Anin vitrofluorescence based study of initiation of RNA synthesis by influenza B polymerase

Abstract

Influenza polymerase replicates, via a complementary RNA intermediate (cRNA), and transcribes the eight viral RNA (vRNA) genome segments. To initiate RNA synthesis it is bound to the conserved 5΄ and 3΄ extremities of the vRNA or cRNA (the ‘promoter’). 5΄-3΄ base-pairing in the distal promoter region is essential to position the template RNA at the polymerase active site, as shown by a new crystal structure with the 3΄ end threading through the template entry tunnel. We develop fluorescence polarization assays to quantify initiation of cap-primed (transcription) or unprimed (replication) RNA synthesis by recombinant influenza B polymerase bound to the vRNA or cRNA promoter. The rate-limiting step is formation of a primed initiation complex with minimally ApG required to stabilize the 3΄ end of the template within the active-site. Polymerase bound to the vRNA promoter initiates RNA synthesis terminally, while the cRNA promoter directs internal initiation at a significantly lower rate. Progression to elongation requires breaking the promoter 5΄-3΄ base-pairing region and favourable compensation by the emerging template-product base-pairs. The RNA synthesis assay is adaptable to high-throughput screening for polymerase inhibitors. In a pilot study, we find that initiation at the cRNA promoter is unusually susceptible to inhibition by 2΄F-2΄dNTPs.

Real-time analysis of single influenza virus replication complexes reveals large promoter-dependent differences in initiation dynamics

“…Our results also show that in most complexes, the vRNA promoter is highly dynamic, exhibiting rapid transitions between different states. vRNA dynamics may therefore be a requirement for robust RNAP activity, as vRNA initiation complexes have been found to be ~200-fold more efficient in transcription initiation than cRNA initiation complexes (6). Intriguingly, this suggests that the rapid dynamics of the 3' vRNA may be the key to fast initiation, possibly because the dynamic nature of the vRNA promoter gives the 3ˈ RNA a greater chance of accessing the active site, and thus of being stabilized if nucleotides are present.…”

“…Crystal structures of the RNAP have shown that the vRNA 3ˈ end overshoots the active site by 1 nucleotide, meaning that for terminal initiation on the vRNA template to occur, the RNA needs to move back by one nucleotide (Fig. 5A) (6). Sequence comparison of the vRNA and cRNA promoters shows that the proximal 3ˈ cRNA is one nucleotide longer than the 3ˈ vRNA, and the distal promoter duplex region is one nucleotide shorter (compare Fig.…”

“…Replication of the vRNA is carried out by the RNAP, a multi-functional complex that is an attractive target for antiviral drug development. High-resolution structures of the RNAP have become available in recent years, providing intriguing static snapshots of how these enzymes function (3)(4)(5)(6). The RNAP consists of three subunits: PB1 (which contains the canonical polymerase motifs typical of all template-dependent RNAPs), PA, and PB2.…”

“…The structure of the RNAP-bound vRNA promoter has been resolved by x-ray crystallography (Fig. S1A); in the structure, the 5′ proximal region (nucleotides 1-10) forms an intramolecular stem-loop that is tightly bound in a pocket formed between PA and PB1 (3,4,6). Binding of the 5ˈ end results in ordering of the polymerase active site and activates multiple polymerase functions (10)(11)(12).…”

“…The 3′ proximal region (nucleotides 1-9) can adopt two different conformations, either a pre-initiation state in which the 3ˈ vRNA is bound in an arc shape on the outside of the protein (4), or an initiation state in which the 3' terminus of the vRNA threads through the template channel into the RNAP active site ( Fig. S1B) (6,13). The structure of the closely related La Crosse bunyavirus RNAP shows the proximal 3ˈ RNA in a third possible conformation, where the 3ˈ end is tightly and specifically bound in an extended, single-stranded configuration in a narrow cleft on the outside of the protein (10).…”