Structure and Function of the Influenza Virus Transcription and Replication Machinery

Abstract

Transcription and replication of the influenza virus RNA genome is catalyzed by the viral heterotrimeric RNA-dependent RNA polymerase in the context of viral ribonucleoprotein (vRNP) complexes. Atomic resolution structures of the viral RNA synthesis machinery have offered insights into the initiation mechanisms of viral transcription and genome replication, and the interaction of the viral RNA polymerase with host RNA polymerase II, which is required for the initiation of viral transcription. Replication of the viral RNA genome by the viral RNA polymerase depends on host ANP32A, and host-specific sequence differences in ANP32A underlie the poor activity of avian influenza virus polymerases in mammalian cells. A failure to faithfully copy the viral genome segments can lead to the production of aberrant viral RNA products, such as defective interfering (DI) RNAs and mini viral RNAs (mvRNAs). Both aberrant RNA types have been implicated in innate immune responses against influenza virus infection. This review discusses recent insights into the structure-function relationship of the viral RNA polymerase and its role in determining host range and virulence.

Figure 1.

Influenza virus RNAs and viral ribonucleoprotein (vRNP) structure. (A) Influenza virus viral RNAs (vRNAs). The influenza A virus genome consists of eight segments of single-stranded negative sense vRNA. Each segment contains conserved 5′ and 3′ termini, segment-specific untranslated regions (UTRs), and at least one large open-reading frame (ORF). (B) Schematic of influenza virus vRNP and transcription and replication. vRNA segments are assembled into vRNPs with multiple copies of nucleoprotein (NP) and a single copy of the viral RNA-dependent RNA polymerase (RdRp), composed of PB1, PB2, and PA subunits, that binds the conserved 5′ and 3′ vRNA termini. The RNA polymerase, in the context of vRNPs, transcribes vRNA into viral mRNA, which contains a 5′ cap-1 structure (cap) and a 3′ poly(A) tail (A_n), and replicates vRNA through a complementary RNA (cRNA) replicative intermediate, which is assembled with NP and polymerase into complementary ribonucleoprotein (cRNP). (C) Structure of the influenza virus vRNP. NP protomers are shown in surface and cartoon representation (PDB 4BBL).

Figure 2.

Structure of the influenza virus RNA polymerase. (A) Surface presentation of the influenza A virus RNA polymerase (PDB 6RR7) bound to capped RNA primer and 5′ and 3′ vRNA termini. The subunits of the RNA polymerase are colored as in Figure 1. (B) Schematic model of the influenza A virus RNA polymerase showing the core chamber of the polymerase and its connection to the solvent via template and nucleotide triphosphate (NTP) entry channels, and template and product exit channels. The location of the priming loop (PL) as well as the 3′ and 5′ end-binding pockets are indicated.

Figure 3.

Transcription by the influenza virus RNA polymerase. (A) Association of the influenza virus RNA polymerase with the serine-5 phosphorylated carboxy-terminal domain (CTD) of host RNA polymerase II (Pol II) and cap-snatching. (B) Schematic model of the influenza virus RNA polymerase showing the binding of capped RNA primer and template before transcription initiation (top) and elongation and duplex unwinding during transcription (bottom). (PL) Priming loop.

Figure 4.

Replication by the influenza virus RNA polymerase. (A) Synthesis of complementary RNA (cRNA) by the influenza virus RNA polymerase and coreplicative assembly of complementary ribonucleoprotein (cRNP). (B) Synthesis of viral RNA (vRNA) by the influenza virus RNA polymerase and coreplicative assembly of vRNP. Template realignment is promoted by a regulatory polymerase. ANP32A associates with RNA-free RNA polymerase and may regulate dimerization of the RNA polymerase. (C) Schematic model of the influenza virus RNA polymerase showing replication initiation during cRNA synthesis. (D) Schematic model of the influenza virus RNA polymerase showing replication initiation and realignment during vRNA synthesis.

Figure 5.

Aberrant RNA synthesis by the influenza virus RNA polymerase. (A) In addition to full-length viral RNAs (vRNAs), the influenza virus RNA polymerase produces defective interfering (DI) RNAs, mini viral RNAs (mvRNAs), and small viral RNAs (svRNAs). (B) Synthesis of DI RNAs and mvRNAs uses an intramolecular copy-choice mechanism. (C) Host-specific mutation in the viral RNA polymerase are linked to mvRNA levels. mvRNAs can be bound by RIG-I and induce RIG-I activation and cytokine expression.