RNA-Free and Ribonucleoprotein-Associated Influenza Virus Polymerases Directly Bind the Serine-5-Phosphorylated Carboxyl-Terminal Domain of Host RNA Polymerase II

Abstract

Influenza viruses subvert the transcriptional machinery of their hosts to synthesize their own viral mRNA. Ongoing transcription by cellular RNA polymerase II (Pol II) is required for viral mRNA synthesis. By a process known as cap snatching, the virus steals short 5′ capped RNA fragments from host capped RNAs and uses them to prime viral transcription. An interaction between the influenza A virus RNA polymerase and the C-terminal domain (CTD) of the large subunit of Pol II has been established, but the molecular details of this interaction remain unknown. We show here that the influenza virus ribonucleoprotein (vRNP) complex binds to the CTD of transcriptionally engaged Pol II. Furthermore, we provide evidence that the viral polymerase binds directly to the serine-5-phosphorylated form of the Pol II CTD, both in the presence and in the absence of viral RNA, and show that this interaction is conserved in evolutionarily distant influenza viruses. We propose a model in which direct binding of the viral RNA polymerase in the context of vRNPs to Pol II early in infection facilitates cap snatching, while we suggest that binding of free viral polymerase to Pol II late in infection may trigger Pol II degradation.IMPORTANCE Influenza viruses cause yearly epidemics and occasional pandemics that pose a threat to human health, as well as represent a large economic burden to health care systems globally. Existing vaccines are not always effective, as they may not exactly match the circulating viruses. Furthermore, there are a limited number of antivirals available, and development of resistance to these is a concern. New measures to combat influenza are needed, but before they can be developed, it is necessary to better understand the molecular interactions between influenza viruses and their host cells. By providing further insights into the molecular details of how influenza viruses hijack the host transcriptional machinery, we aim to uncover novel targets for the development of antivirals.

Influenza A Virus Polymerase Recruits the RNA Helicase DDX19 to Promote the Nuclear Export of Viral mRNAs

“…Our in cellulo observations are clearly in favor of the second model, which is also supported by the existence of an intranuclear pool of DDX19 that can associate with mRNPs and with the cellular transcription machinery 22 53 . The binding of IAV polymerase to the C-terminal domain of cellular RNA polymerase II early in infection 54 might facilitate its association with DDX19 and thus the loading of DDX19 (possibly co-transcriptionally) onto viral mRNPs.…”

The Battle of RNA Synthesis: Virus versus Host

“…After entry into the cell, the vRNPs are released and transported into the nucleus. After arrival in the nucleus, the C-terminal domain of the FluPol PA subunit will specifically target and bind the phosphorylated Ser5 of actively transcribing RNAPII CTD [ 61 ] ( Figure 2 E). Subsequently, the PB2 cap-binding domain associates with the 5′ end of the cellular mRNA [ 60 ].…”

Initiation, Elongation, and Realignment during Influenza Virus mRNA Synthesis

“…Although it is currently assumed that this process is dependent largely on Watson-Crick base pairing between the primer and the template, transcription initiation without Watson-Crick base pairing has been observed ( 14 – 18 ). To study IAV transcription initiation in detail, we expressed the PB1, PB2, and PA subunits of the influenza A/Northern Territories/60/1968 (H3N2) virus in insect cells ( 9 ) and purified the recombinant enzyme using a tandem affinity purification (TAP) tag on PB2, as described previously ( 5 ). The purified enzyme was analyzed by SDS-PAGE for purity ( Fig.…”

“…Unlike IAV replication, in which the RdRp initiates de novo ( 6 ), the IAV RdRp uses a primer-dependent process for viral transcription initiation. To produce this primer, the IAV RdRp must first bind to the C-terminal domain of an actively transcribing, serine 5-phosphorylated RNA polymerase II (Pol II) complex in the nucleus of an infected cell ( 8 , 9 ). Subsequent binding and cleavage of nascent Pol II transcripts produce 8- to 14-nucleotide (nt)-long capped RNAs ( 10 ) ( Fig.…”