Structural insight into cap-snatching and RNA synthesis by influenza polymerase

Abstract

Influenza virus polymerase uses a capped primer, derived by 'cap-snatching' from host pre-messenger RNA, to transcribe its RNA genome into mRNA and a stuttering mechanism to generate the poly(A) tail. By contrast, genome replication is unprimed and generates exact full-length copies of the template. Here we use crystal structures of bat influenza A and human influenza B polymerases (FluA and FluB), bound to the viral RNA promoter, to give mechanistic insight into these distinct processes. In the FluA structure, a loop analogous to the priming loop of flavivirus polymerases suggests that influenza could initiate unprimed template replication by a similar mechanism. Comparing the FluA and FluB structures suggests that cap-snatching involves in situ rotation of the PB2 cap-binding domain to direct the capped primer first towards the endonuclease and then into the polymerase active site. The polymerase probably undergoes considerable conformational changes to convert the observed pre-initiation state into the active initiation and elongation states.

Identification and characterization of influenza variants resistant to a viral endonuclease inhibitor

“…One explanation, supported by the polymerase assay (Fig. 2B) and the structure of the heterotrimeric (16,17), is that the mutations have less effect in the context of the intact RdRp complex where the RNA substrate is also bound by the CAP-binding domain. Overall, our studies support the notion that the endonuclease domain is an attractive target for influenza antiviral drug discovery.…”

“…An attractive antiinfluenza drug target is the viral RNA-dependent RNA polymerase (RdRp) that synthesizes both genomic RNA and viral mRNA (14,15). The recently determined crystal structure of the heterotrimeric complex comprising subunits PA, PB1 and PB2 has provided key insights into its mechanism (16,17). Early work to identify RdRp inhibitory compounds established that the endonuclease activity is an attractive target for influenza drug development (18).…”

Characterization of the PB2 Cap Binding Domain Accelerates Inhibitor Design

“…Figure 7. The ribbon drawing of influenza A virus polymerase complex using coordinates from PDB 4WSB [27]. PB2cap is colored in orange, and the PA endonuclease domain is colored in blue.…”

The MultiBac Baculovirus/Insect Cell Expression Vector System for Producing Complex Protein Biologics

“…A notable exploit is influenza polymerase, an important drug target to combat the flu, which has remained inaccessible for 40 years since its discovery. Influenza polymerase has been produced, for the first time, successfully using ComplexLink in conjunction with MultiBac, enabling elucidation of its structure and mechanism by X-ray crystallography at near-atomic resolution [55,56] (Figs. 5, 6).…”