Virus-free and live-cell visualizing SARS-CoV-2 cell entry for studies of neutralizing antibodies and compound inhibitors

Abstract

The ongoing COVID-19 pandemic, caused by SARS-CoV-2 infection, has resulted in hundreds of thousands of deaths. Cellular entry of SARS-CoV-2, which is mediated by the viral spike protein and host ACE2 receptor, is an essential target for the development of vaccines, therapeutic antibodies, and drugs. Using a mammalian cell expression system, we generated a recombinant fluorescent protein (Gamillus)-fused SARS-CoV-2 spike trimer (STG) to probe the viral entry process. In ACE2-expressing cells, we found that the STG probe has excellent performance in the live-cell visualization of receptor binding, cellular uptake, and intracellular trafficking of SARS-CoV-2 under virus-free conditions. The new system allows quantitative analyses of the inhibition potentials and detailed influence of COVID-19-convalescent human plasmas, neutralizing antibodies and compounds, providing a versatile tool for high-throughput screening and phenotypic characterization of SARS-CoV-2 entry inhibitors. This approach may also be adapted to develop a viral entry visualization system for other viruses.

Quantatitive Analysis of Conserved Sites on the SARS-CoV-2 Receptor-Binding Domain to Promote Development of Universal SARS-Like Coronavirus Vaccines

“…Although site S4 is far from ACE2 binding site, corresponding mAbs P05-5B6 and P05-6H7 can neutralize SARS-CoV and SARS-CoV-2 infection without blocking S protein binding to ACE2, as reported S309 (47). Majority of potently neutralizing antibodies inhibit viral infection by blocking spike proteins attachment with receptors (53)(54)(55), however, we speculate that the site S4 directed mAbs may inhibit SARS-CoV-2 and SARS-CoV infection by intracellular neutralization pathway, a mechanism inhibiting the release of virus within endosome into cytoplasm, as reported in recent studies (40).…”

“…To investigate whether blocking S protein binding to ACE2 is the key mechanism for NAbs to inhibit infection of SARS-CoV-2 into susceptible cells, such as lung cells and intestinal cells, mAbs were mixed with SARS-CoV-2 S protein at different concentrations and then added into 293T cells expressing human ACE2 to evaluate the capability of blocking S protein entrance into cells, as reported (40). The results showed that 61.0% of the specific mAbs had the ability to block entrance of S protein into cells, IC50 ranging from 4 ng/mL to 100 μg/mL (Fig.…”

Viral infection neutralization tests: A focus on severe acute respiratory syndrome‐coronavirus‐2 with implications for convalescent plasma therapy

“…SARS-CoV-2 Spike trimer fused to a constitutively fluorescent protein (Gamillus, isolated from Olindias formosus) has also been developed, providing a versatile tool for high-throughput screening and phenotypic characterization of SARS-CoV-2 entry inhibitors. 39 The Promega HiBiT/LgBiT ® system can be exploited for HiBiTtagged VNT (hiVNT): genome-free virus-like particles are incorporated with a small luciferase peptide (HiBiT) and their entry into…”

“…SARS‐CoV‐2 Spike trimer fused to a constitutively fluorescent protein (Gamillus, isolated from Olindias formosus ) has also been developed, providing a versatile tool for high‐throughput screening and phenotypic characterization of SARS‐CoV‐2 entry inhibitors 39 …”

A new high-content screening assay of the entire hepatitis B virus life cycle identifies novel antivirals

“…It was previously shown that an actin-dependent receptor-HIV co-localization is essential for viral entry, which is potentially sensitive to cytochalasin D 19 . Furthermore, cytochalasin D was shown to prevent SARS-CoV-2 cell entry in a virus-free, live-cell assay 20 . Importantly, all identified HBV inhibitors show antiviral activities at non-toxic compound concentrations, indicating that no essential cellular house-keeping functions are critically disturbed.…”