Construction of SARS-CoV-2 spike-pseudotyped retroviral vector inducing syncytia formation

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is handled in biosafety level 3 (BSL-3) facilities, whereas the antiviral screening of pseudotype virus is conducted in BSL-2 facilities. In this study, we developed a SARS-CoV-2 spike-pseudotyped virus based on a semi-replication-competent retroviral (s-RCR) vector system. The s-RCR vector system was divided into two packageable vectors, each with gag-pol and env genes. For env vector construction, SARS-CoV-2 SΔ19 env was inserted into the pCLXSN-IRES-EGFP retroviral vector to generate pCLXSN-SΔ19 env-EGFP. When pCLXSN-gag-pol and pCLXSN-SΔ19env-EGFP were co-transfected into HEK293 T cells to generate an s-RCR virus, titers of the s-RCR virus were consistently low in this transient transfection system (1 × 10⁴ TU/mL). However, a three-fold higher amounts of MLV-based SARS-CoV-2 pseudotyped viruses (3 × 10⁴ TU/mL) were released from stable producer cells, and the spike proteins induced syncytia formation in HEK293-hACE2 cells. Furthermore, s-RCR stocks collected from stable producer cells induced more substantial syncytia formation in the Vero E6-TMPRSS2 cell line than in the Vero E6 cell line. Therefore, a combination of the s-RCR vector and the two cell lines (HEK293-hACE2 or Vero E6-TMPRSS2) that induce syncytia formation can be useful for the rapid screening of novel fusion inhibitor drugs.