A Mechanism for Priming and Realignment during Influenza A Virus Replication

Abstract

The influenza A virus genome consists of eight segments of single-stranded RNA. These segments are replicated and transcribed by a viral RNA-dependent RNA polymerase (RdRp) that is made up of the influenza virus proteins PB1, PB2, and PA. To copy the viral RNA (vRNA) genome segments and the cRNA segments, the replicative intermediate of viral replication, the RdRp must use two promoters and two different de novo initiation mechanisms. On the vRNA promoter, the RdRp initiates on the 3′ terminus, while on the cRNA promoter, the RdRp initiates internally and subsequently realigns the nascent vRNA product to ensure that the template is copied in full. In particular, the latter process, which is also used by other RNA viruses, is not understood. Here we provide mechanistic insight into priming and realignment during influenza virus replication and show that it is controlled by the priming loop and a helix-loop-helix motif of the PB1 subunit of the RdRp. Overall, these observations advance our understanding of how the influenza A virus initiates viral replication and amplifies the genome correctly.IMPORTANCE Influenza A viruses cause severe disease in humans and are considered a major threat to our economy and health. The viruses replicate and transcribe their genome by using an enzyme called the RNA polymerases. To ensure that the genome is amplified faithfully and that abundant viral mRNAs are made for viral protein synthesis, the RNA polymerase must work correctly. In this report, we provide insight into the mechanism that the RNA polymerase employs to ensure that the viral genome is copied correctly.

Key Role of the Influenza A Virus PA Gene Segment in the Emergence of Pandemic Viruses

“…These studies also defined the precise conditions for cap-snatching. The median length for cap-snatched primers is 11 or 12 nucleotides depending on the influenza vRNA template, and the endonuclease activity of PA preferentially cleaves after a 3 G. Deep sequencing the cap-snatched primers from influenza mRNAs also provided definitive evidence for a mechanism to rescue suboptimal capped primers that are snatched by the vRdRp [72]. While the median length of cap-snatched primers that are attached to an influenza mRNA is 11 or 12 nucleotides, not all primers that are snatched are initially the appropriate length.…”

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

“…The longer length of the proximal 3ˈ cRNA strand, compared to the proximal 3ˈ vRNA strand, therefore stabilizes the RNA in the RNAP active site. Stable binding of the cRNA 3ˈ end in the active site, compared to the dynamic motions of the vRNA 3ˈ end, may be important for efficient internal initiation and the 'primeand-realign' mechanism, which is crucial for synthesis of a full-length genomic vRNA (16). Similar strategies may exist in members of other virus families, such as the arenaviruses and bunyaviruses (34,35), which also use prime-and-realign mechanisms for the initiation of RNA synthesis.…”

“…Since we hypothesize that the priming loop may stabilize the cRNA 3ˈ strand in the active site, we deleted residues 642-656 of the PB1 subunit of the RNAP, corresponding to the full priming loop (Fig. S11A) (16); the resulting mutant RNAP was previously shown to have a higher activity than wild type RNAP in an in vitro capped primer extension assay (16).…”

“…However, unlike initiation from the vRNA promoter, the priming loop is dispensable for internal initiation on the cRNA promoter (15). Instead, initiation on the cRNA promoter is suggested to use a 'prime-and-realign' mechanism, which starts internally at positions 4 and 5 to produce pppApG, followed by realignment of the pppApG product to bases 1 and 2 of the cRNA 3ˈ terminus, before elongation (14,16). The proximal 3ˈ end of the cRNA promoter must therefore be positioned differently in the active site to the vRNA 3ˈ end, resulting in nucleotides 4 and 5 being positioned correctly for internal initiation (Fig.…”

Structure and Function of the Influenza Virus Transcription and Replication Machinery

“…4D). The dinucleotide next acts as a primer for terminal initiation at positions 1 and 2 of cRNA after backtracking of the cRNA template (Deng et al 2006) with the aid of the priming loop and a regulatory polymerase (Oymans and te Velthuis 2018;Fan et al 2019). The regulatory polymerase forms a dimer with the resident cRNP-associated polymerase, which carries out replication.…”