Fig. 5.

SARS-2 nsp15^mut is attenuated for replication compared to WT SARS-CoV-2 and induces increased IFN signaling and PKR pathway activation in primary nasal epithelial ALI cultures. Primary nasal ALI cultures were infected at MOI 1 with either WT or nsp15^mut SARS-CoV-2. (A) ASL was collected every 48 h and infectious virus was titered by plaque assay. Growth curves shown are the average titers from five independent experiments, each performed in triplicate with a different set of four to six pooled nasal cell donors. (B and C) Intracellular RNA was extracted and analyzed by RT-qPCR at 48, 96 and 192 hpi. (B) Genome copy number was quantified using primers against nsp12 and a standard curve. (C) Relative mRNA expression of IFNB and IFNL1 as well as four representative ISGs: RSAD2, IFIT1 CXCL10, and ISG15. Fold change is expressed as 2^–Δ(ΔCt). (D) Western blot analysis of whole cell lysates collected at 96 and 192 hpi was performed using antibodies against indicated proteins. Data in B–D are from one representative experiment of three total experiments.