A genome-wide CRISPR screen identifies host factors that regulate SARS-CoV-2 entry

Abstract

The global spread of SARS-CoV-2 is posing major public health challenges. One feature of SARS-CoV-2 spike protein is the insertion of multi-basic residues at the S1/S2 subunit cleavage site. Here, we find that the virus with intact spike (Sfull) preferentially enters cells via fusion at the plasma membrane, whereas a clone (Sdel) with deletion disrupting the multi-basic S1/S2 site utilizes an endosomal entry pathway. Using Sdel as model, we perform a genome-wide CRISPR screen and identify several endosomal entry-specific regulators. Experimental validation of hits from the CRISPR screen shows that host factors regulating the surface expression of angiotensin-converting enzyme 2 (ACE2) affect entry of Sfull virus. Animal-to-animal transmission with the Sdel virus is reduced compared to Sfull in the hamster model. These findings highlight the critical role of the S1/S2 boundary of SARS-CoV-2 spike protein in modulating virus entry and transmission and provide insights into entry of coronaviruses.

The SARS-CoV-2 and other human coronavirus spike proteins are fine-tuned towards temperature and proteases of the human airways

“…To discriminate the two S protein activation pathways, we included the . This explains why SARS-CoV-2 passaging in Vero E6 cells regularly leads to emergence of viruses bearing substitutions or deletions in the S1/S2 loop [45][46][47][48][49][50][51][52].…”

“…When unprimed, SARS-2-S pseudoviruses are strongly boosted towards the cathepsin B/L route. This likely explains the replication advantage of loop-deletion SARS-CoV-2 mutants in cathepsin L-rich Vero E6 cells [45][46][47][48][49][50][51][52]. The non-covalently linked S1/S2 form is less stable and a plausible disadvantage for endosomal entry.…”

“…For SARS-2-S, the determinants governing S1/S2 processing are still far from clear. Virus passaging in Vero cells commonly leads to substitutions or deletions in the S1/S2 cleavage loop [45][46][47][48][49][50][51][52]. In animal models, these viruses exhibit reduced transmission [53] or virulence [54], providing an explanation why severe mutations in the S1/S2 loop are only rarely detected in humans [48,53,55] (S1 and S2 Tables).…”

Increased transmission of SARS-CoV-2 lineage B.1.1.7 (VOC 2020212/01) is not accounted for by a replicative advantage in primary airway cells or antibody escape

“…P681H is at the S1/S2 cleavage site and could affect the efficiency of furin cleavage. We and others have previously shown that efficient cleavage of S at this site enhances transmissibility and pathogenicity of SARS-CoV-2 (Johnson et al, 2021;Peacock et al, 2020;Zhu et al, 2021). B.1.1.7 also harbours mutations of interest in other genes including a premature stop codon in ORF8, an accessory gene that likely enables immune evasion (Zhang et al, 2020), and a 3 amino acid deletion in NSP6, one of several proteins associated with virus regulation of the innate immune response (Xia et al, 2020).…”

SARS-CoV-2 Variants Reveal Features Critical for Replication in Primary Human Cells

“…This fusion function of S is dependent on its cleavage by host cell proteases, which occurs either following attachment of virions to the host cell membrane or during virion maturation and egress [3, 7, 8]. Strikingly, SARS-CoV-2 S harbors a furin-cleavage site [9], and pre-activation ‘priming’ of S by furin-like proteases increases fusion-dependent entry efficiency of the virus at the plasma membrane following further cleavage by host TMPRSS2 [10-13]. Other SARS-CoV-2 virion components include the Membrane (M) protein that confers shape and support to the virus particle and interacts with both S and the Nucleoprotein (N; which coats the viral RNA genome [14, 15]), and the Envelope (E) protein, a small membrane protein that both drives virion assembly and budding [16], and has ion channel activity linked to viral pathogenesis [17].…”