Endomembrane remodeling in SARS-CoV-2 infection.

During severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, the viral proteins intimately interact with host factors to remodel the endomembrane system at various steps of the viral lifecycle. The entry of SARS-CoV-2 can be mediated by endocytosis-mediated internalization. Virus-containing endosomes then fuse with lysosomes, in which the viral S protein is cleaved to trigger membrane fusion. Double-membrane vesicles generated from the ER serve as platforms for viral replication and transcription. Virions are assembled at the ER-Golgi intermediate compartment and released through the secretory pathway and/or lysosome-mediated exocytosis. In this review, we will focus on how SARS-CoV-2 viral proteins collaborate with host factors to remodel the endomembrane system for viral entry, replication, assembly and egress. We will also describe how viral proteins hijack the host cell surveillance system-the autophagic degradation pathway-to evade destruction and benefit virus production. Finally, potential antiviral therapies targeting the host cell endomembrane system will be discussed.

DMV biogenesis during ß-coronavirus infection requires autophagy proteins VMP1 and TMEM41B.

Upon entering host cells, ß-coronaviruses specifically induce generation of replication organelles (ROs) from the endoplasmic reticulum (ER) through their nonstructural protein 3 (nsp3) and nsp4 for viral genome transcription and replication. The most predominant ROs are double-membrane vesicles (DMVs). The ER-resident proteins VMP1 and TMEM41B, which form a complex to regulate autophagosome and lipid droplet (LD) formation, were recently shown to be essential for ß-coronavirus infection. Here we report that VMP1 and TMEM41B contribute to DMV generation but function at different steps. TMEM41B facilitates nsp3-nsp4 interaction and ER zippering, while VMP1 is required for subsequent closing of the paired ER into DMVs. Additionally, inhibition of phosphatidylserine (PS) formation by siPTDSS1 partially reverses the DMV and LD defects in VMP1 KO cells, suggesting that appropriate PS levels also contribute to DMV formation. This work provides clues to the mechanism of how host proteins collaborate with viral proteins for endomembrane reshaping to promote viral infection.

Discovery of a novel SARS-CoV-2 main protease inhibitor by a simple and optimized colorimetric screening assay

For rapid discovery of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease (Mpro) inhibitors from a natural product library, a novel colorimetric screening assay was developed. According to the colorimetric principle, the synthetic peptide TSAVLQ-para-nitroanilide (pNA) was used as the Mpro hydrolysis substrate. Subsequently, the working concentration of pNA substrate, Mpro working concentration, hydrolysis time and DMSO tolerance were optimized for the development of a simple and robust colorimetric screening assay. Through these systematic optimizations, we selected 0.4 mumol.L-1 Mpro and 100 mumol.L-1 pNA substrate as the optimal working concentrations in this colorimetric screening assay, and a high Z' factor of 0.9 was achieved. Using this screening assay, natural product ginkgolic acid C13: 0 (GA13: 0) was identified as a novel competitive Mpro inhibitor in vitro. Taken together, we have successfully developed a simple and optimized colorimetric screening assay, which will be vital for the discovery of novel SARS-CoV-2 Mpro inhibitors. Copyright © 2022, Chinese Pharmaceutical Association. All rights reserved.

VMP1 and TMEM41B are essential for DMV formation during ß-coronavirus infection.

ß-coronaviruses reshape host cell endomembranes to form double-membrane vesicles (DMVs) for genome replication and transcription. Ectopically expressed viral nonstructural proteins nsp3 and nsp4 interact to zipper and bend the ER for DMV biogenesis. Genome-wide screens revealed the autophagy proteins VMP1 and TMEM41B as important host factors for SARS-CoV-2 infection. Here, we demonstrated that DMV biogenesis, induced by virus infection or expression of nsp3/4, is impaired in the VMP1 KO or TMEM41B KO cells. In VMP1 KO cells, the nsp3/4 complex forms normally, but the zippered ER fails to close into DMVs. In TMEM41B KO cells, the nsp3-nsp4 interaction is reduced and DMV formation is suppressed. Thus, VMP1 and TMEM41B function at different steps during DMV formation. VMP1 was shown to regulate cross-membrane phosphatidylserine (PS) distribution. Inhibiting PS synthesis partially rescues the DMV defects in VMP1 KO cells, suggesting that PS participates in DMV formation. We provide molecular insights into the collaboration of host factors with viral proteins to remodel host organelles.

Autonomous inference of complex network dynamics from incomplete and noisy data

The availability of empirical data that capture the structure and behaviour of complex networked systems has been greatly increased in recent years;however, a versatile computational toolbox for unveiling a complex system’s nodal and interaction dynamics from data remains elusive. Here we develop a two-phase approach for the autonomous inference of complex network dynamics, and its effectiveness is demonstrated by the tests of inferring neuronal, genetic, social and coupled oscillator dynamics on various synthetic and real networks. Importantly, the approach is robust to incompleteness and noises, including low resolution, observational and dynamical noises, missing and spurious links, and dynamical heterogeneity. We apply the two-phase approach to infer the early spreading dynamics of influenza A flu on the worldwide airline network, and the inferred dynamical equation can also capture the spread of severe acute respiratory syndrome and coronavirus disease 2019. These findings together offer an avenue to discover the hidden microscopic mechanisms of a broad array of real networked systems. © 2022, The Author(s), under exclusive licence to Springer Nature America, Inc.

[Preparation and identification of rat polyclonal antibody against SARS-CoV-2 main protease (Mpro)]

Objective To investigate the immunological functions of SARS-CoV-2 main protease (Mpro) in coronavirus disease 2019 (COVID-19), polyclonal antibody against Mpro was developed. Methods A codon-optimized SARS-CoV-2 Mpro gene was synthesized and ligated into a pET-28a vector for construction of a recombinant plasmid named by pET-28a-Mpro. Subsequently, this plasmid was transformed into E.coli Rosetta (DE3) competent cells for Mpro expression in an optimized condition, and then Mpro was purified using a HisTrap chelating column. The purified Mpro was used as immunogen to inoculate rats and the serum was collected after third immunization cycle. The titer, selectivity and sensitivity of polyclonal antibody against Mpro were analyzed using the ELISA and Western blot analysis. Results An optimized expression condition in E.coli cells for Mpro was determined, and the recombinant Mpro was purified by a HisTrap chelating column. The ELISA and Western blot analysis demonstrated that the highly sensitive polyclonal antibody against Mpro specially recognized the recombinant Mpro, and the titer reached 1:256 000. Conclusion The highly specific polyclonal antibody against SARS-CoV-2 Mpro is successfully prepared, which lays an experimental foundation for investigating the immunological function of Mpro in COVID-19.

Fusion Peptide of SARS-CoV-2 Spike Rearranges into a Wedge Inserted in Bilayered Micelles.

The receptor binding and proteolysis of Spike of SARS-CoV-2 release its S2 subunit to rearrange and catalyze viral-cell fusion. This deploys the fusion peptide for insertion into the cell membranes targeted. We show that this fusion peptide transforms from intrinsic disorder in solution into a wedge-shaped structure inserted in bilayered micelles, according to chemical shifts, 15N NMR relaxation, and NOEs. The globular fold of three helices contrasts the open, extended forms of this region observed in the electron density of compact prefusion states. In the hydrophobic, narrow end of the wedge, helices 1 and 2 contact the fatty acyl chains of phospholipids, according to NOEs and proximity to a nitroxide spin label deep in the membrane mimic. The polar end of the wedge may engage and displace lipid head groups and bind Ca2+ ions for membrane fusion. Polar helix 3 protrudes from the bilayer where it might be accessible to antibodies.

A Social Network Under Social Distancing: Risk-Driven Backbone Management During COVID-19 and Beyond

As the COVID-19 pandemic reshapes our social landscape, its lessons have far-reaching implications on how online service providers manage their infrastructure to mitigate risks. This paper presents Facebook's risk-driven backbone management strategy to ensure high service performance throughout the COVID-19 pandemic. We describe Risk Simulation System (RSS), a production system that identifies possible failures and quantifies their potential severity with a set of metrics for network risk. With a year-long risk measurement from RSS we show that our backbone resiliently withstood the COVID-19 stress test, achieving high service availability and low route dilation while efficiently handling traffic surges. We also share our operational practices to mitigate risk throughout the pandemic. Our findings give insights to further improve risk-driven network management. We argue for incorporating short-term failure statistics in modeling failures. Common failure prediction models based on long-term modeling achieve stable output at the cost of assigning low significance to unique short-term events of extreme importance such as COVID-19. Furthermore, we advocate augmenting network management techniques with non-networking signals. We support this by identifying and analyzing the correlation between network traffic and human mobility.

Thermophoretic collection of virus-laden (SARS-CoV-2) aerosols.

Detecting the existence of SARS-CoV-2 in the indoor atmosphere is a practical solution to track the prevalence and prevent the spread of the virus. In this work, a thermophoretic approach is presented to collect the novel coronavirus-laden aerosols from the air and accumulate to high concentrations adequate for the sensitivity of viral RNA detection. Among the factors, the density and particle size have negligible effects on particle trajectory, while the vertical coordinates of particles increase with the rise in heating source temperature. When the heating temperature is higher than 355 K , all of the particles exit the channel from one outlet; thus, the collecting and accumulating of virus-laden aerosols can be realized. This study provides a potential approach to accelerate the detection of SARS-CoV-2 and avoid a false negative in the following RNA test.

Control Strategy for Urban Public Transit in Response to Large-scale Emergent Epidemic

The relatively small and confined space inside public transportation vehicles, which induces frequent and close contact between passengers, is a high-risk environment for the propagation of infectious disease. In the face of a sudden, large-scale epidemic such as the COVID-19, the strategy usually adopted by traffic managers is to shut down all bus lines, thus completely blocking disease transmission through the public transport system. However, for bus passengers, the lack of alternative means of public transport seriously reduces their mobility and even affects their basic living conditions. Here, we have explored the characteristics of individual passenger travel and contact among passengers, and have reconstructed dynamic contact networks based on data from bus IC cards in the city of Beijing. Experimental data show that the average contact time between any pair of bus passengers on a single trip is 17 min, and the average cumulative contact times between one commuter and other passengers during weekdays is 123 We used the Susceptible-Exposed-Infected (SEI) and Susceptible-Infected-Susceptible (SIS) models to simulate the dynamics of disease spread on such temporal networks, which led to the discovery of an efficient control strategy. Compared with a simulation on random networks, we found that the periodic occurrence of contact between bus passengers significantly promoted the rapid spread of disease. For conventional bus networks, we designed a partial shutdown scheme by aggregating the individual transmission influence into the bus lines, and used the average transfer distance between bus stations as an indicator of the reduced accessibility of the conventional bus system due to partial shutdown. The scale of the epidemic before and after the implementation of the partial shutdown scheme were compared. We found that disease outbreaks could be contained by stopping a small number of bus lines, and that the overall accessibility of the bus transportation system did not decrease significantly. © 2020, Editorial Department of China Journal of Highway and Transport. All right reserved.