Influenza Virus Hemagglutinin Delays Endosomal Acidification - a Strategy for Successful Delivery of the Viral Genome?

Abstract

Upon endocytic uptake of influenza virus, acidification of the endosomal lumen triggers a conformational change of the virus spike protein hemagglutinin (HA) leading to fusion between the endosomal and the viral membrane. For efficient infection, release of the viral genome favorably occurs in the vicinity of the nucleus to prevent lysosomal degradation of the viral RNA and activation of the cellular antiviral response. How influenza viruses ensure optimal duration of endosomal residence to avoid premature as well as delayed fusion and release of the genome is not understood.The tight packing of HA in the viral envelope represents a remarkably high intra-endosomal protein concentration with high buffering potential for incoming protons. By using pH sensitive fluorescent markers we could show for the first time that the presence of a virus inside an endosome drastically slows down the acidification kinetics. We investigated the effect of cytoskeletal inhibitors on virus fusion and infection using a combination of single virus tracking and an intracellular fusion assay. In control cells, fusion mostly occurs in the perinuclear region. Inhibition of endosomal transport along microtubules by nocodazole did not change the numbers of fusion events, but randomized their localization within the cell. Interestingly, this dislocation correlates with strongly reduced infection efficiency, confirming that the site of virus-endosome fusion indeed plays an important role in the delivery of the viral genome.Taken together, our results demonstrate that influenza virus HA delays the endosomal acidification to ensure timely as well as locally optimal release of its genome. This suggests a general function of the high-density packing of spike proteins that is characteristic of enveloped viruses infecting via the endocytic route.