Characterization of SARS-CoV-2 variants B.1.617.1 (Kappa), B.1.617.2 (Delta) and B.1.618 on cell entry, host range, and sensitivity to convalescent plasma and ACE2 decoy receptor (preprint)

Recently, highly transmissible SARS-CoV-2 variants B.1.617.1 (Kappa), B.1.617.2 (Delta) and B.1.618 were identified in India with mutations within the spike proteins. The spike protein of Kappa contains four mutations E154K, L452R, E484Q and P681R, and Delta contains L452R, T478K and P681R, while B.1.618 spike harbors mutations {Delta}145-146 and E484K. However, it remains unknown whether these variants have altered in their entry efficiency, host tropism, and sensitivity to neutralizing antibodies as well as entry inhibitors. In this study, we found that Kappa, Delta or B.1.618 spike uses human ACE2 with no or slightly increased efficiency, while gains a significantly increased binding affinity with mouse, marmoset and koala ACE2 orthologs, which exhibits limited binding with WT spike. Furthermore, the P618R mutation leads to enhanced spike cleavage, which could facilitate viral entry. In addition, Kappa, Delta and B.1.618 exhibits a reduced sensitivity to neutralization by convalescent sera owning to the mutation of E484Q, T478K, {Delta}145-146 or E484K, but remains sensitive to entry inhibitors-ACE2-lg decoy receptor. Collectively, our study revealed that enhanced human and mouse ACE2 receptor engagement, increased spike cleavage and reduced sensitivity to neutralization antibodies of Kappa, Delta and B.1.618 may contribute to the rapid spread of these variants and expanded host range. Furthermore, our result also highlighted that ACE2-lg could be developed as broad-spectrum antiviral strategy against SARS-CoV-2 variants.

Mutation Y453F in the spike protein of SARS-CoV-2 enhances interaction with the mink ACE2 receptor for host adaption (preprint)

COVID-19 patients transmitted SARS-CoV-2 to minks in the Netherlands in April 2020. Subsequently, the mink-associated virus (miSARS-CoV-2) spilled back over into humans. Genetic sequences of the miSARS-CoV-2 identified a new genetic variant known as "Cluster 5" that contained mutations in the spike protein. However, the functional properties of these "Cluster 5" mutations have not been well established. In this study, we found that the Y453F mutation located in the RBD domain of miSARS-CoV-2 is an adaptive mutation that enhances binding to mink ACE2 and other orthologs of Mustela species without compromising, and even enhancing, its ability to utilize human ACE2 as a receptor for entry. Structural analysis suggested that despite the similarity in the overall binding mode of SARS-CoV-2 RBD to human and mink ACE2, Y34 of mink ACE2 was better suited to interact with a Phe rather than a Tyr at position 453 of the viral RBD due to less steric clash and tighter hydrophobic-driven interaction. Additionally, the Y453F spike exhibited resistance to convalescent serum, posing a risk for vaccine development. Thus, our study suggests that since the initial transmission from humans, SARS-CoV-2 evolved to adapt to the mink host, leading to widespread circulation among minks while still retaining its ability to efficiently utilize human ACE2 for entry, thus allowing for transmission of the miSARS-CoV-2 back into humans. These findings underscore the importance of active surveillance of SARS-CoV-2 evolution in Mustela species and other susceptible hosts in order to prevent future outbreaks.

Analysis of viral RNA-host protein interactomes enables rapid antiviral drug discovery (preprint)

RNA viruses including SARS-CoV-2, Ebola virus (EBOV), and Zika virus (ZIKV) constitute a major threat to global public health and society. The interactions between viral genomes and host proteins are essential in the life cycle of RNA viruses and thus provide targets for drug development. However, viral RNA-host protein interactions have remained poorly characterized. Here we applied ChIRP-MS to profile the interactomes of human proteins and the RNA genomes of SARS-CoV-2, EBOV, and ZIKV in infected cells. Integrated interactome analyses revealed interaction patterns that reflect both common and virus-specific host responses, and enabled rapid drug screening to target the vulnerable host factors. We identified Enasidenib as a SARS-CoV-2 specific antiviral agent, and Trifluoperazine and Cepharanthine as broad spectrum antivirals against all three RNA viruses.

A novel cell culture system modeling the SARS-CoV-2 life cycle (preprint)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes the global pandemic of COVID-19, and no effective antiviral agents and vaccines are available. SARS-CoV-2 is classified as a biosafety level-3 (BLS-3) agent, impeding the basic research into its biology and the development of effective antivirals. Here, we developed a biosafety level-2 (BSL-2) cell culture system for production of transcription and replication-competent SARS-CoV-2 virus-like-particles (trVLP). This trVLP expresses a reporter gene (GFP) replacing viral nucleocapsid gene (N), which is required for viral genome packaging and virion assembly (SARS-CoV-2-GFP/{Delta}N trVLP). The complete viral life cycle can be achieved and exclusively confined in the cells ectopically expressing SARS-CoV or SARS-CoV-2 N proteins, but not MERS-CoV N. Genetic recombination of N supplied in trans into viral genome was not detected, as evidenced by sequence analysis after one-month serial passages in the N-expressing cells. Moreover, intein-mediated protein trans-splicing approach was utilized to split the viral N gene into two independent vectors, and the ligated viral N protein could function in trans to recapitulate entire viral life cycle, further securing the biosafety of this cell culture model. Based on this BSL-2 SARS-CoV-2 cell culture model, we developed a 96-well format high throughput screening for antivirals discovery. We identified salinomycin, tubeimoside I, monensin sodium, lycorine chloride and nigericin sodium as potent antivirals against SARS-CoV-2 infection. Collectively, we developed a convenient and efficient SARS-CoV-2 reverse genetics tool to dissect the virus life cycle under a BSL-2 condition. This powerful tool should accelerate our understanding of SARS-CoV-2 biology and its antiviral development.

Functional and Genetic Analysis of Viral Receptor ACE2 Orthologs Reveals Broad Potential Host Range of SARS-CoV-2 (preprint)

The pandemic of Coronavirus Disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a major global health threat. Epidemiological studies suggest that bats are the natural zoonotic reservoir for SARS-CoV-2. However, the host range of SARS-CoV-2 and intermediate hosts that facilitate its transmission to humans remain unknown. The interaction of coronavirus with its host receptor is a key genetic determinant of host range and cross-species transmission. SARS-CoV-2 uses angiotensin-converting enzyme 2 (ACE2) as the receptor to enter host cells in a species-dependent manner. It has been shown that human, palm civet, pig and bat ACE2 can support virus entry, while the murine ortholog cannot. In this study, we characterized the ability of ACE2 from diverse species to support viral entry. We found that ACE2 is expressed in a wide range of species, with especially high conservation in mammals. By analyzing amino acid residues of ACE2 critical for virus entry, based on structure of SARS-CoV spike protein interaction with human, bat, palm civet, pig and ferret ACE2, we identified approximately eighty ACE2 proteins from mammals that could potentially mediate SARS-CoV-2 entry. Functional assays showed that 44 of these mammalian ACE2 orthologs, including those of domestic animals, pets, livestock, and animals commonly found in zoos and aquaria, could bind SARS-CoV-2 spike protein and support viral entry. In contrast, New World monkey ACE2 orthologs could not bind SARS-CoV-2 spike protein and support viral entry. We further identified the genetic determinant of New World monkey ACE2 that restricts viral entry using genetic and functional analyses. In summary, our study demonstrates that ACE2 from a remarkably broad range of species can facilitate SARS-CoV-2 entry. These findings highlight a potentially broad host tropism of SARS-CoV-2 and suggest that SARS-CoV-2 might be distributed much more widely than previously recognized, underscoring the necessity to monitor susceptible hosts to prevent future outbreaks.