Skip to main content
. 2022 Apr 12;96(9):e00389-22. doi: 10.1128/jvi.00389-22

FIG 1.

FIG 1

(A) Schematic illustration of construction of the E3L deletion mutant of VACV strain ACAM2000 (ACAM2000ΔE3L). The numbers on the recombinant shuttle vector and the virus genome correspond to the base pair number in the genome of ACAM2000 (accession number AY313847). The transient selection marker mCherry gene is driven by the vaccinia virus late promoter p11. The final ACAM2000ΔE3L virus has the E3L gene disrupted without the transient selection marker mCherry, as colorless plaques were selected during the virus purification. (B) Schematic illustration of construction of the E3L and K3L double deletion mutant (ACAM2000ΔE3LΔK3L). The numbers on the recombinant shuttle vector and the genome of ACAM2000ΔE3L correspond to the base pair number in the genome of ACAM2000 (accession number AY313847). The selection marker EGFP gene is driven by the vaccinia virus late promoter p11. The final ACAM2000ΔE3LΔK3L has both E3L and K3L genes disrupted and has an insertion of EGFP in the locus of the K3L gene. (C) Schematic illustration of construction of recombinant ACAM2000ΔE3LΔK3L expressing SARS-CoV-2 S and N proteins. The shuttle vectors to mediate the integration of the SARS-CoV-2 S and/or N gene into the K3L locus consist of a K3L ortholog gene, taterapox virus 037 (TATV037) as a positive selection marker driven by the promoter of a sheeppox virus K3L ortholog gene (SPPV011), and the SARS-CoV-2 S and N genes driven by the vaccinia virus early and late promoter mH5. The final recombinant ACAM2000ΔE3LΔK3L expressing SARS-CoV-2 spike (S) and nucleocapsid (N) proteins had the EGFP gene replaced by the insertion of the SARS-CoV-2 genes and thus has no fluorescence protein marker. Recombinant ACAM2000 virus naming conventions used are as follows: rACAM2000vc, the empty control recombinant not expressing SARS-CoV-2 protein; rACAM2000N, the recombinant expressing SARS-CoV-2 N protein; rACAM2000S, the recombinant expressing SARS-CoV-2 S protein; rACAM2000SN, expressing both S and N proteins. (D) Western blot analysis of SARS-CoV-2 S and N proteins. Antibodies to the RBD were used to detect the full-length S and S1, and FLAG antibody was used to detect the N, the full-length S, and the S2 proteins. Expression of the proteins was examined in BHK21 and A549 cells. Numbers at left of blots are molecular masses in kilodaltons.