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. 2005 Oct;79(20):13105-15.
doi: 10.1128/JVI.79.20.13105-13115.2005.

Inhibitors of respiratory syncytial virus replication target cotranscriptional mRNA guanylylation by viral RNA-dependent RNA polymerase

Michel Liuzzi  1 , Stephen W Mason, Mireille Cartier, Carol Lawetz, Robert S McCollum, Nathalie Dansereau, Gordon Bolger, Nicole Lapeyre, Yvon Gaudette, Lisette Lagacé, Marie-Josée Massariol, Florence Dô, Paul Whitehead, Lyne Lamarre, Erika Scouten, Josée Bordeleau, Serge Landry, Jean Rancourt, Gulrez Fazal, Bruno Simoneau
Affiliations

Inhibitors of respiratory syncytial virus replication target cotranscriptional mRNA guanylylation by viral RNA-dependent RNA polymerase

Michel Liuzzi et al. J Virol. 2005 Oct.

Abstract

Respiratory syncytial virus (RSV) is a major cause of respiratory illness in infants, immunocompromised patients, and the elderly. New antiviral agents would be important tools in the treatment of acute RSV disease. RSV encodes its own RNA-dependent RNA polymerase that is responsible for the synthesis of both genomic RNA and subgenomic mRNAs. The viral polymerase also cotranscriptionally caps and polyadenylates the RSV mRNAs at their 5' and 3' ends, respectively. We have previously reported the discovery of the first nonnucleoside transcriptase inhibitor of RSV polymerase through high-throughput screening. Here we report the design of inhibitors that have improved potency both in vitro and in antiviral assays and that also exhibit activity in a mouse model of RSV infection. We have isolated virus with reduced susceptibility to this class of inhibitors. The mutations conferring resistance mapped to a novel motif within the RSV L gene, which encodes the catalytic subunit of RSV polymerase. This motif is distinct from the catalytic region of the L protein and bears some similarity to the nucleotide binding domain within nucleoside diphosphate kinases. These findings lead to the hypothesis that this class of inhibitors may block synthesis of RSV mRNAs by inhibiting guanylylation of viral transcripts. We show that short transcripts produced in the presence of inhibitor in vitro do not contain a 5' cap but, instead, are triphosphorylated, confirming this hypothesis. These inhibitors constitute useful tools for elucidating the molecular mechanism of RSV capping and represent valid leads for the development of novel anti-RSV therapeutics.

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Figures

FIG. 1.
FIG. 1.
Molecular structure of RSV polymerase inhibitors. Compounds were synthesized using a method similar to that initially described for compound A (1). Details will be published elsewhere (B. Simoneau, unpublished data).
FIG. 2.
FIG. 2.
Effect of compound A on RSV transcripts. Transcription reactions were performed with 50 nM [α-33P]CTP in the presence of 0 to 75 μM of compound A. Denatured transcripts were run on a 6% polyacrylamide-7 M urea gel. The bracket indicates the position of small RNA transcripts produced only in the presence of compound A. The asterisk indicates nonspecific products by contaminating nucleotidyl transferase. The relative migration of 50- and 100-nucleotide (nt) markers and that of RSV transcripts are indicated.
FIG. 3.
FIG. 3.
Mapping of RSV polymerase inhibitor resistance mutations to a central region of RSV L having similarity to NDKs. (A) Single amino acid changes in three resistant RSV isolates mapped to a central region of RSV L at amino acids 1269, 1381, and 1421. The highlighted motifs have known functions for polymerase activity (black boxes) (32) and methyltransferase activity (yellow diamonds) (11). (B) The L protein sequences from the indicated viruses were used as input for the program MEME. The position of the starting amino acid is in parentheses. Alignment of the 5C and 1C motifs (blue boxes) is shown with the similar region in human NDK (P22392). This NDK sequence (NDKB HUM) is colored according to the conservation of residues found in an alignment of the following NDKs for which an X-ray structure was obtained: P52174, P22887, P15266, and P22392. Residues in red are invariant, residues in blue are highly conserved, and those in green are weakly conserved. Arrows indicate residues that contact nucleotides in NDKs: arginine and threonine contact the β-phosphate and histidine binds the γ-phosphate (31). The circled residue in the RSV sequence is mutated in Cr-19. NDV, Newcastle disease virus; VSV, vesicular stomatitis virus; Pred. S.S., predicted secondary structure; 1NUE (X-ray), experimental secondary structure according to the 1NUE PDB record; H, helix; E, extended.
FIG. 4.
FIG. 4.
RSV polymerase preparations produce both cap 0 and cap 1. HPLC analyses of RNase T2-digested RNA on a Partisil 5 SAX column using a gradient of potassium phosphate (pH 5.5) from 4 to 800 mM. (A) Capped RSV RNA prepared by in vitro transcription in the presence of 0.2 μM [3H]SAM and purified on Oligotex resin followed by treatment with RNase T2 and analysis by HPLC. (B) Capped RSV RNA prepared by in vitro transcription in the presence of 10 μM [3H]SAM was treated as in panel A. (C) Capped NS2 RNA was produced by in vitro transcription using T7 RNA polymerase followed by incubation in the presence of vaccinia virus capping enzyme and [α-32P]GTP. This RNA was then purified on an RNeasy column, treated with RNase T2, and analyzed as in panel A. The peaks labeled cap 0 and cap 1 refer to 7mGpppGp and 7mGpppGmpGp, respectively.
FIG. 5.
FIG. 5.
RSV polymerase inhibitors prevent RSV mRNA guanylylation. RSV transcripts were labeled with [α-32P]GTP in the presence or absence of inhibitor (compound E), digested with nuclease, and analyzed by HPLC as described in the legend of Fig. 4, except that the potassium phosphate gradient used in these experiments (and in those shown in Fig. 6) was 4 to 1,000 mM, instead of 4 to 800 mM. (A) RNase T2 digestion of RSV mRNA in absence of inhibitor. (B) RNase T2 digestion of RSV mRNA in presence of inhibitor. (C) RNase T2 and CIP treatment of RSV mRNA in presence of inhibitor. (D) NP1 digestion of RSV mRNA in presence of inhibitor. (E) T7 polymerase synthesized-NS2 RNA after RNase T2 digestion. (F) T7 polymerase-synthesized NS2 RNA after NP1 digestion. Cap 0 represents 7mGpppGp and cap 1 represents 7mGpppGmpGp. Note that although other inhibitors were more potent, compound E was used in this experiment due to better compound solubility. The y axis, in units of mV, is a measure of radioactivity by the Berthold monitor, which converts radioisotope decay to pulses of electricity (1,000 mV is approximately 8,500 cpm). The retention time of the peak in panel B varied from 48.3 to 50.9 min over seven experiments, whereas the retention time of the peak in panel E varied from 48 to 49 min.
FIG. 6.
FIG. 6.
Analyses of labeled and unlabeled cap and nucleotide standards. (A) Unlabeled commercially available standards (as indicated) were chromatographed on a Partisil 5 SAX column as described in the legend of Fig. 5, except that the peaks were detected by UV absorbance at 254 nm. Each product was analyzed separately in order to determine the identity of the peaks when all standards were chromatographed together as presented in the panel. (B) Capped NS2 RNA was produced by in vitro transcription using T7 RNA polymerase followed by incubation in the presence of human capping enzyme (obtained from A. Shatkin, University of Medicine and Dentistry of New Jersey) and [α-32P]GTP. The RNA was purified on an RNeasy column treated with RNase T2 and analyzed as described in the legend of Fig. 5. (C) Capped NS2 RNA prepared with human capping enzyme, human methyltransferase (obtained from A. Shatkin), and [α-32P]GTP was purified on an RNeasy column and analyzed as in panel B. Note that methylation of the guanylated RNA in panel B is incomplete in panel C. Human methyltransferase only methylates the capping guanine resulting in cap 0 (35). (D) Plot of retention time from HPLC column (x axis) versus calculated charge/mass ratio (y axis) for standards from panels A, B, and C. Also included in the plot is the retention time of the product shown in Fig. 5D. Linear regression of the plot produces the formula shown with R2 = 0.9382, indicating a reasonable fit of the data points.
FIG. 6.
FIG. 6.
Analyses of labeled and unlabeled cap and nucleotide standards. (A) Unlabeled commercially available standards (as indicated) were chromatographed on a Partisil 5 SAX column as described in the legend of Fig. 5, except that the peaks were detected by UV absorbance at 254 nm. Each product was analyzed separately in order to determine the identity of the peaks when all standards were chromatographed together as presented in the panel. (B) Capped NS2 RNA was produced by in vitro transcription using T7 RNA polymerase followed by incubation in the presence of human capping enzyme (obtained from A. Shatkin, University of Medicine and Dentistry of New Jersey) and [α-32P]GTP. The RNA was purified on an RNeasy column treated with RNase T2 and analyzed as described in the legend of Fig. 5. (C) Capped NS2 RNA prepared with human capping enzyme, human methyltransferase (obtained from A. Shatkin), and [α-32P]GTP was purified on an RNeasy column and analyzed as in panel B. Note that methylation of the guanylated RNA in panel B is incomplete in panel C. Human methyltransferase only methylates the capping guanine resulting in cap 0 (35). (D) Plot of retention time from HPLC column (x axis) versus calculated charge/mass ratio (y axis) for standards from panels A, B, and C. Also included in the plot is the retention time of the product shown in Fig. 5D. Linear regression of the plot produces the formula shown with R2 = 0.9382, indicating a reasonable fit of the data points.
FIG. 7.
FIG. 7.
Model for cotranscriptional guanylylation and action of RSV transcriptase inhibitors. See text for details.
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