Mesoionic Heterocyclic Compounds as Candidate Messenger RNA Cap Analogue Inhibitors of the Influenza Virus RNA Polymerase Cap-Binding Activity

2009 ◽  
Vol 19 (5) ◽  
pp. 213-218 ◽  
Author(s):  
Ian Mickleburgh ◽  
Feng Geng ◽  
Laurence Tiley
Keyword(s):  
Influenza Virus ◽  
Rna Polymerase ◽  
Messenger Rna ◽  
Binding Activity ◽  
Endonucleolytic Cleavage ◽  
Virus Rna ◽  
Fused Ring ◽  
Mesoionic Heterocycles ◽  
Potential Inhibitors

Background: An unusual feature of influenza viral messenger RNA (mRNA) synthesis is its dependence upon host cell mRNAs as a source of capped RNA primers. A crucial activity of the influenza polymerase is to steal these primers by binding and cleaving the caps from host mRNAs. The recent structural analysis of the cap-binding fragment of the influenza virus PB2 protein has highlighted the importance of the mesoionic properties of the N7-methylguanine (N7mG) component of the mRNA cap in this interaction. Methods: A series of mesoionic heterocycles with 5,6-fused ring systems analogous to the N7mG component of mRNA cap structures were synthesized and examined for the ability to inhibit the cap-binding activity of the influenza virus RNA polymerase complex using a bead-based in vitro cap-binding assay. Results: None of the compounds tested were able to significantly inhibit binding and subsequent endonucleolytic cleavage of a synthetic radiolabelled capped mRNA substrate by recombinant influenza virus polymerase in vitro. Conclusions: Compounds analogous to the mesoionic N7mG component of mRNA cap structures comprise a large class of potential inhibitors of the influenza virus polymerase. Although this preliminary assessment of a small group of related analogues was unsuccessful, further screening of this class of compounds is warranted.

scholarly journals Analysis of Ribonucleotide 5′-Triphosphate Analogs as Potential Inhibitors of Zika Virus RNA-Dependent RNA Polymerase by Using Nonradioactive Polymerase Assays

2016 ◽  
Vol 61 (3) ◽  
Author(s):  
Gaofei Lu ◽  
Gregory R. Bluemling ◽  
Paul Collop ◽  
Michael Hager ◽  
Damien Kuiper ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Zika Virus ◽  
Nonstructural Protein ◽  
Rna Dependent Rna Polymerase ◽  
Antiviral Compounds ◽  
Double Stranded Dna ◽  
Virus Rna ◽  
Potential Inhibitors

ABSTRACT Zika virus (ZIKV) is an emerging human pathogen that is spreading rapidly through the Americas and has been linked to the development of microcephaly and to a dramatically increased number of Guillain-Barré syndrome cases. Currently, no vaccine or therapeutic options for the prevention or treatment of ZIKV infections exist. In the study described in this report, we expressed, purified, and characterized full-length nonstructural protein 5 (NS5) and the NS5 polymerase domain (NS5pol) of ZIKV RNA-dependent RNA polymerase. Using purified NS5, we developed an in vitro nonradioactive primer extension assay employing a fluorescently labeled primer-template pair. Both purified NS5 and NS5pol can carry out in vitro RNA-dependent RNA synthesis in this assay. Our results show that Mn2+ is required for enzymatic activity, while Mg2+ is not. We found that ZIKV NS5 can utilize single-stranded DNA but not double-stranded DNA as a template or a primer to synthesize RNA. The assay was used to compare the efficiency of incorporation of analog 5′-triphosphates by the ZIKV polymerase and to calculate their discrimination versus that of natural ribonucleotide triphosphates (rNTPs). The 50% inhibitory concentrations for analog rNTPs were determined in an alternative nonradioactive coupled-enzyme assay. We determined that, in general, 2′-C-methyl- and 2′-C-ethynyl-substituted analog 5′-triphosphates were efficiently incorporated by the ZIKV polymerase and were also efficient chain terminators. Derivatives of these molecules may serve as potential antiviral compounds to be developed to combat ZIKV infection. This report provides the first characterization of ZIKV polymerase and demonstrates the utility of in vitro polymerase assays in the identification of potential ZIKV inhibitors.

scholarly journals Mechanism of Action of T-705 against Influenza Virus

2005 ◽  
Vol 49 (3) ◽  
pp. 981-986 ◽  
Author(s):  
Yousuke Furuta ◽  
Kazumi Takahashi ◽  
Masako Kuno-Maekawa ◽  
Hidehiro Sangawa ◽  
Sayuri Uehara ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Rna Polymerase ◽  
Mdck Cells ◽  
Specific Inhibitor ◽  
Polymerase Activity ◽  
Virus Activity ◽  
Virus Rna ◽  
Cellular Dna

ABSTRACT T-705, a substituted pyrazine compound, has been found to exhibit potent anti-influenza virus activity in vitro and in vivo. In a time-of-addition study, it was indicated that T-705 targeted an early to middle stage of the viral replication cycle but had no effect on the adsorption or release stage. The anti-influenza virus activity of T-705 was attenuated by addition of purines and purine nucleosides, including adenosine, guanosine, inosine, and hypoxanthine, whereas pyrimidines did not affect its activity. T-705-4-ribofuranosyl-5′-triphosphate (T-705RTP) and T-705-4-ribofuranosyl-5′-monophosphate (T-705RMP) were detected in MDCK cells treated with T-705. T-705RTP inhibited influenza virus RNA polymerase activity in a dose-dependent and a GTP-competitive manner. Unlike ribavirin, T-705 did not have an influence on cellular DNA or RNA synthesis. Inhibition of cellular IMP dehydrogenase by T-705RMP was about 150-fold weaker than that by ribavirin monophosphate, indicating the specificity of the anti-influenza virus activity and lower level of cytotoxicity of T-705. These results suggest that T-705RTP, which is generated in infected cells, may function as a specific inhibitor of influenza virus RNA polymerase and contributes to the selective anti-influenza virus activity of T-705.

scholarly journals Role of the influenza virus heterotrimeric RNA polymerase complex in the initiation of replication

2006 ◽  
Vol 87 (11) ◽  
pp. 3373-3377 ◽  
Author(s):  
Tao Deng ◽  
Jane L. Sharps ◽  
George G. Brownlee
Keyword(s):  
Influenza Virus ◽  
Rna Polymerase ◽  
Replication Initiation ◽  
Polymerase Complex ◽  
Virus Rna ◽  
Initiation Of Replication ◽  
Rna Genome ◽  
Transcription And Replication

Both transcription and replication of the influenza virus RNA genome are catalysed by a virus-specific RNA polymerase. Recently, an in vitro assay, based on the synthesis of pppApG, for the initiation of replication by recombinant RNA polymerase in the absence of added primer was described. Here, these findings are extended to show that adenosine, AMP and ADP can each substitute for ATP in reactions catalysed by either recombinant ribonucleoprotein or RNA polymerase complexes with either model virion RNA (vRNA) or cRNA promoters. The use of either adenosine or AMP, rather than ATP, provides a convenient, sensitive and easy assay of replication initiation. Moreover, no pppApG was detected when a PB1–PA dimer, rather than the trimeric polymerase, was used to catalyse synthesis, contrasting with a previous report using baculovirus-expressed influenza RNA polymerase. Overall, it is suggested that the heterotrimeric polymerase is essential for the initiation of replication.

scholarly journals Different De Novo Initiation Strategies Are Used by Influenza Virus RNA Polymerase on Its cRNA and Viral RNA Promoters during Viral RNA Replication

2006 ◽  
Vol 80 (5) ◽  
pp. 2337-2348 ◽  
Author(s):  
Tao Deng ◽  
Frank T. Vreede ◽  
George G. Brownlee
Keyword(s):  
Influenza Virus ◽  
Rna Polymerase ◽  
De Novo ◽  
Viral Rna ◽  
Virus Rna ◽  
Initiation Of Replication ◽  
Replicative Intermediates ◽  
Internal Initiation

ABSTRACT Various mechanisms are used by single-stranded RNA viruses to initiate and control their replication via the synthesis of replicative intermediates. In general, the same virus-encoded polymerase is responsible for both genome and antigenome strand synthesis from two different, although related promoters. Here we aimed to elucidate the mechanism of initiation of replication by influenza virus RNA polymerase and establish whether initiation of cRNA and viral RNA (vRNA) differed. To do this, two in vitro replication assays, which generated transcripts that had 5′ triphosphate end groups characteristic of authentic replication products, were developed. Surprisingly, mutagenesis screening suggested that the polymerase initiated pppApG synthesis internally on the model cRNA promoter, whereas it initiated pppApG synthesis terminally on the model vRNA promoter. The internally synthesized pppApG could subsequently be used as a primer to realign, by base pairing, to the terminal residues of both the model cRNA and vRNA promoters. In vivo evidence, based on the correction of a mutated or deleted residue 1 of a cRNA chloramphenicol acetyltransferase reporter construct, supported this internal initiation and realignment model. Thus, influenza virus RNA polymerase uses different initiation strategies on its cRNA and vRNA promoters. To our knowledge, this is novel and has not previously been described for any viral RNA-dependent RNA polymerase. Such a mechanism may have evolved to maintain genome integrity and to control the level of replicative intermediates in infected cells.

scholarly journals Influenza Virion-Derived Viral Ribonucleoproteins Synthesize both mRNA and cRNA In Vitro

2006 ◽  
Vol 81 (5) ◽  
pp. 2196-2204 ◽  
Author(s):  
F. T. Vreede ◽  
G. G. Brownlee
Keyword(s):  
Influenza Virus ◽  
Life Cycle ◽  
Rna Polymerase ◽  
Influenza A ◽  
De Novo ◽  
Early Results ◽  
Virus Rna ◽  
Replicative Intermediates ◽  
Viral Genomic Rna

ABSTRACT The mechanisms regulating the synthesis of mRNA, cRNA, and viral genomic RNA (vRNA) by the influenza A virus RNA-dependent RNA polymerase are not fully understood. Early results suggested that the RNA polymerase “switched” from a transcriptase to a replicase during the viral life cycle in response to the expression of viral proteins. However, recently an alternative model suggesting that replication of influenza virus is regulated by stabilization of the replicative intermediates was proposed. According to this model, the virion-associated polymerase is capable of synthesizing both mRNA and cRNA. We now demonstrate that virion-derived viral ribonucleoproteins (vvRNPs) synthesize both mRNA and cRNA in vitro in the absence of non-virion-associated RNA polymerase or nucleoproteins. The authenticity of the in vitro-transcribed mRNA and cRNA was confirmed by terminal sequence analysis. The addition of non-virion-associated polymerase or NP had no effect on vvRNP activity. De novo synthesis of cRNA was found to be more sensitive than the capped primer-dependent synthesis of mRNA to the concentration of ATP, CTP, and GTP. We conclude that vvRNPs intrinsically possess both transcriptase and replicase activities and that there is no switch in the synthesis of mRNA to cRNA during the influenza virus life cycle.

scholarly journals Expression of Functional Influenza Virus RNA Polymerase in the Methylotrophic Yeast Pichia pastoris

2000 ◽  
Vol 74 (9) ◽  
pp. 4074-4084 ◽  
Author(s):  
Jung-Shan Hwang ◽  
Kazunori Yamada ◽  
Ayae Honda ◽  
Kohji Nakade ◽  
Akira Ishihama
Keyword(s):  
Influenza Virus ◽  
Pichia Pastoris ◽  
Rna Polymerase ◽  
Rna Synthesis ◽  
Methylotrophic Yeast ◽  
Affinity Column ◽  
Kinetic Properties ◽  
Oxidase Gene ◽  
Virus Rna

ABSTRACT Influenza virus RNA polymerase with the subunit composition PB1-PB2-PA is a multifunctional enzyme with the activities of both synthesis and cleavage of RNA and is involved in both transcription and replication of the viral genome. In order to produce large amounts of the functional viral RNA polymerase sufficient for analysis of its structure-function relationships, the cDNAs for RNA segments 1, 2, and 3 of influenza virus A/PR/8, each under independent control of the alcohol oxidase gene promoter, were integrated into the chromosome of the methylotrophic yeast Pichia pastoris. Simultaneous expression of all three P proteins in the yeast P. pastoriswas achieved by the addition of methanol. To purify the P protein complexes, a sequence coding for a histidine tag was added to the PB2 protein gene at its N terminus. Starting from the induced P. pastoris cell lysate, we partially purified a 3P complex by Ni2+-agarose affinity column chromatography. The 3P complex showed influenza virus model RNA-directed and ApG-primed RNA synthesis in vitro but was virtually inactive without addition of template or primer. The kinetic properties of model template-directed RNA synthesis and the requirements for template sequence were analyzed using the 3P complex. Furthermore, the 3P complex showed capped RNA-primed RNA synthesis. Thus, we conclude that functional influenza virus RNA polymerase with the catalytic properties of a transcriptase is formed in the methylotrophic yeast P. pastoris.

scholarly journals Differential Roles of Viral RNA and cRNA in Functional Modulation of the Influenza Virus RNA Polymerase

2001 ◽  
Vol 276 (33) ◽  
pp. 31179-31185 ◽  
Author(s):  
Ayae Honda ◽  
Atsushi Endo ◽  
Kiyohisa Mizumoto ◽  
Akira Ishihama
Keyword(s):  
Influenza Virus ◽  
Rna Polymerase ◽  
Viral Rna ◽  
Virus Rna ◽  
Functional Modulation

scholarly journals Temperature-Sensitive Mutants in the Influenza A Virus RNA Polymerase: Alterations in the PA Linker Reduce Nuclear Targeting of the PB1-PA Dimer and Result in Viral Attenuation

2015 ◽  
Vol 89 (12) ◽  
pp. 6376-6390 ◽  
Author(s):  
Bruno Da Costa ◽  
Alix Sausset ◽  
Sandie Munier ◽  
Alexandre Ghounaris ◽  
Nadia Naffakh ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Rna Polymerase ◽  
Nuclear Import ◽  
Influenza A ◽  
Rational Design ◽  
Hot Spot ◽  
Temperature Sensitive ◽  
Nuclear Targeting ◽  
Virus Rna ◽  
Endonuclease Domain

ABSTRACTThe influenza virus RNA-dependent RNA polymerase catalyzes genome replication and transcription within the cell nucleus. Efficient nuclear import and assembly of the polymerase subunits PB1, PB2, and PA are critical steps in the virus life cycle. We investigated the structure and function of the PA linker (residues 197 to 256), located between its N-terminal endonuclease domain and its C-terminal structured domain that binds PB1, the polymerase core. Circular dichroism experiments revealed that the PA linker by itself is structurally disordered. A large series of PA linker mutants exhibited a temperature-sensitive (ts) phenotype (reduced viral growth at 39.5°C versus 37°C/33°C), suggesting an alteration of folding kinetic parameters. Thetsphenotype was associated with a reduced efficiency of replication/transcription of a pseudoviral reporter RNA in a minireplicon assay. Using a fluorescent-tagged PB1, we observed thattsand lethal PA mutants did not efficiently recruit PB1 to reach the nucleus at 39.5°C. A protein complementation assay using PA mutants, PB1, and β-importin IPO5 tagged with fragments of theGaussia princepsluciferase showed that increasing the temperature negatively modulated the PA-PB1 and the PA-PB1-IPO5 interactions or complex stability. The selection of revertant viruses allowed the identification of different types of compensatory mutations located in one or the other of the three polymerase subunits. Twotsmutants were shown to be attenuated and able to induce antibodies in mice. Taken together, our results identify a PA domain critical for PB1-PA nuclear import and that is a “hot spot” to engineertsmutants that could be used to design novel attenuated vaccines.IMPORTANCEBy targeting a discrete domain of the PA polymerase subunit of influenza virus, we were able to identify a series of 9 amino acid positions that are appropriate to engineer temperature-sensitive (ts) mutants. This is the first time that a large number oftsmutations were engineered in such a short domain, demonstrating that rational design oftsmutants can be achieved. We were able to associate this phenotype with a defect of transport of the PA-PB1 complex into the nucleus. Reversion substitutions restored the ability of the complex to move to the nucleus. Two of thesetsmutants were shown to be attenuated and able to produce antibodies in mice. These results are of high interest for the design of novel attenuated vaccines and to develop new antiviral drugs.

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