Résumé
SARS-CoV-2 amino acid replacements in the receptor binding domain (RBD) occur relatively frequently and some have a consequence for immune recognition. Here we report recurrent emergence and significant onward transmission of a six-nucleotide out of frame deletion in the S gene, which results in loss of two amino acids: H69 and V70. We report that in human infections {Delta}H69/V70 often co-occurs with the receptor binding motif amino acid replacements N501Y, N439K and Y453F, and in the latter two cases has followed the RBD mutation. One of the {Delta}H69/V70+ N501Y lineages, now known as B.1.1.7, has undergone rapid expansion and includes eight S gene mutations: RBD (N501Y and A570D), S1 ({Delta}H69/V70 and {Delta}144) and S2 (P681H, T716I, S982A and D1118H). In vitro, we show that {Delta}H69/V70 does not reduce serum neutralisation across multiple convalescent sera. However, {Delta}H69/V70 increases infectivity and is associated with increased incorporation of cleaved spike into virions. {Delta}H69/V70 is able to compensate for small infectivity defects induced by RBD mutations N501Y, N439K and Y453F. In addition, replacement of H69 and V70 residues in the B.1.1.7 spike reduces its infectivity and spike mediated cell-cell fusion. Based on our data {Delta}H69/V70 likely acts as a permissive mutation that allows acquisition of otherwise deleterious immune escape mutations. Enhanced surveillance for the {Delta}H69/V70 deletion with and without RBD mutations should be considered as a global priority not only as a marker for the B.1.1.7 variant, but potentially also for other emerging variants of concern. Vaccines designed to target the deleted spike protein could mitigate against its emergence as increased selective forces from immunity and vaccines increase globally. HighlightsO_LI{Delta}H69/V70 is present in at least 28 SARS-CoV-2 lineages C_LIO_LI{Delta}H69/V70 does not confer escape from convalescent sera C_LIO_LI{Delta}H69/V70 increases spike infectivity and compensates for RBD mutations C_LIO_LI{Delta}H69/V70 is associated with greater spike cleavage C_LIO_LIB.1.1.7 requires {Delta}H69/V70 for optimal spike cleavage and infectivity C_LI
Résumé
Recently published transcriptomic data of the SARS-CoV-2 coronavirus show that there is a large variation in the frequency and steady state levels of subgenomic mRNA sequences. This variation is derived from discontinuous subgenomic RNA synthesis where the polymerase switches template from a 3 proximal genome body sequence to a 5 untranslated leader sequence. This leads to a fusion between the common 5 leader sequence and a 3 proximal body sequence in the RNA product. This process revolves around a common core sequence (CS) that is present at both the template sites that make up the fusion junction. Base-pairing between the leader CS and the nascent complementary minus strand body CS, and flanking regions (together called the transcription regulating sequence, TRS) is vital for this template switching event. However, various factors can influence the site of template switching within the same TRS duplex. Here, we model the duplexes formed between the leader and complementary body TRS regions, hypothesising the role of the stability of the TRS duplex in determining the major sites of template switching for the most abundant mRNAs. We indicate that the stability of secondary structures and the speed of transcription play key roles in determining the probability of template switching in the production of subgenomic RNAs.