Virion morphology and on-virus spike protein structures of diverse SARS-CoV-2 variants (preprint)

The evolution of SARS-CoV-2 variants with increased fitness has been accompanied by structural changes in the spike (S) proteins that are the major target for the adaptive immune response. Single-particle cryo-EM analysis of soluble S from SARS-CoV-2 variants has revealed this structural adaptation at high-resolution. The analysis of S trimers in situ on intact virions has the potential to provide more functionally relevant insights into S structure and virion morphology. Here, we characterized B.1, Alpha, Beta, Gamma, Delta, Kappa, and Mu variants by cryo-electron microscopy and tomography, assessing S cleavage, virion morphology, S incorporation, "in-situ" high-resolution S structures and the range of S conformational states. We found no evidence for adaptive changes in virion morphology, but describe multiple different positions in the S protein where amino acid changes alter local protein structure. Considered together, our data is consistent with a model where amino acid changes at multiple positions from the top to the base of the spike cause structural changes that can modulate the conformational dynamics of S.

Mutations that adapt SARS-CoV-2 to mustelid hosts do not increase fitness in the human airway. (preprint)

SARS-CoV-2 has a broad mammalian species tropism infecting humans, cats, dogs and farmed mink. Since the start of the 2019 pandemic several reverse zoonotic outbreaks of SARS-CoV-2 have occurred in mink, one of which reinfected humans and caused a cluster of infections in Denmark. Here we investigate the molecular basis of mink and ferret adaptation and demonstrate the spike mutations Y453F, F486L, and N501T all specifically adapt SARS-CoV-2 to use mustelid ACE2. Furthermore, we risk assess these mutations and conclude mink-adapted viruses are unlikely to pose an increased threat to humans, as Y453F attenuates the virus replication in human cells and all 3 mink-adaptations have minimal antigenic impact. Finally, we show that certain SARS-CoV-2 variants emerging from circulation in humans may naturally have a greater propensity to infect mustelid hosts and therefore these species should continue to be surveyed for reverse zoonotic infections.

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 (preprint)

Lineage B.1.1.7 (Variant of Concern 202012/01) is a new SARS-CoV-2 variant which was first sequenced in the UK in September 2020 before becoming the majority strain in the UK and spreading worldwide. The rapid spread of the B.1.1.7 variant results from increased transmissibility but the virological characteristics which underpin this advantage over other circulating strains remain unknown. Here, we demonstrate that there is no difference in viral replication between B.1.1.7 and other contemporaneous SARS-CoV-2 strains in primary human airway epithelial (HAE) cells. However, B.1.1.7 replication is disadvantaged in Vero cells potentially due to increased furin-mediated cleavage of its spike protein as a result of a P681H mutation directly adjacent to the S1/S2 cleavage site. In addition, we show that B.1.1.7 does not escape neutralisation by convalescent or post-vaccination sera. Thus, increased transmission of B.1.1.7 is not caused by increased replication, as measured on HAE cells, or escape from serological immunity.

The furin cleavage site of SARS-CoV-2 spike protein is a key determinant for transmission due to enhanced replication in airway cells. (preprint)

SARS-CoV-2 enters cells via its spike glycoprotein which must be cleaved sequentially at the S1/S2, then the S2' cleavage sites (CS) to mediate membrane fusion. SARS-CoV-2 has a unique polybasic insertion at the S1/S2 CS, which we demonstrate can be cleaved by furin. Using lentiviral pseudotypes and a cell-culture adapted SARS-CoV-2 virus with a S1/S2 deletion, we show that the polybasic insertion is selected for in lung cells and primary human airway epithelial cultures but selected against in Vero E6, a cell line used for passaging SARS-CoV-2. We find this selective advantage depends on expression of the cell surface protease, TMPRSS2, that allows virus entry independent of endosomes thus avoiding antiviral IFITM proteins. SARS-CoV-2 virus lacking the S1/S2 furin CS was shed to lower titres from infected ferrets and was not transmitted to cohoused sentinel animals. Thus, the polybasic CS is a key determinant for efficient SARS-CoV-2 transmission.