Interaction between host G3BP and viral nucleocapsid protein regulates SARS-CoV-2 replication (preprint)

G3BP1/2 are paralogous proteins that promote stress granule formation in response to cellular stresses, including viral infection. G3BP1/2 are prominent interactors of the nucleocapsid (N) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, the functional consequences of the G3BP1-N interaction in the context of viral infection remain unclear. Here we used structural and biochemical analyses to define the residues required for G3BP1-N interaction, followed by structure-guided mutagenesis of G3BP1 and N to selectively and reciprocally disrupt their interaction. We found that mutation of F17 within the N protein led to selective loss of interaction with G3BP1 and consequent failure of the N protein to disrupt stress granule assembly. Introduction of SARS-CoV-2 bearing an F17A mutation resulted in a significant decrease in viral replication and pathogenesis in vivo, indicating that the G3BP1-N interaction promotes infection by suppressing the ability of G3BP1 to form stress granules.

Efflux pump in genomic island GI-M202a mediates transfer of Polymyxin B resistance in Pandoraea pnomenusa M202 (preprint)

Background Polymyxin B was thought to be last-line therapeutic options against multidrug-resistant Gram-negative bacteria, especially in COVID-19 co-infections or other serious infections. The risk of antimicrobial resistance and its spread to the environment should be brought to the forefront.Methods P. pnomenusa M202 was isolated under selection with 8 mg/L polymyxin B from hospital sewage. The genome of M202 was sequenced by PacBio RS II and Illumina HiSeq 4000 platforms. MFS transporter recombinant E. coli strain Mrc-3 was constructed by transferring encoding gene FKQ53_RS21695, a gene in Genomic islands (GIs) of M202, to E. coli 25DN. Mating experiments were performed to evaluate transfer of MFS transporter to Escherichia coli 25DN. And influences of efflux pump inhibitors on MICs were determined. The mechanism of polymyxin B excretion mediated by FKQ53_RS21695 was investigated by Discovery Studio 2.0 based on its homologous model.Results MICs of multidrug-resistant bacterial strain P. pnomenusa M202, isolated from hospital sewage, for polymyxin B is 96 mg/L. Genomic Island GI-M202a with major facilitator superfamily (MFS) transporter encoding gene and conjugative transfer proteins encoding genes of type IV secretion system was identified in strain M202. The mating experiment between M202 and Escherichia coli 25DN reflected the transferability of polymyxin B resistant GI-M202a. Efflux pump inhibitor and heterogeneous expression assays also suggested that MFS transporter gene FKQ53_RS21695 in GI-M202a was responsible to polymyxin B resistance. Molecular docking revealed that polymyxin B fatty acyl group insert into hydrophobic region of transmembrane core with Pi-alkyl and unfavorable bump interactions, and then polymyxin B turns over around Tyr43 to left the peptide group to outside during efflux process, accompanies with conformation change of MFS transporter from inward to outward. Additionally, verapamil and CCCP demonstrated significant inhibition by competing binding sites.Conclusions These findings demonstrated that GI-M202a along with MFS transporter FKQ53_RS21695 in P. pnomenusa M202 could mediate the transmission of polymyxin B resistances.

Both Baicalein and Gallocatechin Gallate Efficaciously Inhibit SARS-CoV-2 Replication by Targeting Mpro and Sepsis in Mice (preprint)

Severe acute syndrome coronavirus 2(SARS-CoV-2) caused the global pandemic of COVID-19 since December 2019. Although most of COVID-19’s patients are mild or common, most of the severe patients have sepsis caused by the cytokine storm, which greatly increases the case fatality rate. Moreover, there is no effective drug that can resist the novel coronavirus so far, so it’s urgent to develop antiviral drug for the SARS-CoV-2. In our research, we screened 29 compounds with a score lower than -6 from 35 flavonoid compounds by molecular docking. (-)-Gallocatechin gallate, (+)-Gallocatechin and Baicalein were identified to have potent inhibit activity with IC50 5.774±0.805μM, 13.14±2.081μM and 5.158±0.928μM by FRET assay. Subsequently, we conducted molecular docking experiments, which showed that (-)-Gallocatechin gallate, (+)-Gallocatechin and Baicalein were non-covalently bound to Mpro through π-π stacking and hydrogen bonds in the Cys145 catalytic site. We further evaluated the effect of (-)-Gallocatechin gallate and Baicalein on cytokine storm use a mouse model of sepsis. (-)-Gallocatechin gallate and Baicalein significant reduced sepsis severity based on weight, murine sepsis score and survival rate and reduced the inflammatory factors level such as TNF-α, IL-1α, IL-4 and IL-10.  Overall, (-)-Gallocatechin gallate and Baicalein may be potential drugs for symptomatic treatment of COVID-19.

Pyroptosis of syncytia formed by fusion of SARS-CoV-2 Spike and ACE2 expressing cells (preprint)

SARS-Cov-2 infected cells fused with the ACE2-positive neighboring cells forming syncytia. However, the effect of syncytia in disease development is largely unknown. We established an in vitro cell-cell fusion system and used it to mimic the fusion of SARS-CoV-2 infected cells with ACE2-expressing cells to form syncytia. We found that Caspase-9 was activated after syncytia formation, and Caspase-3/7 was activated downstream of Caspase-9, but it triggered GSDME-dependent pyroptosis rather than apoptosis. What is more, single cell RNA-sequencing data showed that both ACE2 and GSDME were expression in alveolar type 2 cells in human lung. We propose that pyroptosis is the fate of syncytia formed by SARS-CoV-2 infected host cells and ACE2-positive cells, which indicated that lytic death of syncytia may contribute to the excessive inflammatory responses in severe COVID-19 patients.