Identification of Common Deletions in the Spike Protein of Severe Acute Respiratory Syndrome Coronavirus 2

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel coronavirus first identified in December 2019. Notable features that make SARS-CoV-2 distinct from most other previously identified betacoronaviruses include a receptor binding domain and a unique insertion of 12 nucleotides or 4 amino acids (PRRA) at the S1/S2 boundary. In this study, we identified two deletion variants of SARS-CoV-2 that either directly affect the polybasic cleavage site itself (NSPRRAR) or a flanking sequence (QTQTN). These deletions were verified by multiple sequencing methods. <i>In vitro</i> results showed that the deletion of NSPRRAR likely does not affect virus replication in Vero and Vero-E6 cells; however, the deletion of QTQTN may restrict late-phase viral replication. The deletion of QTQTN was detected in 3 of 68 clinical samples and 12 of 24 <i>in vitro</i>-isolated viruses, while the deletion of NSPRRAR was identified in 3 <i>in vitro</i>-isolated viruses. Our data indicate that (i) there may be distinct selection pressures on SARS-CoV-2 replication or infection <i>in vitro</i> and <i>in vivo</i>; (ii) an efficient mechanism for deleting this region from the viral genome may exist, given that the deletion variant is commonly detected after two rounds of cell passage; and (iii) the PRRA insertion, which is unique to SARS-CoV-2, is not fixed during virus replication <i>in vitro</i> These findings provide information to aid further investigation of SARS-CoV-2 infection mechanisms and a better understanding of the NSPRRAR deletion variant observed here.<b>IMPORTANCE</b> The spike protein determines the infectivity and host range of coronaviruses. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has two unique features in its spike protein, the receptor binding domain and an insertion of 12 nucleotides at the S1/S2 boundary resulting in a furin-like cleavage site. Here, we identified two deletion variants of SARS-CoV-2 that either directly affect the furin-like cleavage site itself (NSPRRAR) or a flanking sequence (QTQTN), and we investigated these deletions in cell isolates and clinical samples. The absence of the polybasic cleavage site in SARS-CoV-2 did not affect virus replication in Vero or Vero-E6 cells. Our data indicate the PRRAR sequence and the flanking QTQTN sequence are not fixed <i>in vitro;</i> thus, there appears to be distinct selection pressures on SARS-CoV-2 sequences <i>in vitro</i> and <i>in vivo</i> Further investigation of the mechanism of generating these deletion variants and their infectivity in different animal models would improve our understanding of the origin and evolution of this virus.