SARS-CoV-2 variants reveal features critical for replication in primary human cells

Since entering the human population, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2; the causative agent of Coronavirus Disease 2019 [COVID-19]) has spread worldwide, causing >100 million infections and >2 million deaths. While large-scale sequencing efforts have identified numerous genetic variants in SARS-CoV-2 during its circulation, it remains largely unclear whether many of these changes impact adaptation, replication, or transmission of the virus. Here, we characterized 14 different low-passage replication-competent human SARS-CoV-2 isolates representing all major European clades observed during the first pandemic wave in early 2020. By integrating viral sequencing data from patient material, virus stocks, and passaging experiments, together with kinetic virus replication data from nonhuman Vero-CCL81 cells and primary differentiated human bronchial epithelial cells (BEpCs), we observed several SARS-CoV-2 features that associate with distinct phenotypes. Notably, naturally occurring variants in Orf3a (Q57H) and nsp2 (T85I) were associated with poor replication in Vero-CCL81 cells but not in BEpCs, while SARS-CoV-2 isolates expressing the Spike D614G variant generally exhibited enhanced replication abilities in BEpCs. Strikingly, low-passage Vero-derived stock preparation of 3 SARS-CoV-2 isolates selected for substitutions at positions 5/6 of E and were highly attenuated in BEpCs, revealing a key cell-specific function to this region. Rare isolate-specific deletions were also observed in the Spike furin cleavage site during Vero-CCL81 passage, but these were rapidly selected against in BEpCs, underscoring the importance of this site for SARS-CoV-2 replication in primary human cells. Overall, our study uncovers sequence features in SARS-CoV-2 variants that determine cell-specific replication and highlights the need to monitor SARS-CoV-2 stocks carefully when phenotyping newly emerging variants or potential variants of concern

Sec61 Inhibitor Apratoxin S4 Potently Inhibits SARS-CoV-2 and Exhibits Broad-Spectrum Antiviral Activity

There is a pressing need for host-directed therapeutics that elicit broad-spectrum antiviral activities to potentially address current and future viral pandemics. Apratoxin S4 (Apra S4) is a potent Sec61 inhibitor that prevents cotranslational translocation of secretory proteins into the endoplasmic reticulum (ER), leading to anticancer and antiangiogenic activity both in vitro and in vivo. Since Sec61 has been shown to be an essential host factor for viral proteostasis, we tested Apra S4 in cellular models of viral infection, including SARS-CoV-2, influenza A virus, and flaviviruses (Zika, West Nile, and Dengue virus). Apra S4 inhibited viral replication in a concentration-dependent manner and had high potency particularly against SARS-CoV-2 and influenza A virus, with subnanomolar activity in human cells. Characterization studies focused on SARS-CoV-2 revealed that Apra S4 impacted a post-entry stage of the viral life-cycle. Transmission electron microscopy revealed that Apra S4 blocked formation of stacked double-membrane vesicles, the sites of viral replication. Apra S4 reduced dsRNA formation and prevented viral protein production and trafficking of secretory proteins, especially the spike protein. Given the potent and broad-spectrum activity of Apra S4, further preclinical evaluation of Apra S4 and other Sec61 inhibitors as antivirals is warranted

Evolution records a Mx tape for anti-viral immunity

Viruses impose diverse and dynamic challenges on host defenses. Diversifying selection of codons and gene copy number variation are two hallmarks of genetic innovation in antiviral genes engaged in host-virus genetic conflicts. The myxovirus resistance (Mx) genes encode interferon-inducible GTPases that constitute a major arm of the cell-autonomous defense against viral infection. Unlike the broad antiviral activity of MxA, primate MxB was recently shown to specifically inhibit lentiviruses including HIV-1. We carried out detailed evolutionary analyses to investigate whether genetic conflict with lentiviruses has shaped MxB evolution in primates. We found strong evidence for diversifying selection in the MxB N-terminal tail, which contains molecular determinants of MxB anti-lentivirus specificity. However, we found no overlap between previously-mapped residues that dictate lentiviral restriction and those that have evolved under diversifying selection. Instead, our findings are consistent with MxB having a long-standing and important role in the interferon response to viral infection against a broader range of pathogens than is currently appreciated. Despite its critical role in host innate immunity, we also uncovered multiple functional losses of MxB during mammalian evolution, either by pseudogenization or by gene conversion from MxA genes. Thus, although the majority of mammalian genomes encode two Mx genes, this apparent stasis masks the dramatic effects that recombination and diversifying selection have played in shaping the evolutionary history of Mx genes. Discrepancies between our study and previous publications highlight the need to account for recombination in analyses of positive selection, as well as the importance of using sequence datasets with appropriate depth of divergence. Our study also illustrates that evolutionary analyses of antiviral gene families are critical towards understanding molecular principles that govern host-virus interactions and species-specific susceptibility to viral infection

Analysis of early mesothelial cell responses to Staphylococcus epidermidis isolated from patients with peritoneal dialysis-associated peritonitis

The major complication of peritoneal dialysis (PD) is the development of peritonitis, an infection within the abdominal cavity, primarily caused by bacteria. PD peritonitis is associated with significant morbidity, mortality and health care costs. Staphylococcus epidermidis is the most frequently isolated cause of PD-associated peritonitis. Mesothelial cells are integral to the host response to peritonitis, and subsequent clinical outcomes, yet the effects of infection on mesothelial cells are not well characterised. We systematically investigated the early mesothelial cell response to clinical and reference isolates of S. epidermidis using primary mesothelial cells and the mesothelial cell line Met-5A. Using an unbiased whole genome microarray, followed by a targeted panel of genes known to be involved in the human antibacterial response, we identified 38 differentially regulated genes (adj. p-value < 0.05) representing 35 canonical pathways after 1 hour exposure to S. epidermidis. The top 3 canonical pathways were TNFR2 signaling, IL-17A signaling, and TNFR1 signaling (adj. pvalues of 0.0012, 0.0012 and 0.0019, respectively). Subsequent qPCR validation confirmed significant differences in gene expression in a number of genes not previously described in mesothelial cell responses to infection, with heterogeneity observed between clinical isolates of S. epidermidis, and between Met-5A and primary mesothelial cells. Heterogeneity between different S. epidermidis isolates suggests that specific virulence factors may play critical roles in influencing outcomes from peritonitis. This study provides new insights into early mesothelial cell responses to infection with S. epidermidis, and confirms the importance of validating findings in primary mesothelial cells

Combined computational and cellular screening identifies synergistic inhibition of SARS-CoV-2 by lenvatinib and remdesivir

Rapid repurposing of existing drugs as new therapeutics for COVID-19 has been an important strategy in the management of disease severity during the ongoing SARS-CoV-2 pandemic. Here, we used high-throughput docking to screen 6000 compounds within the DrugBank library for their potential to bind and inhibit the SARS-CoV-2 3 CL main protease, a chymotrypsin-like enzyme that is essential for viral replication. For 19 candidate hits, parallel in vitro fluorescence-based protease-inhibition assays and Vero-CCL81 cell-based SARS-CoV-2 replication-inhibition assays were performed. One hit, diclazuril (an investigational anti-protozoal compound), was validated as a SARS-CoV-2 3 CL main protease inhibitor in vitro (IC$_{50}$ value of 29 µM) and modestly inhibited SARS-CoV-2 replication in Vero-CCL81 cells. Another hit, lenvatinib (approved for use in humans as an anti-cancer treatment), could not be validated as a SARS-CoV-2 3 CL main protease inhibitor in vitro, but serendipitously exhibited a striking functional synergy with the approved nucleoside analogue remdesivir to inhibit SARS-CoV-2 replication, albeit this was specific to Vero-CCL81 cells. Lenvatinib is a broadly-acting host receptor tyrosine kinase (RTK) inhibitor, but the synergistic effect with remdesivir was not observed with other approved RTK inhibitors (such as pazopanib or sunitinib), suggesting that the mechanism-of-action is independent of host RTKs. Furthermore, time-of-addition studies revealed that lenvatinib/remdesivir synergy probably targets SARS-CoV-2 replication subsequent to host-cell entry. Our work shows that combining computational and cellular screening is a means to identify existing drugs with repurposing potential as antiviral compounds. Future studies could be aimed at understanding and optimizing the lenvatinib/remdesivir synergistic mechanism as a therapeutic option