Influenza A Virus : Spatial analysis of influenza genome trafficking and the evolution of the neuraminidase protein

Sammanfattning: Influenza A viruses (IAVs) are a common infectious agent that seasonally circulates within the human population that causes mild to severe acute respiratory infections. The severity of the infection is often related to how the virus has evolved with respect to the pre-existing immunity in the population. For IAVs, the most common mechanisms to avoid the immune response are to vary the surface antigens, hemagglutinin (HA) and neuraminidase (NA), by processes known as antigenic drift and shift.Antigenic drift refers to point mutations that accumulate in HA and NA as a result of the antibody-mediated selection pressure that exists in the population. The majority of the changes attributed to antigenic drift localize to HA and NA surface exposed regions, however this does not exclude that drift can also result in the selection of residues that are not exposed. One region where non-exposed residues have potentially been selected for is the NA transmembrane domain (TMD) of human H1N1 IAVs, where a temporal bias exists for the accumulation of polar residues. By examining these sequence changes in the NA TMD, we found that the polar residues contribute to the amphipathic characteristic of the NA TMD, which mediates the oligomerization of the N-terminus. As more polar residues became incorporated, the strength of the TMD-TMD interaction increased, presumably to benefit the NA head domain assembly into a functional tetramer. We determined that the amphiphilic drift in the NA TMD is able to bypass the strict hydrophobicity required for membrane insertion at the endoplasmic reticulum because it can utilize the co-translational translocation process to facilitate the insertion and inversion of its non-ideal TMD. The contribution of the TMD to proper NA assembly was traced to the formation of the Ca2+ binding pocket that is located at the center of the tetrameric assembly, as this pocket lies above the stalk linker regions and must be occupied for NA to function.In addition to antigenic drift, NA and HA can also undergo antigenic shift. Antigenic shift occurs when either of the gene segments encoding NA or HA are exchanged with ones from another IAV encoding another subtype of NA or HA. Different from antigenic drift, antigenic shift can only occur when a cell is co-infected and most investigations on the process of reassortment have been made at the protein level due to the methodological issues for labeling the RNA genome in situ. To overcome these technical limitations, we developed an in situ RNA labeling approach that provides highly specific spatial resolution of the IAV genome throughout the infection process. By applying this approach to temporally analyze the co-infection process, we found that the entry of a second IAV is stalled in the cytoplasm if another IAV has begun to replicate. Together, these results provide insight into the low frequency of antigenic shift in nature and provide evidence that non-exposed residues may make an underappreciated contribution to NA antigenic drift in human H1N1 viruses.