Assays to Measure the Activity of Influenza Virus Polymerase

Abstract

Influenza viruses use an RNA-dependent RNA polymerase (RdRp) to transcribe and replicate their segmented negative-stranded RNA genomes. The influenza A virus RdRp consists of a heterotrimeric complex of the proteins PB1, PB2, and PA. The RdRp is associated with the incoming influenza A viral RNA (vRNA) genome bound by the viral nucleoprotein (NP), in complexes called viral ribonucleoproteins, vRNPs. During the viral replication cycle, the RdRp snatches capped primers from nascent host mRNAs to carry out primary viral transcription. Viral mRNA translation produces new copies of the RdRp subunits and NP, which are required to stabilize and encapsidate complementary copies of the genome (cRNAs), forming cRNPs. These cRNPs then use the cRNAs to make new vRNAs, which are encapsidated into new vRNPs. Secondary transcription by new vRNPs results in further viral mRNAs and an increase of the viral protein load in the cell. The activities of the RdRp (mRNA, cRNA, and vRNA synthesis) in the influenza virus replication cycle can be measured on several levels, ranging from assessment of the accumulation of RNA products in virus-infected cells, through in situ reconstitution of the RdRp from cloned cDNAs, to in vitro biochemical assays that allow the dissection of individual functions of the RdRp enzyme. Here we describe these assays and point out the advantages and drawbacks of each.

Keywords: ApG extension assay; Cap-binding assay; Influenza virus; Minigenome; Polymerase purification; Primer extension assay; RNA-dependent RNA polymerase; Replicon; Viral-like RNA.