The SARS-CoV-2 Spike mutation D614G increases entry fitness across a range of ACE2 levels, directly outcompetes the wild type, and is preferentially incorporated into trimers

Abstract

Early in the current pandemic, the D614G mutation arose in the Spike protein of SARS-CoV-2 and quickly became the dominant variant globally. Mounting evidence suggests D614G enhances viral entry. Here we use a direct competition assay with single-cycle viruses to show that D614G outcompetes the wildtype. We developed a cell line with inducible ACE2 expression to confirm that D614G more efficiently enters cells with ACE2 levels spanning the different primary cells targeted by SARS-CoV-2. Using a new assay for crosslinking and directly extracting Spike trimers from the pseudovirus surface, we found an increase in trimerization efficiency and viral incorporation of D614G protomers. Our findings suggest that D614G increases infection of cells expressing a wide range of ACE2, and informs the mechanism underlying enhanced entry. The tools developed here can be broadly applied to study other Spike variants and SARS-CoV-2 entry, to inform functional studies of viral evolution and vaccine development.

Functional importance of the D614G mutation in the SARS-CoV-2 spike protein

“…In line with our own observations on VLPs and MLV PVs, Turonova et al visualized live SARS-CoV-2 G614 virions by cryo-EM and observed much greater spike density than was reported in prior studies with D614 viruses [ 61 , 62 ]. Consistently, increased spike density associated with the D614G mutation was reproduced by Michaud et al in a lentiviral PV system [ 25 ]. The investigators also observed that S(G614), when co-expressed with S(D614), is preferentially incorporated into pseudovirions, and that chimeric spike trimers on pseduovirions contain more S(G614) protomers than S(D614) protomers [ 25 ].…”

“…Pseudovirus (PV) systems based on lentiviral, gamma-retroviral, or vesicular stomatitis virus (VSV) vectors which are coated with the CoV S protein have facilitated rapid advances in understanding the entry of SARS-CoV-2. PVs may be pseudotyped with full-length S protein, although C-terminal truncation has often been observed to improve efficiency of incorporation of SARS-CoV-1 and SARS-CoV-2 S protein into the pseudovirion [ [24] , [25] , [26] ]. Substitution of non-native signal sequences to enhance pseudotyping has also been employed by some groups based on observations with other viral entry proteins, although a recent side-by-side comparison in SARS-CoV-2 S protein did not observe a difference between native and optimized signal peptide [ 27 ].…”

“…At equal copy numbers, GFP reporter-expressing Maloney murine leukemia virus (MLV) pseudotyped with G614 versus D614 resulted in significantly higher transduction of ACE2-overexpressing HEK293T cells with or without TMPRSS2 overexpression, as well as ACE2-overexpressing NCI–H1975 human lung epithelial cells [ 35 ]. Enhanced transduction by lentiviral PVs carrying S(G614) has also been observed in ACE2-overexpressing or ACE2-and TMPRSS2-overexpressing HEK293T cells [ 25 , 28 , 32 , 39 , 40 , 44 , 45 ], ACE2-overexpressing A549 human lung epithelial cells [ 39 ], ACE2-overexpressing Huh7.5 human hepatocytes [ 39 ], Caco-2 human colon epithelial cells [ 39 , 44 ], and Calu-3 human lung epithelial cells [ 44 ]. Similarly, VSV PVs pseudotyped with S(G614) more efficiently infect Vero cells, ACE2-overexpressing HEK293T cells, and ACE2-and TMPRSS2-overexpressing HEK293T cells [ 32 , 45 ].…”

“…Work by Michaud and colleagues further augmented observations on D614G with quantitatively focused approaches. Using a doxycycline-inducible ACE2-expressing HEK293T cell line, investigators observed that S(G614) lentiviral PVs mediated more efficient transduction than S(D614) PVs over a wide range of ACE2 expression levels, which they argue are more representative of physiologically relevant cell types than stable cells that abundantly express the receptor [ 25 ]. The same group also employed a two-color reporter system to observe that GFP-expressing S(G614) PVs outcompete RFP-expressing S(D614) PVs in ACE2-overexpressing HEK293T cells.…”

“…The same group also employed a two-color reporter system to observe that GFP-expressing S(G614) PVs outcompete RFP-expressing S(D614) PVs in ACE2-overexpressing HEK293T cells. Cotransduction over a range of S(D614) to S(G614) PV ratios consistently resulted in a greater proportion of GFP-expressing cells relative to RFP expressing cells, compared to the control in which both RFP- and GFP- expressing vectors were pseudotyped with S(D614) [ 25 ].…”