Cap and Internal Nucleotides of Reovirus mRNA Primers Are Incorporated into Influenza Viral Complementary RNA During Transcription In Vitro

Abstract

Reovirus mRNA's containing a 5′-terminal methylated cap structure (m 7 GpppG m ) were shown to be effective primers for influenza viral RNA transcription in vitro catalyzed by the influenza virion transcriptase. Priming activity required the presence of methyl groups in the cap since reovirus mRNA's with 5′-terminal GpppG were inactive as primers. Both the cap and internal nucleotides were physically transferred from radiolabeled reovirus mRNA to influenza viral complementary RNA (cRNA) during transcription in vitro. By using reovirus mRNA's with methyl - 3 H-labeled caps as primers, we showed that the influenza viral cRNA synthesized in the presence of unlabeled nucleoside triphosphates contained [ methyl - 3 H]m 7 GpppG m , identical to that found in the reovirus mRNA primer. To demonstrate transfer of internal residues, reovirus mRNA's synthesized in the presence of all four α- 32 P-labeled ribonucleoside triphosphates were used as primers. The resulting influenza viral cRNA was 32 P-labeled. Diethyl-aminoethyl-Sephadex chromatography of the RNase T2 digest of this cRNA demonstrated 32 P radiolabel in both internal residues (charge −2) and the cap (charge −4.6). Approximately 25 internal nucleotides along with the cap of reovirus mRNA were transferred to each chain of influenza viral cRNA. Gel electrophoretic analysis indicated that the segments of influenza viral cRNA primed by reovirus mRNA were approximately the same size as those primed by a different mRNA, globin mRNA, strongly suggesting that the influenza virion transcriptase complex transfers approximately the same number of nucleotides plus the cap from different mRNA primers to the 5′ end of influenza viral RNA transcripts.

Introduction to molecular biology of influenza a viruses.

“…During transcription, multimeric viral polymerase interacts with the host polymerase II (Engelhardt et al, 2005). This interaction leads to the phenomenon called "cap-snatching", that is the removal of a cap from newly synthetized cellular mRNAs (Plotch et al, 1979;Bouloy et al, 1979;Bouloy et al, 1980;Krug, 1981). The PB2 subunit of viral polymerase is responsible for cap binding.…”

The 5' ends of Thogoto virus (Orthomyxoviridae) mRNAs are homogeneous in both length and sequence

“…The structure of the 5Ј ends of THO virus mRNAs contrasts with that reported for all other viruses possessing a segmented, negative or ambisense, single-stranded RNA genome, including members of the Orthomyxoviridae (7,11,39), Arenaviridae (15,26,32,35), Bunyaviridae (3,5,13,16,20,29), and tenuiviruses (19,36), in which the 5Ј ends of viral mRNAs are heterogeneous in both length and sequence. For all of these viruses, it has been suggested that these additional nucleotides are generated by a cap snatching mechanism similar to that described for influenza A virus (4,30,31) and Bunyaviridae (29,42).…”

“…These additional nucleotides, which are heterogeneous in sequence, are generated by a virus-encoded endonucleolytic activity that cleaves capped host-cell mRNAs (this mechanism is referred to as cap snatching) (31). The short capped fragments obtained from cellular mRNAs serve as primers for virus mRNA synthesis (4,30) and are elongated until the polymerase reaches a stretch of uridine residues, located at 17 to 22 nucleotides from the 5Ј ends of vRNAs, where transcription terminates and poly(A) is added to mRNA (37).…”

Search for etiology of porcine reproductive and neurologic syndrome: identification and characterization of a novel swine pestivirus

“…Viral cap-dependent endonuclease polymerase cleaves small pieces of capped RNA around 10 to 13 nucleotides long from the 5'end (266). These primers initiate viral mRNA synthesis by DNA-dependent RNA polymerase (35). The viral RNA of influenza virus is bound to four viral proteins: NP protein and three P proteins (PB1, PB2 and PA) (159;326).…”

“…The border of internal exon is the 5' end splice site, and the 3' terminal exon is defined by the presence of cleavage and polyadenylation signal (178;270;293;355 35 binds the PyAg at the 3' splice site in many introns and the complex between SF1-U2AF at the 3' end of the intron and the U1 snRNP(+GSF) complex at the 5' splice site define the border of the intron (134;340;359).…”