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. 2021 Feb 23;184(8):2229–2238.e13. doi: 10.1016/j.cell.2021.02.044

Figure 2.

Figure 2

Adaptive mutations to improve the yield of ΔORF3-E mNG virion production

(A) Viral replication kinetics on Vero-ORF3-E cells. Adaptive mutations (D) were selected by continuously passaging the ΔORF3-E virion on Vero-ORF3-E cells for 10 rounds. For comparing the replication kinetics of the passaged viruses, Vero-ORF3-E cells were infected with the P1 or P10 ΔORF3-E virion, ΔORF3-E virion containing an S mutation in (D) (ΔORF3-E virion mut-S), or ΔORF3-E virion containing all adaptive mutations in nsp1, nsp4, and S in (D) (ΔORF3-E virion mut-All) at an MOI of 0.15. WT mNG SARS-CoV-2 was included as a control. Viral titers in culture supernatants are presented. ANOVA with multiple comparison correction test were performed with ∗p < 0.05; ∗∗p < 0.01. Data are represented as mean ± standard deviation.

(B) mNG-positive cells at 24 and 48 h post-infection from (A). Scale bar, 100 μm.

(C) RT-PCR analysis for single-round infection. For confirming the P10 ΔORF3-E virion remains infectious for only a single round on Vero cells, Vero E6 or Vero-ORF3-E cells were infected with WT mNG SARS-CoV-2 or P10 ΔORF3-E mNG virion for two rounds as described in Figure 1G. Viral RNAs were extracted from the second-round culture fluids and analyzed by RT-PCR. The RT-PCR product, gF8, is indicated in Figure 1B.

(D) Adaptive mutations. Three mutations were identified from whole-genome sequencing of P10 ΔORF3-E mNG virion. No mutation was found in the P1 ΔORF3-E mNG virion.