key: cord-0904429-n2msxkod
authors: Pastorio, Chiara; Zech, Fabian; Noettger, Sabrina; Jung, Christoph; Jacob, Timo; Sparrer, Konstantin M.J.; Kirchhoff, Frank
title: Determinants of Spike Infectivity, Processing and Neutralization in SARS-CoV-2 Omicron subvariants BA.1 and BA.2
date: 2022-04-14
journal: bioRxiv
DOI: 10.1101/2022.04.13.488221
sha: 380efe62070b1db66609399743cb379455f10c82
doc_id: 904429
cord_uid: n2msxkod

The SARS-CoV-2 Omicron variant rapidly outcompeted other variants and currently dominates the COVID-19 pandemic. Its enhanced transmission, immune evasion and pathogenicity is thought to be driven by numerous mutations in the Omicron Spike protein. Here, we examined the impact of amino acid changes that are characteristic for the BA.1 and/or BA.2 Omicron lineages on Spike function, processing and susceptibility to neutralization. Individual mutations of S371F/L, S375F and T376A in the ACE2 receptor-binding domain as well as Q954H and N969K in the hinge region 1 impaired infectivity, while changes of G339D, D614G, N764K and L981F moderately enhanced it. Most mutations in the N-terminal region and the receptor binding domain reduced sensitivity of the Spike protein to neutralization by sera from individuals vaccinated with the BNT162b2 vaccine or therapeutic antibodies. Our results represent a systematic functional analysis of Omicron Spike adaptations that allowed this SARS-CoV-2 variant to overtake the current pandemic. HIGHLIGHTS S371F/L, S373P and S375F impair Spike function and revert in some BA. 1 isolates Changes of Q954H and N969K in HR1 reduce while L981F enhances S-mediated infection Omicron-specific mutations in the NTD and RBD of Spike reduce neutralization N440K, G446S, E484A and Q493K confer resistance to bamlanivimab or imdevimab

24-26 near the N-terminus of BA.2 S markedly reduced VSVpp infection (Figure 2A ), although 140 these residues do not affect known functional domains. 141 To assess infection kinetics and to challenge the above-mentioned infection results, we Cell-to-cell fusion assays showed that co-expression of human ACE2 and the parental Hu-1 218 as well as the S373P, N501Y, D614G, N856K and L981F S proteins resulted in the formation of 219 large syncytia ( Figure 5B, 5C with HR2 to form a six-helix bundle to drive virus-host or cell-to-cell membrane fusion ( Figure   227 5D). In agreement with the functional data, molecular modelling of HR1/HR2 interactions using 228 reactive force field simulations predicted that mutation of L981F significantly enhances 229 interactions between HR1 and HR2 ( Figure 5D ). Taken together, syncytia formation is promoted 230 by D614G found in all VOCs and the Omicron-specific mutation L981F, but almost abrogated 231 by S375F, T376A and the triple SSS to LPF or FPF changes. Omicron-specific mutation, to neutralization by sera from five individuals who received a prime boost vaccination with the mRNA-based BioNTech-Pfizer (BNT162b2) vaccine (Table S1 ). This ). In addition, we also found that mutations of N440K or G446S conferred resistance to 342 imdevimab and changes of E484A or Q493R to bamlanivimab, respectively. This was expected 343 since these mutations are located within the epitopes bound by these antibodies (Figure 6B , 6C). domains. It has been suggested that a shared mutation of N764K and a BA.2-specific substitution 369 of N856K generate potential cleavage sites for SKI-1/S1P protease and might impede the 370 exposition of the fusion peptide for membrane fusion (Maaroufi, 2022). We found that N764K 371 is indeed associated with increased infectivity and increased levels of processed Spike in VSVpp.

In comparison, N856K clearly reduced S-mediated infection despite normal processing.

Limitations of the study 374 In the present study, we used pseudotyped viral particles instead of replication-competent 375 recombinant SARS-CoV-2 variants, which serves as a proxy to assess infectivity, fusion activity 376 and incorporation. In addition, the impact of many changes might be context-dependent and this 377 might explain why some individual changes had disruptive effects on Hu-1 S function although 378 they are found in Omicron S proteins. It is difficult to predict which of the numerous mutations 379 in the Omicron S might compensate for disruptive mutations. For example, we found that the BA.1 S was less effective in mediating infection than the BA.2 S protein (Figure 2 ). This agrees, 381 with accumulating evidence that the BA.2 VOC might be more infectious and more virulent than See also Figure S1 . This study did not generate or analyse datasets or codes. G339D  N440K  Q493R  N679K  L981F  S373P S375F  T478K  D796Y  A67V  S477N  E484A  N501Y Y505H  H655Y  P681H N764K   wt (hu1)  mock  Q498R  T547K  D614G  Q954H  Δ69-70  K417N  S371L  G496S  N969K  T95I Δ142-144  Δ211  L212I  G446S  Y145D  214EPE  N856K  Δ24-26 A27S G142D V213G S371F T376A D405N 

Virological characteristics of SARS-CoV-2 BA.2 825 variant

Structural and Functional Analysis of 828 the D614G SARS-CoV

Structural and functional impact by SARS-CoV-2 Omicron 832 spike mutations

The significant immune escape of pseudotyped SARS-CoV-2 Variant Omicron

SARS-CoV-2 Omicron variant shows less efficient replication 838 and fusion activity when compared with delta variant in TMPRSS2-expressed cells