Transfer of 5′-terminal cap of globin mRNA to influenza viral complementary RNA during transcription in vitro

Abstract

We have recently demonstrated that globin mRNAs are effective primers for influenza viral RNA transcription in vitro catalyzed by the virion transcriptase [Bouloy, M., Plotch, S. J. & Krug, R. M. (1978) Proc. Nati. Acad. Sci. USA 75,[4886][4887][4888][4889][4890]. Here, we present direct evidence that the 5'-terminal methylated cap of the globin mRNAs is transferred to viral complementary RNA (cRNA) during transcription. Chemical (a-elimination) or enzymatic removal of the cap of globin mRNAs eliminated essentially all their priming activity. Much of this activity could be restored by recapping the ,-eliminated globin mRNAs with the vaccinia virus guanylyl and methyl transferases. Globin mRNAs containing 32P label only in the cap (m7G32pppm6A'l-) were prepared by recapping ,-eliminated globin mRNAs with the vaccinia virus enzymes, [a-32PJGTP, and unlabeled S-adenosylmethionine. By using this labeled globin mRNA as primer and unlabeled nucleoside triphosphates as precursors, the viral cRNA segments that were synthesized were shown to contain a 32P-labeled 5'-terminal cap structure. Gel electrophoretic analysis indicated that the globin mRNA-primed cRNA segnments were 10-15 nucleotides longer at their 5' end than ApG-primed cRNA segments, which initiate exactly at the 3' end of the virion RNA templates. This suggests that, in addition to the cap, about 10-15 other nucleotides are also transferred from the globin mRNA to viral cRNA. A mechanism for the priming of influenza viral cRNA synthesis by globin mRNA is proposed.Recently we demonstrated (1) We also presented suggestive evidence that the globin mRNA-primed transcripts contain a 5'-terminal methylated cap structure (1). The translation of this viral cRNA into virus-specific proteins was inhibited 75% by the cap analogue 7-methylguanosine 5'-phosphate, whereas under the same conditions the translation of uncapped viral cRNA (the cRNA primed by the dinucleotide ApG) was not inhibited. Because no detectable de novo synthesis of a methylated cap occurred during globin mRNA-primed viral RNA transcription, we proposed that the cap of the globin mRNA primer was transferred to the viral cRNA during in vitro transcription.In the present report, we provide the evidence that proves this proposal. We demonstrate directly that the 5'-methylated cap of the globin mRNA primer is transferred to newly synthesized viral cRNA. In addition, we show that the globin mRNA-primed cRNA segments are 10-15 nucleotides longer at their 5' end than the ApG-primed cRNA segments, which initiate exactly at the 3' end of the virion RNA (vRNA) segments (9, 10). We assume that these additional nucleotides are also transferred from the globin mRNA to viral cRNA during transcription. Based on these results, we propose a mechanism for the priming of influenza viral cRNA synthesis by globin mRNA.METHODS AND MATERIALS The procedures for the synthesis of viral cRNA by detergenttreated purified influenza virus, for the isolation of the poly(A)+ in vitro cRNA, and for the purification of 10S...

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

“…termini of the cleaved fragments from various capped RNAs so far examined in other laboratories (9,16). If the viral endonuclease cleaves the 3' phospho= diester bond of purine residues, viral mRNA itself should be a substrate for the viral endonuclease because the first G residue at the primer-transcript junction is within the recognition distance from the CAP-1 structure.…”

“…Various RNAs with CAP structures such as globin mRNA, reovirus mRNA, CPV (cytoplasmic polyhedrosis virus) mRNA, several plant virus RNAs recapped with the CAP-1 structure, and synthetic polymers with the CAP-1 structure are effective primers for the influenza virus-associated RNA polymerase reaction in vitro and transfer short 5' RNA fragments containing the CAP-1 structure into viral mRNA (3,(9)(10)(11)(12). Evidence is being accumulated indicating that the host cell mRNAs are utilized as primers for in vivo initiation of viral transcription (4,(13)(14)(15).…”

Involvement of the influenza A virus PB2 protein in the regulation of viral gene expression

“…Plotch et al, 1979;Beaton & Krug, 1981 ;Ulmanen et al, 1983;. The PB2 protein has been shown to have a site(s) that recognizes and binds to the type 1 cap structure of the heterogeneous nuclear RNA (Braam et al, 1983).…”

Discovery of Novel Antiviral Agents Directed Against the Influenza A Virus Nucleoprotein

“…After uncoating, the vRNPs are transported into the nucleus through importin / transport systems, where they undergo transcription and replication (Herz et al, 1981;Martin & Helenius, 1991). Transcription of vRNA requires capped RNA primers snartched from cellular pre-mRNAs and premature poly(A) termination of transcripts (Bouloy et al, 1978;Plotch et al, 1979;Robertson et al, www.intechopen.com…”