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

Abstract

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...

The role of template-primer interactions in cleavage and initiation by the influenza virus polymerase

“…Therefore, it can be concluded from these two experiments that cleavage site choice by the polymerase is not influenced by base-pair interactions of the primer and the 3' end of the vRNA template, but may be solely controlled by the mRNA itself. This is in accordance with previous work showing that priming by capped RNA does not require base-pairing with the viral template (Krug et al, 1980).…”

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

“…However, the 9-nt primer exhibited only weak priming ability and produced mostly abortive transcripts corresponding to addition of only 2 nt. The length requirement obtained for priming by this substrate is similar to the 9-to 15-nt heterogenous sequences transferred to viral mRNA previously observed in vivo (3,4,(29)(30)(31)(32) and in vitro (5,7,31). The inability of fragments shorter than 9 nt to prime transcription is presumably due to the lack of a sufficient length by which to span the distance between the cap-binding site and the 3 (10,32), indicating that both the cap and the RNA moieties are critical for inhibition.…”

Role of two of the influenza virus core P proteins in recognizing cap 1 structures (m 7 GpppNm) on RNAs and in initiating viral RNA transcription

“…There were several reasons for using an assay for the cap-recognizing protein in which intact RNAs were probes and specific competitors. First, intact capped RNAs or relatively long 5'-terminal fragments of these RNAs are the actual species that, at low concentrations (0.2 ,AtM), maximally stimulate the viral transcriptase reaction (1,3,18,19). In addition, the 5'-terminal fragments cleaved from these RNAs by the viral endonuclease bind efficiently to cores, whereas the cap 1 structure alone, m7GpppGm, binds very poorly.…”