Polyadenylation-dependent screening assay for respiratory syncytial virus RNA transcriptase activity and identification of an inhibitor

Abstract

RNA-dependent RNA polymerase from respiratory syncytial virus (RSV) is a multi-subunit ribonucleoprotein (RNP) complex that, in addition to synthesizing the full 15 222 nt viral genomic RNA, is able to synthesize all 10 viral mRNAs. We have prepared crude RNP from RSV-infected HEp-2 cells, based on a method previously used for Newcastle disease virus, and established a novel polyadenylation-dependent capture [poly(A) capture] assay to screen for potential inhibitors of RSV transcriptase activity. In this homogeneous assay, radiolabeled full-length polyadenylated mRNAs produced by the viral RNP are detected through capture on immobilized biotinylated oligo(dT) in a 96-well streptavidin-coated FlashPlate. Possible inhibitors identified with this assay could interfere at any step required for the production of complete RSV mRNAs, including transcription, polyadenylation and, potentially, co-transcriptional guanylylation. A specific inhibitor of RSV transcriptase with antiviral activity was identified through screening of this assay.

Figure 1

Extraction and activity of RSV RNP. (A) Measurement of RSV RdRp activity present in Total (T), cytosolic (S1) and pellet (P1) fractions derived from the extraction of RSV-infected HEp-2 cells. Incorporation of radiolabeled CTP was monitored by either DE81 filter binding assay (left) or poly(A) capture assay (right). (B) Schematic diagram of extraction procedure for RSV RdRp. Each centrifugation step in the procedure resulted in a supernatant (S) and pellet (P) fraction. T = Total lyso-lecithin permeabilized lysate fraction prior to the first centrifugation. (C) RSV RdRp activity of each fraction is represented relative to the T fraction. Each fraction was added to transcription reactions containing [³³P]CTP and actinomycin D. The mRNA products from each reaction were detected in the poly(A) capture assay. (D) Taking into account the amount of protein recovered at each step, the specific activities of each fraction is represented relative to the T fraction.

Figure 2

Analysis of RSV RNP. (A) Two amounts (5 and 10 μl) of S3 fraction from either RSV-infected (I) or mock (M) cells were analysed on 8% polyacrylamide/SDS gel and stained with coomassie (GelCode Blue; Pierce). For western blot analysis, one-tenth the amount of each fraction in (A) was run on the same type of gel and transferred to polyvinyl difluoride (PVDF) membrane for sequential probing with different antisera in the following western blots probed with (B) polyclonal anti-N (K-126); (C) polyclonal anti-P (K-109); (D) polyclonal anti-M2-1 (K-115); (E) monoclonal anti-actin (C4; Roche). The same blot was sequentially stripped and reprobed. (F) Analysis of transcripts by gel electrophoresis. Transcription reactions contained either total T (lanes 1 and 2) or S3 fractions (lane 3 and 4). The fractions were obtained either from mock-infected cells (M; lanes 1 and 3) or RSV-infected cells (I; lanes 2 and 4). Transcripts produced in the presence of 15 μCi [α-³³P]CTP and 2 μM unlabeled CTP were treated with oligo(dT)/RNaseH prior to denaturation in the presence of glyoxal and run on a 1.5% agarose gel. Labeled transcripts were detected by phosphorimaging. The relative mobility of labeled RNA markers (Gibco) is indicated on the left of the panel. The identity of RSV transcripts, based on expected size (nucleotide length shown in parentheses), is indicated on the right.

Figure 3

Comparison of poly(A) capture and DE81 filter binding assays. (A) Schematic diagram of poly(A) capture assay. In vitro transcription reactions are assembled by adding RSV RNP, unlabeled nucleoside triphosphates (ATP, GTP and UTP), radiolabeled CTP (asterisk) and buffer to the wells of a streptavidin-coated FlashPlate™ that has been pre-coated with biotinylated oligo(dT)₃₀ (black oval with multiple Ts). Nascent transcripts (curved lines) are synthesized resulting in the incorporation of radiolabeled CTP (red circles). Transcription reactions are stopped with buffer that favors hybridization of RNA to DNA bringing the labeled RNA into proximity of the scintillant that is embedded in the plastic well of the plate. Decay of the radiolabel (red lightening bolt) causes the scintillation signal (white lightening bolt) that is detected on a TopCount™ (Beckman) plate reader. In this assay, only RNA that is polyadenylated is captured. Therefore, only full-length RSV mRNA transcripts are detected. (B) Schematic diagram of DE81 filter binding assay. The transcription reactions are essentially the same as in (A). Following synthesis of the RNA, a part of the reaction mixture is spotted onto DE81 filter paper, washed with a phosphate-containing solution, dried and read on a phosphoimager. Since the DE81 filter paper is a weak anion exchanger, all RNA produced in the reaction is trapped and subsequently detected.

Figure 4

Optimization and characterization of poly(A) capture assay. (A) Testing of various ³³P-labeled NTP (UTP, CTP or GTP) with 0, 4 or 20 μM cold NTP. (B) Poly(A) capture assay was co-optimized for time and temperature. (C) Inhibition curves for foscarnet on RSV polymerase activity in DE81 (open circles) and poly(A) capture (closed circles) assays. The percentage inhibition (ordinate) versus the concentration of inhibitor (abscissa) is presented.

Figure 5

Characterization of novel RSV transcriptase inhibitor. (A) Chemical structure of RSV transcriptase inhibitor Compound A (dihydrochloride salt form). In vitro activity (IC₅₀ in μM) of compound in various assays as indicated. (B) Inhibition curves for Compound A on RSV polymerase activity in DE81 assay (closed triangles) and poly(A) capture assay in either washed (closed circles) or unwashed (open circles) assays. The percent inhibition (ordinate) versus the concentration of inhibitor (abscissa) is presented. (C) Mode of inhibition of Compound A and effect on RSV transcripts. Transcription reactions were performed as described in Materials and Methods using the poly(A) capture assay with various CTP concentrations (1.25–10 μM) in the absence (open triangles) or in the presence of 6.25 (closed squares), 12.5 (open diamonds), 25 (open squares) and 50 (closed diamonds) μM Compound A. The y-axis represents the inverse velocity of the reaction expressed in picomoles CTP incorporated per second. (D) Transcription reactions were performed with 15 μCi [α-³³P]CTP and 2 μM unlabeled CTP in the presence of the indicated concentrations (0–50 μM) of Compound A and RSV transcripts were treated with oligo(dT)/RNaseH prior to denaturation in the presence of glyoxal and run on a 1.5% agarose gel. The relative mobility of labeled RNA markers (Gibco) is indicated on the left and the identity of RSV transcripts (based on size) is indicated on the right.