Hairpin Loop Structure in the 3′ Arm of the Influenza A Virus Virion RNA Promoter Is Required for Endonuclease Activity

Abstract

Previous studies have shown that the 5 arm of the influenza A virus virion RNA promoter requires a hairpin loop structure for efficient endonuclease activity of influenza virus RNA polymerase, an activity that is required for the cap-snatching activity of primers from host pre-mRNA. Here we examine whether a hairpin loop is also required in the 3 arm of the viral RNA promoter. We study point mutations at each nucleotide position (1 to 12) within the 3 arm of the promoter as well as complementary "rescue" mutations which restored base pairing in the stem of a potential hairpin loop. Our results suggest that endonuclease activity is absolutely dependent on the presence of a 3 hairpin loop structure. This is the first direct evidence for RNA secondary structure within the 3 arm being required for a specific stage, i.e., endonuclease cleavage, in the influenza virus replicative cycle.Influenza A virus is a segmented, negative-sense RNA virus. The virion RNA of influenza virus serves several functions. During transcription it acts as the template and as a cofactor in endonuclease cleavage of host pre-mRNA (17, 31), leading to the synthesis of viral mRNA which is capped at its 5Ј end and polyadenylated at its 3Ј end (18). Virion RNA (vRNA) also acts as the template for full-length cRNA, which in turn serves as a template for the synthesis of new vRNA molecules. Influenza virus RNA synthesis takes place within the nucleus of infected cells, consistent with the requirement for host cell pre-mRNAs and for splicing (26), suggesting that influenza virus transcription may be coupled to host cell RNA polymerase II transcription (14). Inhibitors selective for either influenza virus endonuclease or RNA polymerase activities suggest that the active site for endonuclease cleavage is separate from the transcription active site (50, 51).The influenza virus RNA polymerase is a heterotrimer formed by the PB1, PB2, and PA subunits (25, 26). All three subunits are required for transcription and replication, although the role of PA is poorly understood (37,44,45). In the virion the polymerase proteins are associated with vRNA and the nucleoprotein to form ribonucleoprotein complexes. It is likely that base pairing between the conserved 3Ј and 5Ј termini of the influenza virus vRNA leads to the formation of an RNA panhandle structure; however, it is probable that the polymerase itself helps to stabilize the interactions between the 5Ј and 3Ј termini (7,19,20,23,31,33,35).PB1 interacts with both PB2 and PA (20,22,52) and is also involved in binding vRNA (11,12,15,31). PB1 also binds cRNA, although the binding sites are different from those for vRNA (16). PB1 contains amino acid motifs present in other RNA-dependent RNA polymerases (38) and is the subunit responsible for polymerization (24). Although it has been reported that the endonuclease site residues in PB2 (32), recent evidence favors its location being in PB1 (31a).PB2 is the cap-binding protein. Cap-binding regions have been mapped, and capped RNA can be cross-linked to PB2 (3, ...

Mutational Analysis of the Influenza Virus cRNA Promoter and Identification of Nucleotides Critical for Replication

“…Interaction between the vRNA 5Ј and 3Ј ends, through base pairing, is required for promoter activity (11,12,24). A "corkscrew" secondary structural model for the vRNA promoter (7) is supported by several more recent studies both in vitro and in vivo (2,6,7,19,20). The main features of the corkscrew model are two short hairpin loop structures, each with a stem of 2 bp and a tetraloop, formed by residues near the 5Ј and 3Ј termini.…”

“…However, Lee et al (21) have recently proposed that both the 5Ј and 3Ј ends of the vRNA promoter are needed for efficient cap binding. Subsequently, polymerase bound to the 5Ј and 3Ј ends of the vRNA activates an endonuclease activity that results in the cleavage of host pre-mRNAs (14,19,20). However, another recent report suggests that when the capped RNA substrate contains a CA cleavage site, the 5Ј end of vRNA alone is apparently sufficient for endonuclease activation (30).…”

“…of the vRNA (2,7,19,20). The cRNA promoter is complementary to the vRNA promoter, and recent data have suggested that it may also adopt a corkscrew configuration (1).…”

“…The main features of the corkscrew model are two short hairpin loop structures, each with a stem of 2 bp and a tetraloop, formed by residues near the 5Ј and 3Ј termini. In vitro experiments suggest that a hairpin loop at the 5Ј end is required for ApG-primed transcription (11) and for polyadenylation of mRNA (29), whereas hairpin loops at both the 5Ј and 3Ј ends of the vRNA are required for endonuclease activity (19,20) and for stability (2). However, Rao et al (30) have recently reported that when the capped RNA substrate contains a CA cleavage site, the 3Ј end of vRNA functions only as a template for mRNA synthesis and does not require a hairpin loop structure.…”

Influenza A Virus Polymerase Inhibits Type I Interferon Induction by Binding to Interferon β Promoter Stimulator 1

“…2C). Because it has been reported that the conserved flanking sequence lying on viral genomic RNA is required for the activation of the cap-snatching function of the influenza A virus polymerase (37)(38)(39), it was assumed that stimulation by both HCV 5Ј-UTR and poly(I-C) could not activate cap-snatching activity. Therefore, we concluded that the inhibitory effect of a viral polymerase on RIG-I-mediated IFN␤ promoter activation was independent of its cap-snatching function.…”