Figure S3.

Selection of ΔORF3-E mNG virion capable of inefficiently infecting Vero E6 cells for more than one round, related to Figure 2

Four independently selected P5 ΔORF3-E mNG virions (generated from five rounds of passaging ΔORF3-E mNG virion on Vero-ORF3-E cells) were used to infect naive Vero E6 cells for two rounds as described in Figure 1F.

(A) The P5 ΔORF3-E mNG virion-infected Vero E6 cells were analyzed for mNG signals. Scale bar, 100 μm.

(B) The extracellular RNA from the second-round infected cells were examined for viral RNA by RT-PCR.

(C) Selection IV P5 ΔORF3-E (S-IV-P5) mNG virion could infect Vero cells for multiple rounds. To remove the single-round virion from the multi-round virion in the S-IV-P5 stock, the S-IV-P5 stock was passaged on Vero E6 cells for two rounds, resulting in S-IV-P5-Vero-P2 virion capable of multi-round infection. The replication kinetics of WT mNG SARS-CoV-2 and S-IV-P5-Vero-P2 mNG virion were compared on Vero E6 cells. The cells were inoculated at an MOI of 0.001. Limit of detection, L.O.D. Data are represented as mean ± SD.

(D) Adaptive mutations were identified from the S-IV-P5-Vero-P2 mNG virion.

(E) The T130N mutation from the M protein was engineered into ΔORF3-E mNG virion. The resulting ΔORF3-E mNG M T130N virion was used to infect Vero E6 cells for two rounds. Fluorescence and phase contrast images of the infected cells are shown. Scale bar, 100 μm.

(F) Sequence alignment shows that the M proteins from SARS-CoV and SARS-CoV-2 share the same T130 residue. Red arrow indicates the T130 residue of SARS-CoV-2.