Anticancer Activity and Mechanisms of Action of MAPK pathway inhibitors

Broad sarbecovirus neutralizing antibodies define a key site of vulnerability around the SARS-CoV-2 spike protein

Broad sarbecovirus neutralizing antibodies define a key site of vulnerability around the SARS-CoV-2 spike protein. generated Hydrocortisone acetate and used such rVSVs to safely and effectively study entry by lethal viruses that require high biocontainment (Ca et al., 2019; Jae et al., 2013; Jangra et al., 2018; Kleinfelter et al., 2015; Maier Hydrocortisone acetate et al., 2016; Raaben et al., 2017; Whelan et al., 1995; Wong et al., 2010). Although rVSVs bearing the S glycoprotein from SARS-CoV(Fukushi et al., 2006a, 2006b; Kapadia et al., 2005, 2008) and the Middle East respiratory syndrome coronavirus (MERS-CoV) (Liu et al., 2018) have been developed, no such systems have been described to date for SARS-CoV-2. Here, we generate a rVSV encoding SARS-CoV-2 S and identify key passage-acquired mutations in the S glycoprotein that facilitate strong rVSV replication. We show that this entry-related properties of rVSV-SARS-CoV-2 S resemble those of the authentic agent and use a large panel of COVID-19 convalescent sera to demonstrate that this neutralization of the rVSV and authentic SARS-CoV-2 by spike-specific antibodies is usually highly correlated. Our findings underscore the power of rVSV-SARS-CoV-2 S for the development of spike-specific vaccines and antivirals and for mechanistic studies of viral entry and its inhibition. Results Identification of S gene mutations that facilitate strong rVSV-SARS-CoV-2 S replication. To generate a Igf2r replication-competent rVSV expressing SARS-CoV-2 S, we replaced the open-reading frame of the native VSV entry glycoprotein gene, (Wuhan-Hu-1 isolate) (Fig. 1A). We also introduced a sequence encoding the enhanced green fluorescent protein (eGFP) as an independent transcriptional unit at the first position of the VSV genome. Plasmid-based rescue of rVSV-SARS-CoV-2 S generated a slowly replicating computer virus bearing the wild-type S sequence. Five serial passages yielded viral populations that Hydrocortisone acetate displayed enhanced spread. This was associated with a dramatic increase in the formation of syncytia (Fig. 1B and Fig. S1) driven by S-mediated membrane fusion (data not shown). Sequencing of this viral population identified nonsense mutations that introduced stop codons in the glycoprotein gene (amino acid position C1250* and C1253*), causing 24- and 21-amino acid deletions in the S cytoplasmic tail, respectively. S24 and S21 were maintained in the viral populations upon further passage, and S21 in all plaque-purified isolates, highlighting their likely importance as adaptations for viral growth. Viral populace sequencing after four more passages identified two additional mutations, L517S and P812R in S1 and S2, respectively, whose emergence coincided with more rapid viral spread and the appearance of non-syncytium-forming infectious centers (Fig. 1B, passage 5). Pelleted viral particles from clarified infected-cell supernatants incorporated the S glycoprotein, as determined by an S-specific ELISA (Fig 1C). Open in a separate windows Fig 1. Generation of a recombinant vesicular stomatitis computer virus (rVSV) bearing the SARS-CoV-2 spike (S) glycoprotein. (A) Schematic representation of the VSV genome, in which its native glycoprotein gene has been replaced by that encoding the SARS-CoV-2 S protein. The VSV genome has been further altered to encode an enhanced green fluorescent protein (eGFP) reporter to easily score for contamination. (B) Infectious center formation assay on Vero cells at 24 h post-infection showing growth of the rVSV-SARS-CoV-2 S after the indicated number of rounds of serial passage of the passage #1 computer virus (carrying wild-type (WT) S sequences) on Huh7.5.1 cell line (scale bar.