Translation Initiation During Influenza Virus Infection and Its Role in Cellular Immunit

There are numerous factors that direct and constrain influenza evolution. On the one hand influenza virus is constrained to encode proteins that allow it to complete the viral life cycle, but the immune system also recognizes those same viral proteins to mount an immune response. The interplay between selection for viral propagation and escape from immunity shapes the rapid evolution of influenza virus.;In Chapter 2, I explore one of the immune pressures that drives influenza evolution. It is well known that antibodies are a driver of the rapid evolution of influenza. However, it was unknown whether CD8 T-cells, which are also a component of adaptive immunity to influenza, also shape influenza evolution. There is experimental support that CD8 T-cells help control influenza infection and there are even documented instances of mutations that confer escape from CD8 T-cells. However, conventional tests of selection consistently fail to find evidence that CD8 T-cells drive influenza evolution. Since CD8 T-cells tend to be found in regions that are fairly conserved in influenza, I hypothesized that CD8 T-cells are a selective pressure but that functional constraint masked our ability to detect such selection by conventional means. I therefore employed two novel statistical methods that have increased sensitivity to detect selection in influenza. I leveraged the existence of parallel lineages of influenza that infect different hosts with varying immune pressures to examine whether CD8 T-cells drive influenza evolution. I find statistical support that one influenza protein, nucleoprotein, is under selective pressure to evade the CD8 T-cell response. This means that the role of CD8 T-cells is sufficiently strong to shape the evolutionary trajectory of influenza virus. There is strong interest in generating vaccines with broad responses to influenza. Since T-cells are capable of eliciting broad responses, the finding that the biological role of T-cells is strong enough to shape influenza evolution validates the use of T-cells in the development of broad-acting vaccines.;In Chapter 3, I explore translation initiation in the context of influenza virus infection. Because CD8 T-cells shape influenza evolution and non-canonical translation initiation can generate novel CD8 T-cell epitopes, I examined whether there was an evolutionary signature consistent with selection against alternate translation initiation in influenza virus. I find evolutionary support that is consistent with selection against alternate translation initiation of CTG codons in mammalian lineages of influenza. I then performed ribosome profiling, a deep sequencing technique that enables capture of ribosome protected fragments, to annotate sites of translation initiation in the influenza genome. I did not find evidence of CTG initiation in the influenza viral genome, but I did find a small number of alternate translation initiation sites at ATG codons in the influenza viral genome. One of these alternate initiation sites generates an N-terminally truncated form of neuraminidase (NA), the influenza protein that mediates viral egress. This N-terminally truncated NA lacks the first fourteen amino acids, but is enzymatically active, supports viral growth, and is broadly conserved in the N1 lineage. In the Discussion, I speculate about the possible discordance of the evolutionary results and ribosome profiling data. Overall, this work increases our understanding of the range of viral proteins that are generated in the compact influenza genome and uncovers new evolutionary signatures of influenza virus.