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

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© 2021 Elsevier Inc.

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Generation of single-round infectious ΔORF3-E mNG virion

(A) A trans-complementation system for SARS-CoV-2. Vero-ORF3-E cells are electroporated with ΔORF3-E mNG RNA. Trans-complementation produces ΔORF3-E mNG virion (left panel), which can infect naive Vero E6 cells for only single round (right panel).

(B) ΔORF3-E mNG virion genome. Both the full-length mNG SARS-CoV-2 genome (top panel) and the ΔORF3-E mNG virion genome (bottom panel) are shown. The genomic fragment 8 (gF8) of reverse transcription PCR (RT-PCR) analysis is indicated above both genomes. The ORF3-E deletion junction is indicated. The WT (green box) and mutant (red box) transcription regulatory sequences (TRSs) are also depicted. The mutated TRS (red) is also engineered at the 5′ end of each of the downstream ORFs.

(C) ORF3-E RNA expression in Vero-ORF3-E cells. Doxycycline (Dox) was used to induce the expression of ORF3-E RNA. RT-PCR analyses were performed on Vero-ORF3-E cells with or without Dox induction as well as on naive Vero E6 cells.

(D) Induction of mCherry expression in Vero-ORF3-E cells. Passage 1 (P1) and 20 (P20) of Vero-ORF3-E cells were induced by Dox to express mCherry fluorescence. Scale bar, 100 μm.

(E) Production of ΔORF3-E mNG virion after electroporation. After electroporating ΔORF3-E mNG RNA into Vero-ORF3-E cells (with Dox), infectious titers of ΔORF3-E mNG virion were measured by infecting Vero-ORF3-E cells. Three sets of repeated experiments are presented with bars representing standard deviations.

(F) Analysis of ΔORF3-E mNG virion infection. Vero E6 or Vero-ORF3-E cells were incubated with WT mNG SARS-CoV-2 or ΔORF3-E mNG virion for 2 h. The cells were washed three times with PBS to remove residual input virus. At 48 h post-infection, the supernatants of the infected cells were transferred to fresh Vero E6 or Vero-ORF3-E cells for a second round of infection. The mNG signals from both rounds of infected cells are presented. Scale bar, 100 μm.

(G) RT-PCR analysis.

Extracellular RNA from the second-round infection from (F) was harvested at 48 h post-infection. Fragment 8 of the viral genome, depicted in (B), was amplified by RT-PCR to confirm the ORF3-E deletion and mNG retention.