Generation of recombinant SARS-CoVs with E protein PBM truncated or mutated by reverse genetics and growth kinetics in cell culture.

<p>(A) Top, representation of SARS-CoV E protein sequence and its corresponding domains. Below, sequences corresponding to the end of E protein are shown in boxes for the different viruses. SARS-CoV-E-wt represents the wild type sequence. In SARS-CoV-E-ΔPBM and SARS-CoV-E-mutPBM, E protein PBM was eliminated by deletion or point mutations, reducing or maintaining the full-length protein, respectively. In SARS-CoV-E-potPBM, four amino acids of E protein were replaced to alanine, to generate a non-native new potential PBM. PBM⁺ and PBM⁻ represent the presence or absence of a PBM within E protein sequence, respectively. Red boxes highlight PBMs within E protein. (B) Subconfluent monolayers of Vero E6 and DBT-mACE2 cells were infected with wt, ΔE, ΔPBM, mutPBM and potPBM viruses at an MOI of 0.05. Culture supernatants collected at 4, 24, 48 and 72 hpi were titrated by plaque assay. Error bars represent standard deviations of the mean of results from three experiments. Statistically significant data are indicated with one (<i>P</i><0.05) or two (<i>P</i><0.01) asterisks.