Rapid SARS-CoV-2 Adaptation to Available Cellular Proteases

Abstract

Recent emergence of SARS CoV-2 variants demonstrates the potential of this virus for targeted evolution, despite its overall genomic stability. Here we show the dynamics and the mechanisms behind the rapid adaptation of SARS-CoV-2 to growth in Vero E6 cells. The selective advantage for growth in Vero E6 cells is due to increased cleavage efficiency by cathepsins at the mutated S1/S2 site. S1/S2 site also constitutes a heparan sulfate (HS) binding motif that influenced virus growth in Vero E6 cells, but HS antagonist did not inhibit virus adaptation in these cells. The entry of Vero E6-adapted virus into human cells is defective, because the mutated spike variants is poorly processed by furin or TMPRSS2. Minor subpopulation that lack the furin cleavage motif in the spike protein rapidly become dominant upon passaging in Vero E6 cells, but wildtype sequences are maintained at low percentage in the virus swarm and mediate a rapid reverse adaptation if the virus is passaged again on TMPRSS2 + human cells. Our data show that the spike protein of SARS-CoV-2 can rapidly adapt itself to available proteases and argue for deep sequence surveillance to identify the emergence of novel variants. IMPORTANCE Recently emerging SARS-CoV-2 variants B.1.1.7 (alpha variant), B.1.617.2 (delta variant) and B.1.1.529 (omicron variant) harbor spike mutations and have been linked to increased virus pathogenesis. The emergence of these novel variants highlights coronavirus adaptation and evolution potential, despite the stable consensus genotype of clinical isolates. We show that subdominant variants maintained in the virus population enable the virus to rapidly adapt to selection pressure. Although these adaptations lead to genotype change, the change is not absolute and genomes with original genotype are maintained in the virus swarm. Thus, our results imply that the relative stability of SARS-CoV-2 in numerous independent clinical isolates belies its potential for rapid adaptation to new conditions.

Assessing the Reliability of SARS-CoV-2 Neutralization Studies That Use Post-Vaccination Sera

“…Confirmation by sequencing SARS-CoV-2 can acquire adaptational mutations in cell culture passaging [38][39][40][41]. Because it is not yet known if these mutations might affect neutralization titers, the virus sequence should be confirmed for the passage used in neutralization assays.…”

Collection of Monoclonal Antibodies Targeting SARS-CoV-2 Proteins

“…SARS-CoV-2 viruses used for infection of ACE-2 transfected HEK 293T cells were USA-WA1/2020 (NR-52281, here named as WT1), obtained from BEI resources; B.1.1.7 (Alpha) and B.1.617.2 (Delta) strains, isolated from oropharyngeal swabs and B.1.351 (Beta), kindly provided by Dr. Alex Sigal (Nelson R Mandela School of Medicine, UKZN). SARS-CoV-2 virus used for infection of Vero E6 cells was a SARS-CoV-2/297/20 Zagreb (here named as WT2), isolate derived from a positively tested nasopharyngeal swab in Zagreb, Croatia (GISAID database ID: EPI_ISL_451934) passage 5 [10,11] or 0707*149 (B.1.617.2, Delta) passage 3 isolated from a nasopharyngeal swab. All virus stocks were propagated (four passages) and tittered on Vero E6 cells.…”

Capturing a Crucial ‘Disorder-to-Order Transition’ at the Heart of the Coronavirus Molecular Pathology—Triggered by Highly Persistent, Interchangeable Salt-Bridges

“…Apart from Furin, SARS-CoV-2 entry is also primed by other proteases, for example, TMPRSS2, lysosomal cathepsins [36], as well as by proteases (NE, PR3, CatG, NSP4) released by activated neutrophils [37] swarming around the invaded pathogen to elicit an immune response. In fact, adaptability to available cellular proteases is found to be a salient feature that is also conserved in the rapidly growing variants [38]. Host protease priming also helps the virus to escape host immune surveillance.…”