Figure 4.
Pyrimidine biosynthesis regulates NS1-targeted host mRNA nuclear export. (A) A luciferase gene expression assay was performed in 293T cells by cotransfection of reporter plasmids and plasmids encoding NS1. Cells were treated with compounds 1 (5 µM) or 1-14 (1 µM) in the presence or absence of uridine or cytidine, as indicated, and luciferase activity was measured. Cell lysates as in A were subjected to immunoblot analysis with antibodies against the indicated proteins. (B) HeLa cells transfected with plasmid alone or plasmid expressing NS1 were incubated with DMSO, 1 µM compound 1-14 alone, or compound 1-14 in the presence of 500 µM uridine. Cells were then subjected to oligo d(T) in situ hybridization and immunofluorescence with anti-NS1 antibodies. Bar, 15 µM. (C) Quantification of cells that presented nuclear retention of poly(A)+ RNA in B. The percentage of cells presenting nuclear retention of poly(A)+ RNA was calculated with respect to cells that showed normal distribution of poly(A)+ RNA—in the nucleus and in the cytoplasm. 500 cells were counted for each condition. Results represent mean values ± SD; **, P < 0.01.