The polyadenylation signal of influenza virus RNA involves a stretch of uridines followed by the RNA duplex of the panhandle structure

Abstract

Appropriate RNAs are transcribed and amplified and proteins are expressed after transfection into cells of in vitro-reconstituted RNA-protein complexes and infection with influenza virus as the helper. This system permits us to study the signals involved in transcription of influenza virus RNAs. For the analysis we used a plasmid-derived RNA containing the reporter gene for chloramphenicol acetyltransferase (CAT) flanked by the noncoding sequences of the NS RNA segment of influenza A/WSN/33 virus. Mutations were then introduced into both the 5' and 3' ends, and the resulting RNAs were studied to determine their transcription in vitro and their CAT expression activity in the RNA-protein transfection system. The results reveal that a stretch of uninterrupted uridines at the 5' end of the negative-strand RNA is essential for mRNA synthesis. Also, a double-stranded RNA "panhandle" structure generated by the 5'-and 3'-terminal nucleotides appears to be required for polyadenylation, since opening up of these base pairs diminished mRNA synthesis and eliminated expression of CAT activity by the mutant RNAs. Finally, it was shown that this double-stranded RNA structural requirement is not sequence specific, since a synthetic GC clamp can replace the virus-coded RNA duplex. The data suggest that the viral RNA polymerase adds poly(A) by a slippage (stuttering) mechanism which occurs when it hits the double-stranded RNA barrier next to the stretch of uridines.

Replication of Synthetic Defective Interfering RNAs Derived from Coronavirus Mouse Hepatitis Virus-A59

“…Among the more at play. With respect to the first, the host cells used for the characterization of the deletion mutants of both vi-thoroughly studied of these are alphaviruses, containing a 5 stem-loop structure involved in replication con-ruses are different: the JHM-derived DIs were all tested in DBT cells, whereas we and Masters et al (1994) per-served in structure but not in sequence (reviewed by Strauss and Strauss, 1994); poliovirus, which requires a formed the experiments in L cells or 17Cl1 cells.…”

“…W.L. is supported by a grant (Luo et al, 1991; Fodor et al, 1994). However, the putative from the Royal Dutch Academy of Sciences.…”

Chapter 3 Virus Versus Host Cell Translation

“…7). Thus, Influenza virus mRNAs that are capped via capsnatching and polyadenylated (Herz et al, 1981;Krug et al, 1979;Luo et al, 1991) are translated efficiently under conditions of partial inactivation of eIF4F (Feigenblum and Schneider, 1993) when host protein synthesis is blocked (Katze and Krug, 1990). Several studies have shown that the NS1 viral protein selectively promotes translation of viral mRNAs by increasing their rate of initiation (de la Luna et al, 1995;Enami et al, 1994;Katze et al, 1986;Park and Katze, 1995) and interacts with PABP and eIF4GI (one of the two isoforms of eIF4G in animals) in viral mRNA translation initiation complexes (Aragó n et al, 2000;Burgui et al, 2003).…”

Examples of expression systems based on animal RNA viruses: Alphaviruses and influenza virus

“…Messenger RNA transcripts are initiated using 10-13base 5'-capped oligonucleotide primers derived by endonucleolytic cleavage of host RNA polymerase II transcripts. Influenza mRNAs terminate with a 3' terminal poly (A) tract derived from a short poly (U) template [44]. Transcripts therefore lack the 3' terminal conserved element found on genome-length RNAs and are transported to the cytoplasm for translation and are not packaged into nucleocapsid structures or virions.…”

“…This technology has had tremendous impact for basic studies on influenza virus replication and may also be useful for developing improved influenza virus vaccine strains. Studies dealing with analysis of influenza cis-acting RNA elements [41,44] and engineering of…”