Biochemical studies on capped RNA primers identify a class of oligonucleotide inhibitors of the influenza virus RNA polymerase.

Chung, Thomas D.Y.; Cianci, Christopher; Hagen, Moira; Terry, Brian; Matthews, James T.; Krystal, Mark; Colonno, Richard J.

doi:10.1073/pnas.91.6.2372

Cited by 50 publications

(58 citation statements)

References 30 publications

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“…These capped short RNA fragments are potential inhibitors of cap-dependent transcription in vitro. A recent study has confirmed earlier data (Bouloy et al, 1978;Ulmanen et al, 1981;Braam et al, 1983;Kawakami et al, 1983;Kato et al, 1985) that suggest that priming by capped oligonucleotides can be uncoupled from the endonuclease activity of the influenza RNA polymerase, and has suggested the use of such inhibitors as decoys of cap-dependent transcription in vitro (Chung et al, 1994) …”

supporting

confidence: 74%

“…That is to say, the 5′-capped phosphorothioate RNA fragment was not cleaved by the endonuclease, probably because the influenza virus polymerase could not hydrolyze the phosphorothioate internucleotidic bonds between the bases in the RNA tail, for this capped RNA is long enough to be used as a primer. However, the unmodified capped substrates containing RNA chains of more 13 nt could be cleaved to the primer fragment (Chung et al, 1994;Hatta et al, 1998). Therefore, there is a minimal RNA chain length of the unmodified 5′-capped RNA fragments (Gm) required for priming activity.…”

Section: Discussionmentioning

confidence: 99%

“…An inhibitor based on the viral RNA polymerase (PB1, PB2 and PA) of influenza virus A would be ideal for both potency and specifcity. A few studies have investigated the inhibition of influenza virus replication in vitro and in vivo by Cap decoy as a potent inhibitors of capped-RNA primed transcription (Chung et al, 1994;Hatta et al, 1998).…”

Section: Discussionmentioning

confidence: 99%

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Inhibitory Effect of Modified 5′-Capped Short RNA Fragments on Influenza Virus RNA Polymerase Gene Expression

Tado

Abe

Hatta

et al. 2001

Antivir Chem Chemother

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Influenza virus employs a novel mechanism for the synthesis of its viral mRNAs. Viral mRNA synthesis requires initiation by host cell primers, specifically capped (m7Gppp Nm-containing) RNA fragments derived from host cell RNA polymerase II transcripts (Plotch et al., 1979;Krug et al., , 1981. This occurs in the nucleus of the infected cells. Viral mRNA synthesis requires the continuous functioning of the cellular RNA polymerase II, and is therefore inhibited by α-amanitin. The host cell primers are generated by a viral cap-dependent endonuclease that cleaves the capped cellular RNAs 10-13 nucleotides from their 5′ ends, preferentially at a purine residue. Priming does not require hydrogen bonding between the capped primer fragments and the 3′-ends of the viral (v)RNA templates. Rather, priming requires the presence of a 5′-methylated cap structure. In fact, each of the methyl groups in the cap, the 2′-O-methyl on the penultimate base as well as the 7-methyl on the terminal G, strongly increase priming activity. Transcription is initiated by the incorporation of a G residue onto the 3′ end of the resulting fragments, directed by the penultimate C residue of the vRNAs. Viral mRNA chains are then elongated until a stretch of 4-7 uridine residues is reached, 17 to 22 nucleotides before the 5′ ends of the vRNAs, where transcription terminates and polyadenylate is added to the mRNAs. These capped short RNA fragments are potential inhibitors of cap-dependent transcription in vitro. A recent study has confirmed earlier data (Bouloy et al., 1978;Ulmanen et al., 1981;Braam et al., 1983;Kawakami et al., 1983;Kato et al., 1985) that suggest that priming by capped oligonucleotides can be uncoupled from the endonuclease activity of the influenza RNA polymerase, and has suggested the use of such inhibitors as decoys of cap-dependent transcription in vitro (Chung et al., 1994

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supporting

confidence: 74%

Section: Discussionmentioning

confidence: 99%

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Inhibitory Effect of Modified 5′-Capped Short RNA Fragments on Influenza Virus RNA Polymerase Gene Expression

Tado

Abe

Hatta

et al. 2001

Antivir Chem Chemother

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“…6). Chung et al (2) have shown that this synthetic mRNA-like substrate is cleaved after the guanosine residue, 11 nucleotides from the cap, by the endonuclease activity of the polymerase complex to produce a single 5Ј-capped, 11-nucleotide primer termed G11 (2, 4) which can act as a primer for influenza virus transcription. The addition of [ 32 P]CTP in the absence of other nucleotides resulted in a labelled, capped, 12-nucleotide product through incorporation of one [ 32 P]CMP residue to the 3Ј end of the G11 primer (Fig.…”

mentioning

confidence: 99%

Inhibition of influenza virus transcription by 2'-deoxy-2'-fluoroguanosine

Tisdale

Ellis

Klumpp

et al. 1995

Antimicrob Agents Chemother

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The nucleoside analog 2-deoxy-2-fluoroguanosine (2-fluorodGuo) is phosphorylated by cellular enzymes and reversibly inhibits influenza virus replication in chick embryo cells within the first 4 h of infection. RNA hybridization studies revealed that primary and secondary transcription of influenza virus RNA were blocked at a compound concentration of 10 M, but no inhibition of cell protein synthesis was seen even at high compound concentrations (200 M). In vitro, the triphosphate of 2-fluorodGuo is a competitive inhibitor of influenza virus transcriptase activity from disrupted virus, with a K i of 1.0 M. The cellular polymerases DNA polymerase ␣ and RNA polymerase II were only weakly inhibited or were insusceptible to 2-fluorodGTP. In kinetic studies with the influenza virus transcriptase, 2-fluorodGTP, in the absence of GTP, blocked elongation of the virus RNA chain. Similarly, by using purified ribonucleoprotein complexes it was found that the addition of a single nucleotide of 2-fluorodGTP to the virus RNA caused chain termination, which resulted in the blockage of further virus transcription. Furthermore, the specificity for influenza virus transcriptase was confirmed when the transcriptase from partially resistant virus was found to be 10-fold less susceptible to 2-fluorodGTP (K i ‫؍‬ 13.1 M).The 2Ј-fluororibosides are potent inhibitors of influenza virus A and B strains in cell culture, in the mouse pneumonia model (16), and in the ferret model (6). All of the influenza virus strains examined were susceptible to 2Ј-deoxy-2Ј-fluoroguanosine (2Ј-fluorodGuo), but the level of inhibition varies with cell type and appears to be related to the levels of phosphorylation in these different cells. In human tracheal cells, growth of influenza virus A/England/40/83 (H3N2) was reduced by 4 log 10 units, suggesting that 2Ј-fluorodGuo is phosphorylated very efficiently in the human target cell. Furthermore, evaluation in human respiratory epithelial explants showed that 2Ј-fluorodGuo was extremely potent against both influenza virus A and B strains (90% effective concentration, Ͻ 0.1 g/ml [Ͻ0.35 M]), and no toxicity was observed at 100 g/ml (350 M) (15). These results indicate that the compound may be particularly effective when it is delivered directly to the respiratory tract.Deoxycytidine kinase is one enzyme which has been shown to be able to phosphorylate both pyrimidine and purine analogs to their 5Ј-monophosphates (17). Furthermore, 2Ј-fluorodGuo monophosphate is a substrate for GMP kinase (K m , 53 M, compared with a K m of 7 to 9 M for GMP [11a]). Correlations between intracellular 2Ј-fluorodGuo triphosphate (2Ј-fluorodGTP) levels and influenza virus inhibition suggest that the compound may be targeting influenza virus RNA replication possibly by inhibiting the influenza virus polymerase complex.The present study was undertaken to elucidate further the mechanism of inhibition of influenza virus replication by 2Ј-fluorodGuo. The triphosphate of 2Ј-fluorodGuo was synthesized for enzyme kinetic studies, including...

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“…It was shown that a 67 nucleotide RNA substrate with a 32 Plabelled type 1 cap structure (m 7 G 32 pppGm) was specifically cleaved by influenza virus endonuclease, resulting in the production of an 11 nucleotide RNA fragment that was capable of priming transcription (Chung et al, 1994). In an elegant series of experiments, the RNA substrate was systematically truncated and it was demonstrated that only one nucleotide beyond the cleavage site was required for cleavage; only nine nucleotides were required for cleavage whereas only four nucleotides were required for binding to influenza RNA polymerase.…”

Section: Inhibition Of Transcription Translation and Replicationmentioning

confidence: 99%

Approaches and Strategies for the Treatment of Influenza Virus Infections

Colacino

Staschke

Laver

1999

Antivir Chem Chemother

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Influenza A and B viruses belong to theOrthomyxoviridae family of viruses. These viruses are responsible for severe morbidity and significant excess mortality each year. Infection with influenza viruses usually leads to respiratory involvement and can result in pneumonia and secondary bacterial infections. Vaccine approaches to the prophy-laxis of influenza virus infections have been problematic owing to the ability of these viruses to undergo antigenic shift by exchanging genomic segments or by undergoing antigenic drift, consisting of point mutations in the haemagglutinin (HA) and neuraminidase (NA) genes as a result of an error-prone viral polymerase. Historically, antiviral approaches for the therapy of both influenza A and B viruses have been largely unsuccessful until the elucidation of the X-ray crystallographic structure of the viral NA, which has permitted structure-based drug design of inhibitors of this enzyme. In addition, recent advances in the elucidation of the structure and complex function of influenza HA have resulted in the discovery of a number of diverse compounds that target this viral protein. This review article will focus largely on newer antiviral agents including those that inhibit the influenza virus NA and HA. Other novel approaches that have entered clinical trials or been considered for their clinical utility will be mentioned. Keywords: Influenza virus; haemagglutinin; neuraminidase; amantadine; ribavirin; zanamivir; GS 4104Infection with influenza viruses can result in an infectious disease for which there is no adequate control. Killed influenza vaccines against this virus have been available for nearly 70 years but, on a community-wide basis, the use of these vaccines has not resulted in a significant decrease in the morbidity or mortality of this disease and has not alleviated the severe financial loss which occurs as a result of hospitalizations and lost productivity. Currently, the only compounds approved by the US FDA for the treatment of influenza infections are amantadine and rimantadine, both of which target the M2 ion channel protein of the virus (Pinto et al., 1992). However, these compounds are not effective against influenza B virus, which does not have an M2 ion channel. Also, virus mutants resistant to amantadine or rimantadine can be isolated readily from individuals treated with these drugs Hayden et al., 1989). Recently, it has been suggested that resistance to amantadine can be overcome by mixing amantadine with other amantadine derivatives that interfere with the ion channel function of M2 from amantadine-resistant viruses (Scholtissek et al., 1998). Ribavirin, a broad-spectrum antiviral agent Witkowski et al., 1972;Huffman et al., 1973;Oxford, 1975) has been considered for use against influenza A and B viruses, but the clinical trials of this compound have not been encouraging (Cohen et al., 1976;Togo & McCracken, 1976;Magnussen et al., 1977;Smith et al., 1980;Stein et al., 1987) and it has not been approved for the treatment of influenza virus infection.Hist...

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Biochemical studies on capped RNA primers identify a class of oligonucleotide inhibitors of the influenza virus RNA polymerase.

Cited by 50 publications

References 30 publications

Inhibitory Effect of Modified 5′-Capped Short RNA Fragments on Influenza Virus RNA Polymerase Gene Expression

Inhibitory Effect of Modified 5′-Capped Short RNA Fragments on Influenza Virus RNA Polymerase Gene Expression

Inhibition of influenza virus transcription by 2'-deoxy-2'-fluoroguanosine

Approaches and Strategies for the Treatment of Influenza Virus Infections

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