FIG 6.

Generation of ISG20-resistant BUNV and demonstration that endogenous ISG20 possesses antibunyaviral activity. (A and B) Plaque phenotypes (on Vero and Vero-ISG20 cells) (A) and fold inhibition (B) of the parental virus and the P20 and P40 ISG20-resistant viruses by ISG20 (fold reductions in PFU per milliliter for comparisons of naive and Vero-ISG20 cells). (C) Comparison of growth curves for the parental virus and the P40 virus on Vero-ISG20 and naive Vero cells. Cells were infected at an MOI of 0.01 (data are means ± SD; n = 3). (D) Infectious yields (36 h after infection at an MOI of 0.01) of wild-type (wt) BUNV and reassortant mutants (mutant segments are denoted by “′”) from Vero-ISG20 or Vero-D94G cells (data are means and ranges; n = 2). (E) Biological IFN assays to assess the ability of supernatants from HeLa cells infected with the indicated viruses to inhibit the replication of EMCV in A549-Npro cells. (F) Western blot analysis of basal and inducible expression in naive HeLa cells, HeLa-shGFP cells, and HeLa-shISG20 cells. Basal expression or universal interferon (500 U/ml)-induced ISG20 expression was stably detected in naive HeLa cells and HeLa-shGFP cells, whereas both basal and inducible ISG20 expression was undetectable in HeLa-shISG20 cells. (G) Growth curves for parental BUNV, P40 Vero-EMPTY-passaged BUNV, and P40 ISG20-resistant BUNV on HeLa cells (MOI = 0.01) (data are means ± SD; n = 3). (H) Growth curves for parental BUNV and P40 ISG20-resistant BUNV on HeLa-shGFP (control) and HeLa-shISG20 cells.