Inhibition of influenza virion transcriptases by polynucleotides

Smith, J. C.; Raper, Robert H.; Bell, L.D.; Stebbing, Nowell; McGeoch, Duncan J.

doi:10.1016/0042-6822(80)90144-0

Cited by 13 publications

(3 citation statements)

References 16 publications

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“…Thus, poly(A,U) and poly(C,G), which have highly ordered secondary structures (22,23), and multistranded poly(A)-poly(U) did not inhibit the transcriptase, whereas poly(U) and especially poly(s4U) [which probably has less secondary structure than poly(U) (24)] were effective inhibitors of both mRNA and ApG-primed RNA synthesis. Inhibition of ApG-primed influenza viral RNA transcription by poly(U) and poly(s4U) was reported also by others (25).…”

Section: Discussionmentioning

confidence: 91%

Priming and inhibitory activities of RNAs for the influenza viral transcriptase do not require base pairing with the virion template RNA.

Krug¹,

Broni²,

LaFiandra³

et al. 1980

Proc. Natl. Acad. Sci. U.S.A.

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Capped ribopolymers lacking a sequence complementary to the common 3' end of the influenza virion RNA segments effectively stimulated transcription of these RNAs by the virion-associated transcriptase. Thus, stimulation of transcription results not from hydrogen bonding between the capped RNA and the 3' end of the virion RNA but resumably from a specific interaction of the capped RNA with protein (s) in the transcriptase complex. Althou no specific nucleotide sequence was required for priming activity, capped mRNAs wi diminished secondary structure were preieffffeas primers.Inosine-substituted or bisulfite-modified capped reovirus mRNAs were at least 3-to 5-fod more effective as primers than were the native capped mRNAs. On the other hand, inosine substitution or bisuffite treatment of the uncapped form of reovirus mRNAs converted them from essentially inactive species to potent inhibitors of the transcriptase reaction primed by either ApG or globin mRNA. These effects of reduced secondary structure also most probably reflect an interaction of the exogenous RNAs with transcriptase protein(s). The results obtained from screening a series of native uncapped ribopolymers were consistent with inhibitory activity requiring the absence of most hydrogen bonding in the nibpolymer and also suggested that specific structural feature(s) of the nucleotides in the chain were important.Capped eukaryotic mRNAs strongly stimulate influenza viral RNA transcription in vitro (1, 2). During this reaction, the methylated cap structure and a short stretch of 5'-terminal nucleotides are transferred from the stimulating primer mRNA to the influenza viral RNA transcripts (2-4). Although the presence of a methylated cap structure in the exogenous mRNA clearly is required for priming activity (2, 3, 5), other RNA structural features also may affect priming. In terms of understanding the mechanism of priming, it is of particular interest to determine whether the capped primer must also contain one or more sequences complementary to the 3' terminus [3' U-C-G-U (6,7)] of the virion RNA (vRNA) in order to allow hydrogen bonding between the primer and template RNAs.To answer this question, we tested the priming activity of various capped ribopolymers of different base composition. In this report we show that capped ribopolymers lacking a sequence complementary to the 3' terminus of vRNA are effective primers for influenza viral RNA transcription. This indicates that stimulation of the initiation of transcription does not require specific hydrogen bonding between the primer and template RNAs. Although no specific nucleotide sequence is apparently necessary, sequence alterations that affect the secondary structure of capped RNAs markedly influence priming activity. Experiments involving inosine-substituted and bisulfite-modified reovirus mRNAs indicate that the influenza virion transcriptase prefers capped RNAs with diminished secondary structure as primers. In the course of these experiments, it was also found that decreasing the se...

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Section: Discussionmentioning

confidence: 91%

Priming and inhibitory activities of RNAs for the influenza viral transcriptase do not require base pairing with the virion template RNA.

Krug¹,

Broni²,

LaFiandra³

et al. 1980

Proc. Natl. Acad. Sci. U.S.A.

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“…A pentose modified oligomcleotide, 13-4'-thio-oligouridylate, has been shown recently o interact with HIV-1 RT [16]. It was shown earlier that poly(s4U), a base modified polyribonucleotide, inhibits the virion-associated transcriptase of influenza A [17].…”

Section: • Introductionmentioning

confidence: 99%

(s⁴dU)₃₅: a novel, highly potent oligonucleotide inhibitor of the human immunodeficiency virus type 1 reverse transcriptase

Tőkés

Aradi

1996

FEBS Letters

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.~ey wordr." Inhibition; Reverse transcriptase; Human ~mmunodeficiency virus; Oligo 4-thio-2'-deoxyuridylate: ~mtitemplate • IntroductionReverse transcriptase (EC 2.7.7.49) represents one of the main targets in the development of the chemotherapy against HIV, the etiological agent of the acquired immunodeficiency .yndrome [1]. Therefore, a large number of nucleoside and non-nucleoside RT inhibitors have been studied recently as i~otential agents against AIDS [2][3][4][5].Chemically modified oligo-and polynucleotides have also ~een studied and characterized as inhibitory template (or '~rimer) analogs for DNA polymerases including RT. They rove been termed antitemplates [6]. The antitemplates interact vith proteins (i.e. template dependent nucleic acid polynerases) rather than nucleic acids (antisense effect). Their acivities are not strictly dependent on specific nucleic acid seluences. In addition to the nucleoside and non-nucleoside 'ype inhibitors the modified oligonucleotides are another class ,~f RT inhibitors.The most thoroughly studied inhibitory template analogs tre 5-mercaptopyrimidine-containing polynucleotides [7][8][9].['hey are potent inhibitors of the replication of HIV in prinary human lymphocytes [10]. The poly(U60,hsSU40) proved o be a potent competitive inhibitor of RT [11]. The phos~horothioate-linked and phosphorodithioate-linked oligoleoxycytidylate analogs are also potent competitive inhibitors ff viral DNA polymerases [12][13][14][15]. A pentose modified oligomcleotide, 13-4'-thio-oligouridylate, has been shown recently o interact with HIV-1 RT [16]. It was shown earlier that poly(s4U), a base modified polyribonucleotide, inhibits the virion-associated transcriptase of influenza A [17].In this paper we report the HIV-1 RT inhibitory activity of oligonucleotides containing s4dUMP. Materials and methods MaterialsThe unlabeled deoxyribonucleotides dTTP, dGTP ('Ultrapure') as well as poly(A) and poly(C) were Pharmacia products. The labelled nucleotides [aH]dTTP and [3H]dGTP were from Amersham. All other reagents were of the highest quality commercially available.HIV-1 RT was purchased from Worthington (Freehold, NJ). Its concentration was 8 U/gl, Lot No. 63E792N. The alkaline phosphatase (grade I) and phosphodiesterase (snake venom nuclease from Crotalus durissus) were purchased from Boehringer Mannheim (Germany).The template-primers were poly(A)'(dT)a~i and poly(C)'(dGh6, prepared as described earlier [1 I].The oligodeoxycytidylates were synthesized on a Pharmacia Gene Assembler Plus DNA synthesizer in our laboratory and purified by two precipitations with n-butanol. Synthes& and character&ation of s4dU containing oligonucleotides,( dCx,s4 dUv ),~ The thiolated oligonucleotides were prepared by treating 1.5-3.5 mg of (dC)n (n = 10, 20, 25, 30, 35, 40), dissolved in aqueous pyridine, with liquid H2S [18,19] for 2-10 days at 35°C or 50°C, depending on the desired degree of thiolation. The modified oligonucleotides were isolated by ethanol precipitation as described for modified 5S rRNA [20], or ...

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“…The CBP-24 competed only when prereacted with the primer and inhibited partially even in molar excess (sixfold relative to mRNA caps and to the estimated amount [8] of influenza P proteins). It therefore seems likely that, under these conditions, the influenza transcriptase complex has a greater affinity for caps than does purified CBP-24 or has additional internal binding sites on the primer mRNA (12,19).…”

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confidence: 99%

mRNA 5'-cap binding activity in purified influenza virus detected by simple, rapid assay

Kroath¹,

Shatkin²

1982

J Virol

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Reovirus mRNA 5'-terminal caps were 3'-radiolabeled with pCp and as affinity probes for proteins with cap binding activity. A rapid, simple, and sensitive blot assay was devised that could detect cellular cap binding protein in a complex polypeptide mixture. By using this method, cap binding activity was found in detergent-treated influenza virus but not in reovirus or vaccinia virus. Preincubation of capped reovirus mRNA with purified cellular cap binding protein reduced its primer effect on influenza transcriptase, whereas priming by ApG was not affected. The results indicate that influenza transcriptase complexes include cap-recognizing proteins that are involved in the formation of chimeric mRNAs.

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Inhibition of influenza virion transcriptases by polynucleotides

Cited by 13 publications

References 16 publications

Priming and inhibitory activities of RNAs for the influenza viral transcriptase do not require base pairing with the virion template RNA.

Priming and inhibitory activities of RNAs for the influenza viral transcriptase do not require base pairing with the virion template RNA.

(s⁴dU)₃₅: a novel, highly potent oligonucleotide inhibitor of the human immunodeficiency virus type 1 reverse transcriptase

mRNA 5'-cap binding activity in purified influenza virus detected by simple, rapid assay

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Inhibition of influenza virion transcriptases by polynucleotides

Cited by 13 publications

References 16 publications

Priming and inhibitory activities of RNAs for the influenza viral transcriptase do not require base pairing with the virion template RNA.

Priming and inhibitory activities of RNAs for the influenza viral transcriptase do not require base pairing with the virion template RNA.

(s4dU)35: a novel, highly potent oligonucleotide inhibitor of the human immunodeficiency virus type 1 reverse transcriptase

mRNA 5'-cap binding activity in purified influenza virus detected by simple, rapid assay

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(s⁴dU)₃₅: a novel, highly potent oligonucleotide inhibitor of the human immunodeficiency virus type 1 reverse transcriptase