The D614G mutation redirects SARS-CoV-2 spike to lysosomes and suppresses deleterious traits of the furin cleavage site insertion mutation

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) egress occurs by lysosomal exocytosis. We show that the Spike D614G mutation enhances Spike trafficking to lysosomes, drives Spike-mediated reprogramming of lysosomes, and reduces cell surface Spike expression by ~3-fold. D614G is not a human-specific adaptation. Rather, it is an adaptation to the earlier furin cleavage site insertion (FCSI) mutation that occurred at the genesis of SARS-CoV-2. While advantageous to the virus, furin cleavage of spike has deleterious effects on spike structure and function, inhibiting its trafficking to lysosomes and impairing its infectivity by the transmembrane serine protease 2(TMPRSS2)-independent, endolysosomal pathway. D614G restores spike trafficking to lysosomes and enhances the earliest events in SARS-CoV-2 infectivity, while spike mutations that restore SARS-CoV-2’s TMPRSS2-independent infectivity restore spike’s trafficking to lysosomes. Together, these and other results show that D614G is an intragenic suppressor of deleterious traits linked to the FCSI and lend additional support to the endolysosomal model of SARS-CoV-2 egress and entry.

Antigen-display exosomes provide adjuvant-free protection against SARS-CoV-2 disease at nanogram levels of spike protein

“…As a first step towards testing whether exosome display of SARS-CoV-2 spike would enhance its immunogenicity, we asked whether spike, a type-1 integral membrane protein, is normally secreted from the cell in exosomes. For these experiments, we used Tet-on 293 cell lines designed to inducibly express the original form of spike or a D614G form of spike, which we used previously to show that spike is primarily a lysosomal protein and that the D614G mutation enhances the lysosomal sorting of spike ( 24 ) (the spike D614G mutation was the first mutation in SARS-CoV-2 to show a distinct fitness advantage for the virus, is present in all variants of concern, and acts primarily to suppress the deleterious effects of the pandemic-triggering furin cleavage site, resulting in restoration of spike’s trafficking to lysosomes( 24 ) and the ‘up’ conformation of the receptor binding domain that promotes infectivity( 25, 26 )). These spike-expressing cell lines were grown in the presence of doxycycline for three days, after which we collected cell and exosome fractions, and examined them by immunoblot (IB).…”

“…Spike’s sorting to lysosomes is atypical, as it’s not mediated by a canonical lysosomal sorting signal in its C-terminal tail, but rather, by ill-defined structural information in the spike extracellular domain (ECD)( 24 ). Moreover, we’ve found that structure-altering mutations in spike’s ECD can interfere with spike’s trafficking to lysosomes( 24 ), leading us to ask whether the structure-altering diproline (2P) substitution (986KV987-to-986PP987)( 30, 31 ) might inhibit the lysosomal sorting of spike, as inhibition of a protein’s lysosomal sorting almost always results in its delivery to the cell surface( 32 ). Furthermore, since retention of spike in the endoplasmic reticulum (ER) is also likely to inhibit spike’s plasma membrane accumulation, we also deleted the ER retrieval signal present in its short cytoplasmic, carboxy-terminal tail( 33, 34 ), replaced it with an ER export signal( 35, 36 ), and combined these…”

“…Doxycycline-inducible, Tet-on derivatives of these cell lines (HtetZ, FtetZ, and FtetP) were generated by transfection with rtTAv16 expression vectors pJM1463 (expresses rtTAv16-2a-BleoR) or pJM1464 (expresses rtTAv16-2a-PuroR), followed by selection with either zeocin or puromycin, respectively. Derivatives of HtetZ, FtetZ, and FtetP cells were created by transfecting the cells lines with pITRSB-based Sleeping Beauty vectors carrying (i) an EFS-driven selectable marker gene and (ii) a TRE3G-driven gene encoding a form of SARS-CoV-2 Spike, as previously described( 24 ). Transfections were performed using Lipofectamine 3000 (Invitrogen Cat#L3000001) according to the manufacturer’s instructions, and the transgenic cell lines were selected as previously described (17).…”

“…Membrane proteins are loaded into exosomes most efficiently when they're localized to the plasma membrane (12)(13)(14)(27)(28)(29), and thus, our first goal was to redirect spike from lysosomes to the plasma membrane. Spike's sorting to lysosomes is atypical, as it's not mediated by a canonical lysosomal sorting signal in its C-terminal tail, but rather, by ill-defined structural information in the spike extracellular domain (ECD) (24). Moreover, we've found that structurealtering mutations in spike's ECD can interfere with spike's trafficking to lysosomes (24), leading us to ask whether the structure-altering diproline (2P) substitution (986KV987-to-986PP987) (30,31) might inhibit the lysosomal sorting of spike, as inhibition of a protein's lysosomal sorting almost always results in its delivery to the cell surface (32).…”

Self-Aligning Nanojunctions for Integrated Single-Molecule Circuits

“…For these experiments, we test whether we can detect the presence of a specific oligonucleotide related to the D614G mutation (aspartic acid swapped for glycine at amino acid residue 614 on the spike protein) in the SARS-CoV-2 virus. The D614G modification was an early mutation that greatly increased the virulence of SARS-CoV-2, , and by focusing on this coding sequence with a known point mutation difference between the wild type (WT) (GAT codon) and the variant (GGT codon), we can examine the specificity of this detection approach and its resilience when challenged with potentially interfering targets.…”