scholarly journals Optimization of Potent and Selective Quinazolinediones: Inhibitors of Respiratory Syncytial Virus That Block RNA-Dependent RNA-Polymerase Complex Activity

2014 ◽  
Vol 57 (24) ◽  
pp. 10314-10328 ◽  
Author(s):  
Daljit S. Matharu ◽  
Daniel P. Flaherty ◽  
Denise S. Simpson ◽  
Chad E. Schroeder ◽  
Donghoon Chung ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
Rna Polymerase ◽  
Complex Activity ◽  
Rna Dependent Rna Polymerase ◽  
Polymerase Complex ◽  
Syncytial Virus

Biochemical Characterization of Respiratory Syncytial Virus RNA-Dependent RNA Polymerase Complex

2020 ◽  
Vol 6 (10) ◽  
pp. 2800-2811
Author(s):  
Anand Balakrishnan ◽  
Edmund Price ◽  
Catherine Luu ◽  
Jacob Shaul ◽  
Charles Wartchow ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
Rna Polymerase ◽  
Biochemical Characterization ◽  
Rna Dependent Rna Polymerase ◽  
Polymerase Complex ◽  
Virus Rna ◽  
Syncytial Virus

scholarly journals Nonnucleoside Inhibitor of Measles Virus RNA-Dependent RNA Polymerase Complex Activity

2007 ◽  
Vol 51 (7) ◽  
pp. 2293-2303 ◽  
Author(s):  
Laura K. White ◽  
Jeong-Joong Yoon ◽  
Jin K. Lee ◽  
Aiming Sun ◽  
Yuhong Du ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
High Throughput Screening ◽  
Measles Virus ◽  
Specific Inhibitor ◽  
Cross Resistance ◽  
Complex Activity ◽  
Rna Dependent Rna Polymerase ◽  
Developmental Potential ◽  
Polymerase Complex ◽  
Fusion Inhibitors

ABSTRACT Paramyxoviruses comprise several major human pathogens. Although a live-attenuated vaccine protects against measles virus (MV), a member of the paramyxovirus family, the virus remains a principal cause of worldwide mortality and accounts for approximately 21 million cases and 300,000 to 400,000 deaths annually. The development of novel antivirals that allow improved case management of severe measles and silence viral outbreaks is thus highly desirable. We have previously described the development of novel MV fusion inhibitors. The potential for preexisting or emerging resistance in the field constitutes the rationale for the identification of additional MV inhibitors with a diverse target spectrum. Here, we report the development and implementation of a cell-based assay for high-throughput screening of MV antivirals, which has yielded several hit candidates. Following confirmation by secondary assays and chemical synthesis, the most potent hit was found to act as a target-specific inhibitor of MV replication with desirable drug-like properties. The compound proved highly active against multiple primary isolates of diverse MV genotypes currently circulating worldwide, showing active concentrations of 35 to 145 nM. Significantly, it does not interfere with viral entry and lacks cross-resistance with the MV fusion inhibitor class. Mechanistic characterization on a subinfection level revealed that the compound represents a first-in-class nonnucleoside inhibitor of MV RNA-dependent RNA polymerase complex activity. Singly or in combination with the fusion inhibitors, this novel compound class has high developmental potential as a potent therapeutic against MV and will likely further the mechanistic characterization of the viral polymerase complex.

scholarly journals Inhibitors of Respiratory Syncytial Virus Replication Target Cotranscriptional mRNA Guanylylation by Viral RNA-Dependent RNA Polymerase

2005 ◽  
Vol 79 (20) ◽  
pp. 13105-13115 ◽  
Author(s):  
Michel Liuzzi ◽  
Stephen W. Mason ◽  
Mireille Cartier ◽  
Carol Lawetz ◽  
Robert S. McCollum ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
Rna Polymerase ◽  
High Throughput Screening ◽  
Antiviral Agents ◽  
Respiratory Illness ◽  
The Elderly ◽  
Transcriptase Inhibitor ◽  
Rna Dependent Rna Polymerase ◽  
Syncytial Virus

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.

Intrinsic Disorder to Order Transitions in the Scaffold Phosphoprotein P from the Respiratory Syncytial Virus RNA Polymerase Complex

Biochemistry ◽  
2016 ◽  
Vol 55 (10) ◽  
pp. 1441-1454 ◽  
Author(s):  
María G. Noval ◽  
Sebastian A. Esperante ◽  
Ivana G. Molina ◽  
Lucía B. Chemes ◽  
Gonzalo de Prat-Gay
Keyword(s):  
Respiratory Syncytial Virus ◽  
Rna Polymerase ◽  
Intrinsic Disorder ◽  
Polymerase Complex ◽  
Virus Rna ◽  
Syncytial Virus

scholarly journals Generation of Bovine Respiratory Syncytial Virus (BRSV) from cDNA: BRSV NS2 Is Not Essential for Virus Replication in Tissue Culture, and the Human RSV Leader Region Acts as a Functional BRSV Genome Promoter

1999 ◽  
Vol 73 (1) ◽  
pp. 251-259 ◽  
Author(s):  
Ursula J. Buchholz ◽  
Stefan Finke ◽  
Karl-Klaus Conzelmann
Keyword(s):  
Cell Culture ◽  
Respiratory Syncytial Virus ◽  
Rna Polymerase ◽  
Virus Replication ◽  
Nonstructural Protein ◽  
Bovine Respiratory Syncytial Virus ◽  
T7 Rna Polymerase ◽  
Leader Region ◽  
Successful Recovery ◽  
Syncytial Virus

ABSTRACT In order to generate recombinant bovine respiratory syncytial virus (BRSV), the genome of BRSV strain A51908, variant ATue51908, was cloned as cDNA. We provide here the sequence of the BRSV genome ends and of the entire L gene. This completes the sequence of the BRSV genome, which comprises a total of 15,140 nucleotides. To establish a vaccinia virus-free recovery system, a BHK-derived cell line stably expressing T7 RNA polymerase was generated (BSR T7/5). Recombinant BRSV was reproducibly recovered from cDNA constructs after T7 RNA polymerase-driven expression of antigenome sense RNA and of BRSV N, P, M2, and L proteins from transfected plasmids. Chimeric viruses in which the BRSV leader region was replaced by the human respiratory syncytial virus (HRSV) leader region replicated in cell culture as efficiently as their nonchimeric counterparts, demonstrating that allcis-acting sequences of the HRSV promoter are faithfully recognized by the BRSV polymerase complex. In addition, we report the successful recovery of a BRSV mutant lacking the complete NS2 gene, which encodes a nonstructural protein of unknown function. The NS2-deficient BRSV replicated autonomously and could be passaged, demonstrating that NS2 is not essential for virus replication in cell culture. However, growth of the mutant was considerably slower than and final infectious titers were reduced by a factor of at least 10 compared to wild-type BRSV, indicating that NS2 provides a supporting factor required for full replication capacity.

scholarly journals Cryo-EM structure of the respiratory syncytial virus RNA polymerase

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Dongdong Cao ◽  
Yunrong Gao ◽  
Claire Roesler ◽  
Samantha Rice ◽  
Paul D’Cunha ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
Rna Polymerase ◽  
Virus Rna ◽  
Syncytial Virus

scholarly journals The Respiratory Syncytial Virus M2-1 Protein Forms Tetramers and Interacts with RNA and P in a Competitive Manner

2009 ◽  
Vol 83 (13) ◽  
pp. 6363-6374 ◽  
Author(s):  
Thi-Lan Tran ◽  
Nathalie Castagné ◽  
Virginie Dubosclard ◽  
Sylvie Noinville ◽  
Emmanuelle Koch ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
Amino Acid ◽  
Rna Binding ◽  
Amino Acid Residues ◽  
Polymerase Complex ◽  
Bacterial Rna ◽  
Infected Cells ◽  
Syncytial Virus ◽  
Α Helix ◽  
Zinc Binding Domain

ABSTRACT The respiratory syncytial virus (RSV) M2-1 protein is an essential cofactor of the viral RNA polymerase complex and functions as a transcriptional processivity and antitermination factor. M2-1, which exists in a phosphorylated or unphosphorylated form in infected cells, is an RNA-binding protein that also interacts with some of the other components of the viral polymerase complex. It contains a CCCH motif, a putative zinc-binding domain that is essential for M2-1 function, at the N terminus. To gain insight into its structural organization, M2-1 was produced as a recombinant protein in Escherichia coli and purified to >95% homogeneity by using a glutathione S-transferase (GST) tag. The GST-M2-1 fusion proteins were copurified with bacterial RNA, which could be eliminated by a high-salt wash. Circular dichroism analysis showed that M2-1 is largely α-helical. Chemical cross-linking, dynamic light scattering, sedimentation velocity, and electron microscopy analyses led to the conclusion that M2-1 forms a 5.4S tetramer of 89 kDa and ∼7.6 nm in diameter at micromolar concentrations. By using a series of deletion mutants, the oligomerization domain of M2-1 was mapped to a putative α-helix consisting of amino acid residues 32 to 63. When tested in an RSV minigenome replicon system using a luciferase gene as a reporter, an M2-1 deletion mutant lacking this region showed a significant reduction in RNA transcription compared to wild-type M2-1, indicating that M2-1 oligomerization is essential for the activity of the protein. We also show that the region encompassing amino acid residues 59 to 178 binds to P and RNA in a competitive manner that is independent of the phosphorylation status of M2-1.

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