Comparison of RNA synthesis initiation properties of non-segmented negative strand RNA virus polymerases.

It is generally thought that the promoters of non-segmented, negative strand RNA viruses (nsNSVs) direct the polymerase to initiate RNA synthesis exclusively opposite the 3´ terminal nucleotide of the genome RNA by a de novo (primer independent) initiation mechanism. However, recent studies have revealed that there is diversity between different nsNSVs with pneumovirus promoters directing the polymerase to initiate at positions 1 and 3 of the genome, and ebolavirus polymerases being able to initiate at position 2 on the template. Studies with other RNA viruses have shown that polymerases that engage in de novo initiation opposite position 1 typically have structural features to stabilize the initiation complex and ensure efficient and accurate initiation. This raised the question of whether different nsNSV polymerases have evolved fundamentally different structural properties to facilitate initiation at different sites on their promoters. Here we examined the functional properties of polymerases of respiratory syncytial virus (RSV), a pneumovirus, human parainfluenza virus type 3 (PIV-3), a paramyxovirus, and Marburg virus (MARV), a filovirus, both on their cognate promoters and on promoters of other viruses. We found that in contrast to the RSV polymerase, which initiated at positions 1 and 3 of its promoter, the PIV-3 and MARV polymerases initiated exclusively at position 1 on their cognate promoters. However, all three polymerases could recognize and initiate from heterologous promoters, with the promoter sequence playing a key role in determining initiation site selection. In addition to examining de novo initiation, we also compared the ability of the RSV and PIV-3 polymerases to engage in back-priming, an activity in which the promoter template is folded into a secondary structure and nucleotides are added to the template 3´ end. This analysis showed that whereas the RSV polymerase was promiscuous in back-priming activity, the PIV-3 polymerase generated barely detectable levels of back-primed product, irrespective of promoter template sequence. Overall, this study shows that the polymerases from these three nsNSV families are fundamentally similar in their initiation properties, but have differences in their abilities to engage in back-priming.

Mechanism for de novo initiation at two sites in the respiratory syncytial virus promoter.

The respiratory syncytial virus (RSV) RNA dependent RNA polymerase (RdRp) initiates two RNA synthesis processes from the viral promoter: genome replication from position 1U and mRNA transcription from position 3C. Here, we examined the mechanism by which a single promoter can direct initiation from two sites. We show that initiation at 1U and 3C occurred independently of each other, and that the same RdRp was capable of precisely selecting the two sites. The RdRp preferred to initiate at 3C, but initiation site selection could be modulated by the relative concentrations of ATP versus GTP. Analysis of template mutations indicated that the RdRp could bind ATP and CTP, or GTP, independently of template nucleotides. The data suggest a model in which innate affinity of the RdRp for particular NTPs, coupled with a repeating element within the promoter, allows precise initiation of replication at 1U or transcription at 3C.

Discovery of novel benzothienoazepine derivatives as potent inhibitors of respiratory syncytial virus.

The development of novel non-nucleoside inhibitors of the RSV polymerase complex is of significant clinical interest. Compounds derived from the benzothienoazepine core, such as AZ-27, are potent inhibitors of RSV viruses of the A-subgroup, but are only moderately active against the B serotype and as yet have not demonstrated activity in vivo. Herein we report the discovery of several novel families of C-2 arylated benzothienoazepine derivatives that are highly potent RSV polymerase inhibitors and reveal an exemplary structure, compound 4a, which shows low nanomolar activity against both RSV A and B viral subtypes. Furthermore, this compound is effective at suppressing viral replication, when administered intranasally, in a rodent model of RSV infection. These results suggest that compounds belonging to this chemotypes have the potential to provide superior anti-RSV agents than those currently available for clinical use.

Activation Pathway of a Nucleoside Analog Inhibiting Respiratory Syncytial Virus Polymerase.

Human respiratory syncytial virus (RSV) is a negative-sense RNA virus and a significant cause of respiratory infection in infants and the elderly. No effective vaccines or antiviral therapies are available for the treatment of RSV. ALS-8176 is a first-in-class nucleoside prodrug inhibitor of RSV replication currently under clinical evaluation. ALS-8112, the parent molecule of ALS-8176, undergoes intracellular phosphorylation, yielding the active 5'-triphosphate metabolite. The host kinases responsible for this conversion are not known. Therefore, elucidation of the ALS-8112 activation pathway is key to further understanding its conversion mechanism, particularly given its potent antiviral effects. Here, we have identified the activation pathway of ALS-8112 and show it is unlike other antiviral cytidine analogs. The first step, driven by deoxycytidine kinase (dCK), is highly efficient, while the second step limits the formation of the active 5'-triphosphate species. ALS-8112 is a 2'- and 4'-modified nucleoside analog, prompting us to investigate dCK recognition of other 2'- and 4'-modified nucleosides. Our biochemical approach along with computational modeling contributes to an enhanced structure-activity profile for dCK. These results highlight an exciting potential to optimize nucleoside analogs based on the second activation step and increased attention toward nucleoside diphosphate and triphosphate prodrugs in drug discovery.

Discovery of ß-D-2'-deoxy-2'-α-fluoro-4'-α-cyano-5-aza-7,9-dideaza adenosine as a potent nucleoside inhibitor of respiratory syncytial virus with excellent selectivity over mitochondrial RNA and DNA polymerases.

Novel 4'-substituted ß-d-2'-deoxy-2'-α-fluoro (2'd2'F) nucleoside inhibitors of respiratory syncytial virus (RSV) are reported. The introduction of 4'-substitution onto 2'd2'F nucleoside analogs resulted in compounds demonstrating potent cell based RSV inhibition, improved inhibition of the RSV polymerase by the nucleoside triphosphate metabolites, and enhanced selectivity over incorporation by mitochondrial RNA and DNA polymerases. Selectivity over the mitochondrial polymerases was found to be extremely sensitive to the specific 4'-substitution and not readily predictable. Combining the most potent and selective 4'-groups from N-nucleoside analogs onto a 2'd2'F C-nucleoside analog resulted in the identification of ß-D-2'-deoxy-2'-α-fluoro-4'-α-cyano-5-aza-7,9-dideaza adenosine as a promising nucleoside lead for RSV.

Respiratory Syncytial Virus Inhibitor AZ-27 Differentially Inhibits Different Polymerase Activities at the Promoter.

UNLABELLED: Respiratory syncytial virus (RSV) is the leading cause of pediatric respiratory disease. RSV has an RNA-dependent RNA polymerase that transcribes and replicates the viral negative-sense RNA genome. The large polymerase subunit (L) has multiple enzymatic activities, having the capability to synthesize RNA and add and methylate a cap on each of the viral mRNAs. Previous studies (H. Xiong et al., Bioorg Med Chem Lett, 23:6789-6793, 2013, http://dx.doi.org/10.1016/j.bmcl.2013.10.018; C. L. Tiong-Yip et al., Antimicrob Agents Chemother, 58:3867-3873, 2014, http://dx.doi.org/10.1128/AAC.02540-14) had identified a small-molecule inhibitor, AZ-27, that targets the L protein. In this study, we examined the effect of AZ-27 on different aspects of RSV polymerase activity. AZ-27 was found to inhibit equally both mRNA transcription and genome replication in cell-based minigenome assays, indicating that it inhibits a step common to both of these RNA synthesis processes. Analysis in an in vitro transcription run-on assay, containing RSV nucleocapsids, showed that AZ-27 inhibits synthesis of transcripts from the 3' end of the genome to a greater extent than those from the 5' end, indicating that it inhibits transcription initiation. Consistent with this finding, experiments that assayed polymerase activity on the promoter showed that AZ-27 inhibited transcription and replication initiation. The RSV polymerase also can utilize the promoter sequence to perform a back-priming reaction. Interestingly, addition of AZ-27 had no effect on the addition of up to three nucleotides by back-priming but inhibited further extension of the back-primed RNA. These data provide new information regarding the mechanism of inhibition by AZ-27. They also suggest that the RSV polymerase adopts different conformations to perform its different activities at the promoter. IMPORTANCE: Currently, there are no effective antiviral drugs to treat RSV infection. The RSV polymerase is an attractive target for drug development, but this large enzymatic complex is poorly characterized, hampering drug development efforts. AZ-27 is a small-molecule inhibitor previously shown to target the RSV large polymerase subunit (C. L. Tiong-Yip et al., Antimicrob Agents Chemother, 58:3867-3873, 2014, http://dx.doi.org/10.1128/AAC.02540-14), but its inhibitory mechanism was unknown. Understanding this would be valuable both for characterizing the polymerase and for further development of inhibitors. Here, we show that AZ-27 inhibits an early stage in mRNA transcription, as well as genome replication, by inhibiting initiation of RNA synthesis from the promoter. However, the compound does not inhibit back priming, another RNA synthesis activity of the RSV polymerase. These findings provide insight into the different activities of the RSV polymerase and will aid further development of antiviral agents against RSV.

Discovery of 4'-chloromethyl-2'-deoxy-3',5'-di-O-isobutyryl-2'-fluorocytidine (ALS-8176), a first-in-class RSV polymerase inhibitor for treatment of human respiratory syncytial virus infection.

Respiratory syncytial virus (RSV) is a leading pathogen of childhood and is associated with significant morbidity and mortality. To date, ribavirin is the only approved small molecule drug, which has limited use. The only other RSV drug is palivizumab, a monoclonal antibody, which is used for RSV prophylaxis. Clearly, there is an urgent need for small molecule RSV drugs. This article reports the design, synthesis, anti-RSV activity, metabolism, and pharmacokinetics of a series of 4'-substituted cytidine nucleosides. Among tested compounds 4'-chloromethyl-2'-deoxy-2'-fluorocytidine (2c) exhibited the most promising activity in the RSV replicon assay with an EC50 of 0.15 µM. The 5'-triphosphate of 2c (2c-TP) inhibited RSV polymerase with an IC50 of 0.02 µM without appreciable inhibition of human DNA and RNA polymerases at 100 µM. ALS-8176 (71), the 3',5'-di-O-isobutyryl prodrug of 2c, demonstrated good oral bioavailability and a high level of 2c-TP in vivo. Compound 71 is a first-in-class nucleoside RSV polymerase inhibitor that demonstrated excellent anti-RSV efficacy and safety in a phase 2 clinical RSV challenge study.

Factors affecting de novo RNA synthesis and back-priming by the respiratory syncytial virus polymerase.

Respiratory syncytial virus RNA dependent RNA polymerase (RdRp) initiates RNA synthesis from the leader (le) and trailer-complement (trc) promoters. The RdRp can also add nucleotides to the 3' end of the trc promoter by back-priming, but there is no evidence this occurs at the le promoter in infected cells. We examined how environmental factors and RNA sequence affect de novo RNA synthesis versus back-priming using an in vitro assay. We found that replacing Mg(2+) with Mn(2+) in the reaction buffer increased de novo initiation relative to back-priming, and different lengths of trc sequence were required for the two activities. Experiments with le RNA showed that back-priming occurred with this sequence in vitro, but less efficiently than with trc RNA. These findings indicate that during infection, the RdRp is governed between de novo RNA synthesis and back-priming by RNA sequence and environment, including a factor missing from the in vitro assay.

Discovery of a potent respiratory syncytial virus RNA polymerase inhibitor.

Targeting viral polymerases has been a proven and attractive strategy for antiviral drug discovery. Herein we describe our effort in improving the antiviral activity and physical properties of a series of benzothienoazepine compounds as respiratory syncytial virus (RSV) RNA polymerase inhibitors. The antiviral activity and spectrum of this class was significantly improved by exploring the amino substitution of the pyridine ring, resulting in the discovery of the most potent RSV A polymerase inhibitors reported to date.

Hsp90 inhibitors exhibit resistance-free antiviral activity against respiratory syncytial virus.

Respiratory syncytial virus (RSV) is a major cause of respiratory illness in young children, leading to significant morbidity and mortality worldwide. Despite its medical importance, no vaccine or effective therapeutic interventions are currently available. Therefore, there is a pressing need to identify novel antiviral drugs to combat RSV infections. Hsp90, a cellular protein-folding factor, has been shown to play an important role in the replication of numerous viruses. We here demonstrate that RSV requires Hsp90 for replication. Mechanistic studies reveal that inhibition of Hsp90 during RSV infection leads to the degradation of a viral protein similar in size to the RSV L protein, the viral RNA-dependent RNA polymerase, implicating it as an Hsp90 client protein. Accordingly, Hsp90 inhibitors exhibit antiviral activity against laboratory and clinical isolates of RSV in both immortalized as well as primary differentiated airway epithelial cells. Interestingly, we find a high barrier to the emergence of drug resistance to Hsp90 inhibitors, as extensive growth of RSV under conditions of Hsp90 inhibition did not yield mutants with reduced sensitivity to these drugs. Our results suggest that Hsp90 inhibitors may present attractive antiviral therapeutics for treatment of RSV infections and highlight the potential of chaperone inhibitors as antivirals exhibiting high barriers to development of drug resistance.

Respiratory syncytial virus polymerase can initiate transcription from position 3 of the leader promoter.

The mechanisms by which the respiratory syncytial virus (RSV) RNA-dependent RNA polymerase (RdRp) initiates mRNA transcription and RNA replication are poorly understood. A previous study, using an RSV minigenome, suggested that the leader (Le) promoter region at the 3' end of the genome has two initiation sites, one at position +1, opposite the 3' terminal nucleotide of the genome, and a second site at position +3, at a sequence that closely resembles the gene start (GS) signal of the RSV L gene. In this study, we show that the +3 initiation site of the Le is utilized with apparently high frequency in RSV-infected cells and yields small RNA transcripts that are heterogeneous in length but mostly approximately 25 nucleotides (nt) long. Experiments with an in vitro assay in which RSV RNA synthesis was reconstituted using purified RdRp and an RNA oligonucleotide showed that nt 1 to 14 of the Le promoter were sufficient to signal initiation from +3 and that the RdRp could access the +3 initiation site without prior initiation at +1. In a minigenome assay, nucleotide substitutions within the Le to increase its similarity to a GS signal resulted in more-efficient elongation of the RNA initiated from position +3 and a reduction in RNA initiated from the NS1 gene start signal at +45. Taken together, these data suggest a new model for initiation of sequential transcription of the RSV genes, whereby the RdRp initiates the process from a gene start-like sequence at position +3 of the Le.

The respiratory syncytial virus polymerase has multiple RNA synthesis activities at the promoter.

Respiratory syncytial virus (RSV) is an RNA virus in the Family Paramyxoviridae. Here, the activities performed by the RSV polymerase when it encounters the viral antigenomic promoter were examined. RSV RNA synthesis was reconstituted in vitro using recombinant, isolated polymerase and an RNA oligonucleotide template representing nucleotides 1-25 of the trailer complement (TrC) promoter. The RSV polymerase was found to have two RNA synthesis activities, initiating RNA synthesis from the +3 site on the promoter, and adding a specific sequence of nucleotides to the 3' end of the TrC RNA using a back-priming mechanism. Examination of viral RNA isolated from RSV infected cells identified RNAs initiated at the +3 site on the TrC promoter, in addition to the expected +1 site, and showed that a significant proportion of antigenome RNAs contained specific nucleotide additions at the 3' end, demonstrating that the observations made in vitro reflected events that occur during RSV infection. Analysis of the impact of the 3' terminal extension on promoter activity indicated that it can inhibit RNA synthesis initiation. These findings indicate that RSV polymerase-promoter interactions are more complex than previously thought and suggest that there might be sophisticated mechanisms for regulating promoter activity during infection.

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

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.

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

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.

Sequence analysis of the polymerase L gene of human respiratory syncytial virus and predicted phylogeny of nonsegmented negative-strand viruses.

The complete nucleotide sequence of the large (L) polymerase gene of human respiratory syncytial virus (RSV) strain A2 was determined by analysis of cloned-cDNAs representing the entire gene and confirmed in part by dideoxy sequencing of genomic RNA. The RSV L gene is 6578 nucleotides in length and contains a single major open reading frame that encodes a protein of 2165 amino acids. The molecular weight (250,226) and amino acid composition of the deduced RSV L protein are similar to those of other negative-strand RNA viruses. Regions of statistically significant amino acid sequence similarity were identified in pairwise global alignments of the RSV L protein with its counterparts in four paramyxoviruses (parainfluenza virus type 3, Sendai virus, measles virus, Newcastle disease virus) and two rhabdoviruses (rabies virus, vesicular stomatitis virus). In addition, amino acid sequence alignments showed that the RSV L protein has a 70-amino acid amino-terminal extension relative to the others. This is suggested to be due to the acquisition of gene overlap of the RSV L gene with its upstream neighbor, the 22K (M2) gene and the use of a new translational start site. The most highly related region among these seven proteins is located within the amino-terminal half, representing approximately 20% of each protein sequences. This region contains six discrete segments that are colinear and highly conserved in each paramyxovirus and rhabdovirus L protein, and three of these overlapped with sequence motifs found previously in other RNA-dependent RNA and DNA polymerases. A phylogenetic tree was constructed from the paramyxovirus and rhabdovirus L protein sequences to further define their relationships. The branching order indicates that RSV represents a lineage within the paramyxovirus family which is relatively distinct from the others, which in turn are more closely interrelated. Among these other members of the family Paramyxoviridae, the branching order does not entirely conform to their current taxonomic organization, providing support for its reevaluation.

Gene overlap and site-specific attenuation of transcription of the viral polymerase L gene of human respiratory syncytial virus.

Sequence analysis of the gene encoding polymerase L protein of respiratory syncytial virus showed that L-gene transcription initiates within its upstream neighbor, the gene encoding the 22-kDa protein 22K. This is an exception to the canonical mode of sequential transcription of nonoverlapping genes described for other nonsegmented negative-strand RNA viruses. As a consequence of the gene overlap, the termination/polyadenylylation signal for the 22K gene is located at nucleotides 56-68 within the L gene. L-gene transcription yielded two RNAs: an abundant, truncated, polyadenylylated transcript resulting from termination at the internal signal and a markedly less abundant large polyadenylylated transcript representing the complete L gene. This result showed that the internal termination/polyadenylylation signal is an attenuator of L-gene transcription.

DNA synthesis by RNA-containing tumor viruses.

Murine leukemia (Rauscher and Moloney strains) and sarcoma (Kirsten strain) virions, as well as the mammary tumor virus of mice, contain an RNA-dependent DNA polymerase. Optimal incorporation of deoxyribonucleoside triphosphates occurs at a critical detergent (Triton X-100) concentration (0.010-0.014%). At higher than optimal detergent concentrations the virion is seen to be disrupted and enzyme activity is lost. The virion, enzymatic activity, and newly synthesized DNA all cosediment in a sucrose gradient. Thus far the enzymatic activity has been found only in RNA viruses that have oncogenic properties.