SARS-CoV-2 variants reveal features critical for replication in primary human cells

Abstract

Since entering the human population, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2; the causative agent of Coronavirus Disease 2019 [COVID-19]) has spread worldwide, causing >100 million infections and >2 million deaths. While large-scale sequencing efforts have identified numerous genetic variants in SARS-CoV-2 during its circulation, it remains largely unclear whether many of these changes impact adaptation, replication, or transmission of the virus. Here, we characterized 14 different low-passage replication-competent human SARS-CoV-2 isolates representing all major European clades observed during the first pandemic wave in early 2020. By integrating viral sequencing data from patient material, virus stocks, and passaging experiments, together with kinetic virus replication data from nonhuman Vero-CCL81 cells and primary differentiated human bronchial epithelial cells (BEpCs), we observed several SARS-CoV-2 features that associate with distinct phenotypes. Notably, naturally occurring variants in Orf3a (Q57H) and nsp2 (T85I) were associated with poor replication in Vero-CCL81 cells but not in BEpCs, while SARS-CoV-2 isolates expressing the Spike D614G variant generally exhibited enhanced replication abilities in BEpCs. Strikingly, low-passage Vero-derived stock preparation of 3 SARS-CoV-2 isolates selected for substitutions at positions 5/6 of E and were highly attenuated in BEpCs, revealing a key cell-specific function to this region. Rare isolate-specific deletions were also observed in the Spike furin cleavage site during Vero-CCL81 passage, but these were rapidly selected against in BEpCs, underscoring the importance of this site for SARS-CoV-2 replication in primary human cells. Overall, our study uncovers sequence features in SARS-CoV-2 variants that determine cell-specific replication and highlights the need to monitor SARS-CoV-2 stocks carefully when phenotyping newly emerging variants or potential variants of concern.

Manipulation of the unfolded protein response: A pharmacological strategy against coronavirus infection

“…Upon reaching 70–80% confluence, 17 Cl-1 cells were infected with MHV-A59 at MOI 5 as described [ 9 ]. Vero CCL81 and Calu3 cells were infected with SARS-CoV-2 (SARS-CoV-2/human/Switzerland/ZH-UZH-IMV5/2020) at two MOIs (1 and 5) for 24 or 48 has previously described [ 107 , 108 ]. Caco2 cells were infected with SARS-CoV-2 (isolate hCoV-19/Edinburgh/2/2020, a kind gift from Dr Christine Tait-Burkhard and Dr Juergen Haas) at MOI 0.01 and incubated for 48 h in MEM containing 1% L-glutamine, 1% non-essential aminoacids, 1% penicillin/streptomycin and supplemented with 2% FBS.…”

Mechanisms of SARS-CoV-2 entry into cells

“…It was suggested that the acquisition of the furin-cleavage site in the SARS-CoV-2 S protein was essential for zoonotic transfer to humans 9,163 , and experimental data confirmed that SARS-CoV-2 pseudoviruses lacking this cleavage site in the S protein are incapable of facilitating entry into human airway cells 9 . Notably, the furin site is not essential for infection of mammalian epithelial cells generally, as it is lost after a few passages of the virus in Vero cells (African green monkey kidney epithelial cells) 28,[164][165][166][167] . However, recent preliminary studies show virus passage in TMPRSS2overexpressing Vero cells or in human lung cells prevents deletion or mutation of this site [167][168][169] .…”

Structural Evaluation of the Spike Glycoprotein Variants on SARS-CoV-2 Transmission and Immune Evasion

“…Most mutations are neutral or detrimental to virus fitness, while some may provide selective advantages, such as increased infectivity, transmissibility and reduced effectiveness of the immune response. There have been thousands of mutations recorded so far in SARS-CoV-2, with D614G being the most common mutation: it is found in a high number of isolates worldwide and has been suggested to increase replication and human-to-human transmission [ 96 , 97 , 98 ]. D614G enhances viral replication by increasing the stability of virions in human lung epithelial cells and airway tissues [ 99 ].…”