SARS-CoV-2 Spike Pseudoviruses: A Useful tool to study virus entry and address emerging neutralization escape phenotypes

Kalkeri, Raj; Cai, Zhaohui; Lin, Shuling; Farmer, John; Kuzmichev, Yury V.; Koide, Fusataka

doi:10.1101/2021.07.16.452709

Cited by 5 publications

(1 citation statement)

References 38 publications

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“…Hence, their applications were well popularized in the pandemic period. To mimic the natural infectivity of the virus, recent protocols have been used for the production of pseudoviruses expressing spike glycoprotein of SARS-CoV-2 in other viral backbones such as murine leukemia virus (MLV), vesicular stomatitis virus (VSV), and human immunodeficiency virus (HIV) (Almahboub et al, 2020;Condor Capcha et al, 2020;Crawford et al, 2020;Hyseni et al, 2020;Johnson et al, 2020;Nie et al, 2020;Schmidt et al, 2020;Donofrio et al, 2021;Kalkeri et al, 2021;Lu et al, 2021;Neerukonda et al, 2021). The efficiency of pseudovirus production critically depends on the backbone used in making the pseudovirus and the efficiency of the transfection reagent in delivering the viral plasmids into the cells.…”

Section: Introductionmentioning

confidence: 99%

Optimization of SARS-CoV-2 Pseudovirion Production in Lentivirus Backbone With a Novel Liposomal System

et al. 2022

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Due to the fast mutating nature of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the development of novel therapeutics, vaccines, and evaluating the efficacies of existing one’s against the mutated strains is critical for containing the virus. Pseudotyped SARS-CoV-2 viruses are proven to be instrumental in evaluating the efficiencies of therapeutics, owing to their ease in application and safety when compared to handling the live virus. However, a comprehensive protocol that includes selecting transfection reagents, validating different packaging systems for high-throughput screening of neutralizing antibodies, is still a requisite. To this end, we designed and synthesized amide linker-based cationic lipids with varying hydrophilic head groups from dimethyl (Lipo-DME) to methyl, ethylhydroxyl (Lipo-MeOH), and diethylhydroxyl (Lipo-DOH) keeping the hydrophobic tail, stearic acid, as constant. Among the liposomal formulations of these lipids, Lipo-DOH was found to be superior in delivering plasmids and demonstrated comparable transfection efficiencies with commercial standard Lipofectamine 3000. We further used Lipo-DOH for lentivirus and SARS-CoV-2 pseudovirion preparation. For comparing different lentivirus packaging systems, we optimized conditions using Addgene and BEI systems and found that the BEI lenti plasmid system was found to be efficient in making lentiviruses using Lipo-DOH. Using the optimized transfection reagent and the lentivirus system, we developed a robust protocol for the generation of SARS-CoV-2 pseudovirions and characterized their infectivity in human ACE2 expressing HEK-293T cells and neutralizing properties in IgG against spike protein of SARS-CoV-2 positive human sera from individuals recovered from COVID-19.

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Section: Introductionmentioning

confidence: 99%

Optimization of SARS-CoV-2 Pseudovirion Production in Lentivirus Backbone With a Novel Liposomal System

et al. 2022

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Herbal Compounds Dauricine and Isoliensinine Impede SARS-CoV-2 Viral Entry

Dabrell,

Li,

Yamaguchi

et al. 2023

Biomedicines

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Targeting viral entry has been the focal point for the last 3 years due to the continued threat posed by SARS-CoV-2. SARS-CoV-2’s entry is highly dependent on the interaction between the virus’s Spike protein and host receptors. The virus’s Spike protein is a key modulator of viral entry, allowing sequential cleavage of ACE2 at the S1/S2 and S2 sites, resulting in the amalgamation of membranes and subsequent entry of the virus. A Polybasic insertion (PRRAR) conveniently located at the S1/S2 site can also be cleaved by furin or by serine protease, TMPRSS2, at the cell surface. Since ACE2 and TMPRSS2 are conveniently located on the surface of host cells, targeting one or both receptors may inhibit receptor-ligand interaction. Here, we show that Dauricine and Isoliensinine, two commonly used herbal compounds, were capable of inhibiting SARS-CoV-2 viral entry by reducing Spike-ACE2 interaction but not suppressing TMPRSS2 protease activity. Further, our biological assays using pseudoviruses engineered to express Spike proteins of different variants revealed a reduction in infection rates following treatment with these compounds. The molecular modeling revealed an interconnection between R403 of Spike protein and both two compounds. Spike mutations at residue R403 are critical, and often utilized by ACE2 to gain cell access. Overall, our findings strongly suggest that Dauricine and Isoliensinine are effective in blocking Spike-ACE2 interaction and may serve as effective therapeutic agents for targeting SARS-CoV-2′s viral entry.

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