ABSTRACT
ABSTRACT SARS-CoV-2 remained genetically stable during the first three months of the pandemic, before acquiring a D614G spike mutation that rapidly spread worldwide, and then generating successive waves of viral variants with increasingly high transmissibility. We set out to evaluate possible epistatic interactions between the early occurring D614G mutation and the more recently emerged cleavage site mutations present in spike of the Alpha, Delta, and Omicron variants of concern. The P681H/R mutations at the S1/S2 cleavage site increased spike processing and fusogenicity but limited its incorporation into pseudoviruses. In addition, the higher cleavage rate led to higher shedding of the spike S1 subunit, resulting in a lower infectivity of the P681H/R-carrying pseudoviruses compared to those expressing the Wuhan wild-type spike. The D614G mutation increased spike expression at the cell surface and limited S1 shedding from pseudovirions. As a consequence, the D614G mutation preferentially increased the infectivity of P681H/R-carrying pseudoviruses. This enhancement was more marked in cells where the endosomal route predominated, suggesting that more stable spikes could better withstand the endosomal environment. Taken together, these findings suggest that the D614G mutation stabilized S1/S2 association and enabled the selection of mutations that increased S1/S2 cleavage, leading to the emergence of SARS-CoV-2 variants expressing highly fusogenic spikes. AUTHOR SUMMARY The successive emergence of SARS-CoV-2 variants is fueling the COVID pandemic, thus causing a major and persistent public health issue. The parameters involved in the emergence of variants with higher pathogenic potential remain incompletely understood. The first SARS-CoV-2 variant that spread worldwide in early 2020 carried a D614G mutation in the viral spike, making this protein more stable in its cleaved form at the surface of virions, and resulting in viral particles with higher infectious capacity. The Alpha and the Delta variants that spread in late 2020 and early 2021, respectively, proved increasingly transmissible and pathogenic when compared to the original SARS-CoV-2 strain. Interestingly, Alpha and Delta both carried mutations in a spike cleavage site that needs to be processed by cellular proteases prior to viral entry. The cleavage site mutations P681H/R made the Alpha and Delta spikes more efficient at viral fusion, by generating a higher fraction of cleaved spikes subunits S1 and S2. We show here that the early D614G mutation and the late P681H/R mutations act synergistically to increase the fusion capacity of SARS-CoV-2 variants. Specifically, viruses with increased spike cleavage due to P681H/R were even more dependent on the stabilizing effect of D614G mutation, which limited the shedding of cleaved S1 subunits from viral particles. These findings suggest that the worldwide spread of the D614G mutation was a prerequisite to the emergence of more pathogenic SARS-CoV-2 variants with highly fusogenic spikes.
ABSTRACT
Robust severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection in nasal turbinate (NT) accounts for high viral transmissibility, yet whether neutralizing IgA antibodies can control it remains unknown. Here, we evaluated receptor binding domain (RBD)-specific monomeric B8-mIgA1 and B8-mIgA2, and dimeric B8-dIgA1 and B8-dIgA2 against intranasal SARS-CoV-2 challenge in Syrian hamsters. These antibodies exhibited comparably potent neutralization against authentic virus by competing with human angiotensin converting enzyme-2 (ACE2) receptor for RBD binding. While reducing viruses in lungs, pre-exposure intranasal B8-dIgA1 or B8-dIgA2 led to 81-fold more infectious viruses and severer damage in NT than placebo. Virus-bound B8-dIgA1 and B8-dIgA2 could engage CD209 as an alternative receptor for entry into ACE2-negative cells and allowed viral cell-to-cell transmission. Cryo-EM revealed B8 as a class II neutralizing antibody binding trimeric RBDs in 3-up or 2-up/1-down conformation. Therefore, RBD-specific neutralizing dIgA engages an unexpected action for enhanced SARS-CoV-2 nasal infection and injury in Syrian hamsters.
Subject(s)
Severe Acute Respiratory SyndromeABSTRACT
SARS-CoV-2 B.1.1.7 (variant Alpha) and B.1.351 (variant Beta) have supplanted pre-existing strains in many countries. Severe COVID-19 is characterized by lung abnormalities, including the presence of syncytial pneumocytes. Syncytia form when infected cells fuse with adjacent cells. The fitness, cytopathic effects and type-I interferon (IFN) sensitivity of the variants remain poorly characterized. Here, we assessed B.1.1.7 and B.1.351 spread and fusion in cell cultures. B.1.1.7 and B.1.351 replicated similarly to D614G reference strain in Vero, Caco-2, Calu-3 and primary airway cells and were similarly sensitive to IFN. The variants formed larger and more numerous syncytia. Variant Spikes, in the absence of any other viral proteins, resulted in faster fusion relative to D614G. B.1.1.7 and B.1.351 fusion was similarly inhibited by interferon induced transmembrane proteins (IFITMs). Individual mutations present in the variant Spikes modified fusogenicity, binding to ACE2 and recognition by monoclonal antibodies. Also, B.1.1.7 and B.1.351 variants remain sensitive to innate immunity components. The mutations present in the two variants globally enhance viral fusogenicity and allow for antibody evasion.