Sequence specific determinants, and impacts, of NS2 interactions with the influenza A virus polymerase

Influenza A virus (IAV) remains a threat. With high pandemic potential, and two circulating strains in human populations, IAV has contributed to yearly morbidity and mortality despite its identification and isolation nearly 100 years ago in 1930. In addition to intense study of its surface proteins, hemagglutinin and neuraminidase (and the surface-exposed loop of the viral ion channel) to identify relevant targets of adaptive immunity, there have been considerable efforts to characterize the viral replication and transcription machinery. Such work has recently led to the development of new antivirals, and using this information has also led to an improved ability to leverage IAV as a vector in oncolytic therapy. In this proposal, we will expand on this prior work with a particular focus on the interactions with, and effects of, NS2 on viral sequence. The minimal IAV replication machinery consists of a heterotrimeric polymerase (PB1, PB2, and PA), and viral nucleoprotein (NP). Transfection into cells of this machinery with an appropriate RNA template (or polymerase I-driven plasmid expressing an appropriate RNA template) is sufficient to recapitulate both viral transcription and genome replication. This results in the production of three molecules, vRNA (- sense viral genomes), cRNA (+ sense viral antigenomes), and mRNA (+ sense viral transcripts). For IAV, mRNA possesses a host-derived cap, snatched by the endonuclease subunit, PA, and a polyA tail that is generated through a stuttering mechanism. While the minimal machinery is sufficient to produce all three molecules, it does so in amounts that are quite different than during infection. One of the largest differences is that during infection, each of the eight RNA segments comprising the IAV genome produces dramatically different amounts of mRNA, which is not the case during transfection. As an explanation for this divergence in behavior, it has been determined that expression of an additional IAV protein, NS2, modulates transcription and replication. Specifically, NS2 broadly decreases transcription in favor of replication. However, it does so unequally across the eight genomic segments of IAV, more consistent with what is observed during infection. As the characterized, minimal, viral promoter is identical across the eight IAV segments, this suggests NS2 must interact with an extended, uncharacterized, promoter sequence. Moreover, whether NS2 plays any role beyond regulating this switch is unclear. In this grant we will identify the sequence interface regulating NS2 interactions using a high-throughput mutation approach that we have already trialed successfully, explore our preliminary results that NS2 may modulate viral polymerase processivity, and, lastly, follow up on recent results from our group that NS2 may also impact viral polymerase fidelity. By the completion of this grant, we will have redefined the replication and transcription dynamics of IAV, providing novel interfaces for potential intervention, as well as providing essential information to those seeking to develop artificial viral platforms for therapeutics.