Figure S2.

Single-round infection of ΔORF3-E mNG virion, related to Figure 1

(A) Negative-staining electron microscopic image of ΔORF3-E mNG virion. Scale bar, 50 nm.

(B) Calu-3 and A549-hACE2 cells (MOI of 1 and 10 for mNG SARS-CoV-2 and ΔORF3-E mNG virion, respectively; viral titers determined on Vero-ORF3-E cells) were infected with mNG SARS-CoV-2 or ΔORF3-E mNG virion for 2 h, after which the cells were washed and cultured in fresh medium. At day 2 post-infection, supernatants of the infected cells were transferred to infect naive Calu-3 and A549-hACE2 for the second round. Fluorescence and phase contrast images for the infected cells are presented. Scale bar, 100 μm.

(C) RT-PCR analysis of viral RNA. Extracellular RNAs from the second round of infection from (B) were harvested at day 2 post-infection and subjected to RT-PCR analysis of viral RNA.

(D) WT mNG SARS-CoV-2 and P10 ΔORF3-E mNG virion (derived from 10 rounds of passaging of ΔORF3-E mNG virion on Vero-ORF3-E cells) were used to infect Vero E6 cells for two rounds as described in Figure 1F. Fluorescence and phase contrast images of infected cells are presented for both the first and second rounds of infections. Scale bar, 100 μm.

(E) RT-PCR analysis of viral RNA extracted from the culture fluids from the second-round infected cells in (D).