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.

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.

Molecular characterization of SARS-CoV-2 from Bangladesh: Implications in genetic diversity, possible origin of the virus, and functional significance of the mutations (preprint)

In a try to understand the pathogenesis, evolution, and epidemiology of the SARS-CoV-2 virus, scientists from all over the world are tracking its genomic changes in real-time. Genomic studies can be helpful in understanding the disease dynamics. We have downloaded 324 complete and near-complete SARS-CoV-2 genomes submitted in the GISAID database from Bangladesh which were isolated between 30 March to 7 September 2020. We then compared these genomes with the Wuhan reference sequence and found 4160 mutation events including 2253 missense single nucleotide variations, 38 deletions, and 10 insertions. The C>T nucleotide change was most prevalent possibly due to selective mutation pressure to reduce CpG sites to evade CpG targeted host immune response. The most frequent mutation that occurred in 98% of the isolates was 3037C>T which is a synonymous change that almost always accompanied 3 other mutations that include 241C>T, 14408C>T (P323L in RdRp), and 23403A>G (D614G in spike protein). The P323L was reported to increase mutation rate and D614G is associated with increased viral replication and currently the most prevalent variant circulating all over the world. We identified multiple missense mutations in B-cell and T-cell predicted epitope regions and/or PCR target regions (including R203K and G204R that occurred in 86% of the isolates) that may impact immunogenicity and/or RT-PCR based diagnosis. Our analysis revealed 5 large deletion events in ORF7a and ORF8 gene products that may be associated with less severity of the disease and increased viral clearance. Our phylogeny analysis identified most of the isolates belonged to the Nextstrain clade 20B (86%) and GISAID clade GR (88%). Most of our isolates shared common ancestors either directly with European countries or jointly with middle eastern countries as well as Australia and India. Interestingly, the 19B clade (GISAID S clade) was unique to Chittagong which was originally prevalent in China. This reveals possible multiple introductions of the virus in Bangladesh via different routes. Hence more genome sequencing and analysis with related clinical data are needed to interpret the functional significance and better predict the disease dynamics that may be helpful for policymakers to control the COVID-19 pandemic in Bangladesh.

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.

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.

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.