Uncapped, polyadenylated viral transcript induced <i>IFNB</i> gene expression.

Abstract

<p><b>(A-C)</b> Total RNA of mock treated or NDV-infected (12 hpi, MOI = 1) HeLa cells (A), RSV-infected (60 hpi, MOI = 1) HEp-2 cells (B), or VSV-infected (12 hpi, MOI = 1) HeLa cells (C) was separated into poly(A)^- and poly(A)⁺ RNA fractions by oligo(dT)-combined latex beads and transfected into MEFs (1×10⁵ cells were transfected with 200 ng RNA). <i>Ifnb</i> mRNA expression levels were measured by RT-qPCR. Data are represented as means ±SD. <b>(D and E)</b> The Poly(A)⁺ RNA fraction from NDV-infected (12 hpi, MOI = 1) HeLa cells was mock treated (NT) or treated with RNase III, DNase I, CIAP (D), or 5’-capping enzyme of Vaccinia virus (E) and then transfected to MEFs (1×10⁵ cells were transfected with 200 ng RNA). <i>Ifnb</i> mRNA expression levels were measured by RT-qPCR. Data are represented as means ±SD (t-test: **p<0.05, NS = not significant). <b>(F)</b> NDV vRNA(+) detection by strand-specific northern blotting. Total, poly(A)^-, and poly(A)⁺ RNA (each 0250 ng) from mock treated or NDV-infected (12 hpi, MOI = 1) HeLa cells were separated on a denaturing agarose gel. An ethidium bromide (EtBr)-stained gel was shown in the bottom. vRNA(+) was detected by blotting with an N-specific RNA probe (N(-)). Positions of N vmRNA (N(+)) and N-P read-through RNA (N-P(+) RT) are shown. knt = kilo nucleotide. RNA probe and target vRNA(+) are illustrated alongside. <b>(G)</b> Poly(A)⁺ RNA from mock treated or NDV-infected (12 hpi, MOI = 1) HeLa cells were subjected to strand-specific northern blotting using a leader-specific RNA probe (Le(-)). The position of Le-N read-through RNA is shown (Le-N(+) RT). *non-specific band. RNA probe and target vRNA(+) are illustrated alongside. <b>(H and I)</b> Total, poly(A)^-, and poly(A)⁺ RNA (each 200 ng) from mock treated or NDV-infected (12 hpi, MOI = 1) HeLa cells were subjected to strand-specific RT-qPCR (ssRT-qPCR) targeting Le-N(+) read-through RNA (H) or targeting vRNA(-) (I). Primers used are listed in Table in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005444#ppat.1005444.s014" target="_blank">S1 Table</a>. Data are represented as means ±SD. Target vRNAs in the assay are illustrated below.</p