RNA polymerase of influenza virus. IV. Catalytic properties of the capped RNA endonuclease associated with the RNA polymerase

Abstract

Catalytic properties of the capped RNA-specific endonuclease associated with the influenza virus RNA polymerase were analyzed with use of synthetic hetero-and homopolymers containing 32P-labeled CAP structures at their 5' termini. The endonuclease displays its intrinsic activity provided that substrate RNA contains both the CAP-1 structure (m7GpppGm) and either A or U residues at 9 to 11 nucleotides distant from the CAP structure. Independent recognition of multiple RNA signals by the endonuclease was further supported by the findings that dinucleotide ApG, free CAP structures and RNA without the CAP structure inhibited the endonuclease activity to different extents.

Two separate sequences of PB2 subunit constitute the RNA cap‐binding site of influenza virus RNA polymerase

“…The electrophoretic patterns of endonucleolytic cleavage products were essentially the same as observed in our previous study (Kawakami et al 1983). All the cap monomers examined as above inhibited the endonucleolytic cleavage of globin RNA to various extents, but the stronger inhibition was again observed with the cap-1 monomers (m 7 GpppGm and m 7 GpppAm) than the cap-0 monomers (m 7 GpppG and m 7 GpppA) without ribose-associated methyl (data not shown).…”

“…UV-crosslinking of RNA cap-1 structure to the PB2 protein In¯uenza virus RNA polymerase possesses a unique endonuclease activity, which cleaves host cell RNA with the cap-1 structure at 10±12 nucleotide positions from the 5 H -terminal cap structure (Plotch et al 1981;Kawakami et al 1983). For identi®cation of the cap-1 recognition site within the RNA polymerase, we crosslinked either globin RNA or synthetic RNAs with 32 P only at the 5 H -terminal cap-1 structure to the virus-associated RNA polymerase.…”

“…Poly(A, 2 U, 2G) was synthesized using polynucleotide phosphorylase as described previously (Kawakami et al 1983), while globin mRNA was decapped by treatment with 0.3 M aniline (Moss & Koczot 1976). Both RNAs were re-capped using vaccinia virus capping enzyme (BRL, USA) and [a-32 P]GTP in a reaction mixture which contained 50 mM Tris-HCl (pH 7.9), 2 mM MgCl 2 , 40mM NaCl, 20 mM DTT and 0.5 mM Sadenosylmethionine (SAM) (Sigma, USA) at 37 8C for 2 h. The cap structures thus formed were analysed by electrophoresis on DE81 after treatment with P1 nuclease (Boehringer Mannheim, Germany) and alkaline phosphatase (NEB, USA).…”

“…The capped RNA endonuclease activity was measured essentially according to the published procedure (Kawakami et al 1983). In brief, RNP was incubated for 30 min at 30 8C in 50 mL of the transcription reaction mixture (Honda et al 1987) which contained 50 mM HEPES (N-2-hydroxyethylpiperazine-N H -2-ethanesulphonic acid) buffer (pH 7.9), 0.1 M NaCl, 5 mM Mg acetate, 2 mM DTT, 0.25 mM ATP, 10 mg tRNA and capped RNA with radioactive cap-1.…”

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

“…Kawakami et al, 1983;Krug et al, 1979;Plotch et al, 1979;Caton & Robertson, 1980;Dhar et al, 1980;Beaton & Krug, 1981;Plotch et al, 1981;Blaas et al, 1982a, b;Shaw & Lamb, 1984). Recently, there have been several in vitro systems developed in order to study the transcriptional properties of the influenza polymerase (Nagata et al, 1989;Honda et al, 1990;Huang et al, 1990;Kimura et al, 1992;Kobayashi et al, 1992;Martin et al, 1992;Seong & Brownlee, 1992a).…”