACE2, significantly reduces infection of pulmonary cells by the SARS-CoV-2 virus pseudotype. Soluble human recombinant AXL, but not ACE2, blocks SARS-CoV-2 virus pseudotype infection in pulmonary cells. Taken together, our findings suggest AXL may play an important role in promoting SARS-CoV-2 infection of the human respiratory system and is a potential target in future clinical intervention strategies.

A potent neutralizing human antibody reveals the N-terminal domain of the Spike protein of SARS-CoV-2 as a site of vulnerability (preprint)

The pandemic of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) presents a global public health threat. Most research on therapeutics against SARS-CoV-2 focused on the receptor binding domain (RBD) of the Spike (S) protein, whereas the vulnerable epitopes and functional mechanism of non-RBD regions are poorly understood. Here we isolated and characterized monoclonal antibodies (mAbs) derived from convalescent COVID-19 patients. An mAb targeting the N-terminal domain (NTD) of the SARS-CoV-2 S protein, named 4A8, exhibits high neutralization potency against both authentic and pseudotyped SARS-CoV-2, although it does not block the interaction between angiotensin-converting enzyme 2 (ACE2) receptor and S protein. The cryo-EM structure of the SARS-CoV-2 S protein in complex with 4A8 has been determined to an overall resolution of 3.1 Angstrom and local resolution of 3.4 Angstrom for the 4A8-NTD interface, revealing detailed interactions between the NTD and 4A8. Our functional and structural characterizations discover a new vulnerable epitope of the S protein and identify promising neutralizing mAbs as potential clinical therapy for COVID-19.

Structural basis for the recognition of the 2019-nCoV by human ACE2 (preprint)

Angiotensin-converting enzyme 2 (ACE2) has been suggested to be the cellular receptor for the new coronavirus (2019-nCoV) that is causing the coronavirus disease 2019 (COVID-19). Like other coronaviruses such as the SARS-CoV, the 2019-nCoV uses the receptor binding domain (RBD) of the surface spike glycoprotein (S protein) to engage ACE2. We most recently determined the structure of the full-length human ACE2 in complex with a neutral amino acid transporter B0AT1. Here we report the cryo-EM structure of the full-length human ACE2 bound to the RBD of the 2019-nCoV at an overall resolution of 2.9 [A] in the presence of B0AT1. The local resolution at the ACE2-RBD interface is 3.5 [A], allowing analysis of the detailed interactions between the RBD and the receptor. Similar to that for the SARS-CoV, the RBD of the 2019-nCoV is recognized by the extracellular peptidase domain (PD) of ACE2 mainly through polar residues. Pairwise comparison reveals a number of variations that may determine the different affinities between ACE2 and the RBDs from these two related viruses.

Structure of dimeric full-length human ACE2 in complex with B0AT1 (preprint)

Angiotensin-converting enzyme 2 (ACE2) is the surface receptor for SARS coronavirus (SARS-CoV), directly interacting with the spike glycoprotein (S protein). ACE2 is also suggested to be the receptor for the new coronavirus (2019-nCoV), which is causing a serious epidemic in China manifested with severe respiratory syndrome. B0AT1 (SLC6A19) is a neutral amino acid transporter whose surface expression in intestinal cells requires ACE2. Here we present the 2.9 [A] resolution cryo-EM structure of full-length human ACE2 in complex with B0AT1. The complex, assembled as a dimer of ACE2-B0AT1 heterodimers, exhibits open and closed conformations due to the shifts of the peptidase domains (PDs) of ACE2. A newly resolved Collectrin-like domain (CLD) on ACE2 mediates homo-dimerization. Structural modelling suggests that the ACE2-B0AT1 complex can bind two S proteins simultaneously, providing important clues to the molecular basis for coronavirus recognition and infection.