Abstract
ABSTRACTThe RNA genome of influenza A viruses is transcribed and replicated by the viral RNA-dependent RNA polymerase, composed of the subunits PA, PB1, and PB2. High-resolution structural data revealed that the polymerase assembles into a central polymerase core and several auxiliary highly flexible, protruding domains. The auxiliary PB2 cap-binding and the PA endonuclease domains are both involved in cap snatching, but the role of the auxiliary PB2 627 domain, implicated in host range restriction of influenza A viruses, is still poorly understood. In this study, we used structure-guided truncations of the PB2 subunit to show that a PB2 subunit lacking the 627 domain accumulates in the cell nucleus and assembles into a heterotrimeric polymerase with PB1 and PA. Furthermore, we showed that a recombinant viral polymerase lacking the PB2 627 domain is able to carry out cap snatching, cap-dependent transcription initiation, and cap-independent ApG dinucleotide extension in vitro, indicating that the PB2 627 domain of the influenza virus RNA polymerase is not involved in core catalytic functions of the polymerase. However, in a cellular context, the 627 domain is essential for both transcription and replication. In particular, we showed that the PB2 627 domain is essential for the accumulation of the cRNA replicative intermediate in infected cells. Together, these results further our understanding of the role of the PB2 627 domain in transcription and replication of the influenza virus RNA genome.IMPORTANCE Influenza A viruses are a major global health threat, not only causing disease in both humans and birds but also placing significant strains on economies worldwide. Avian influenza A virus polymerases typically do not function efficiently in mammalian hosts and require adaptive mutations to restore polymerase activity. These adaptations include mutations in the 627 domain of the PB2 subunit of the viral polymerase, but it still remains to be established how these mutations enable host adaptation on a molecular level. In this report, we characterize the role of the 627 domain in polymerase function and offer insights into the replication mechanism of influenza A viruses.
Highlights
The RNA genome of influenza A viruses is transcribed and replicated by the viral RNA-dependent RNA polymerase, composed of the subunits polymerase acidic (PA), polymerase basic 1 (PB1), and polymerase basic 2 (PB2)
We first sought to determine whether these PB2 mutants could form a heterotrimeric complex with PB1 and PA and coexpressed them with PB1 and PA fused to a tandem affinity purification tag (PA-TAP) in human HEK-293T cells
Purification of the recombinant polymerases from cell lysates using the protein A tag on PA and analysis of the purified complexes by SDS-PAGE and silver staining showed that PB1 and PA formed a stable dimer in the absence of PB2, in agreement with previous findings [34, 35]
Summary
The RNA genome of influenza A viruses is transcribed and replicated by the viral RNA-dependent RNA polymerase, composed of the subunits PA, PB1, and PB2. We showed that the PB2 627 domain is essential for the accumulation of the cRNA replicative intermediate in infected cells Together, these results further our understanding of the role of the PB2 627 domain in transcription and replication of the influenza virus RNA genome. The influenza virus genome consists of eight single-stranded negative-sense RNA segments These viral RNA (vRNA) segments are coated by nucleoprotein (NP) and bound at their conserved 5= and 3= ends by the viral RNA-dependent RNA polymerase, forming viral ribonucleoprotein (vRNP) complexes. Transcription involves “cap snatching,” in which cellular capped RNA is bound by the PB2 cap-binding domain and cleaved by the PA endonuclease domain of the viral polymerase 8 to 14 nucleotides (nt) downstream of the 5= m7G cap [2,3,4]. Published crystal structures of influenza virus RNA polymerases have revealed that jvi.asm.org 2
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