Phenotypic size heterogeneity is a viral mechanism of persistence

Many enveloped animal viruses, including emerging human pathogens Ebola, Nipah, and Hendra viruses, produce a mixture of virus-particle sizes including very long, filamentous members. However, the function of the filamentous particles is unknown. The main impediments to characterizing viral filaments are their phenotypic origin (a single infecting particle gives rise to a range of particle sizes), difficulty of purifying particles to homogeneity according to their size, and the apparent lack of filament advantage in vitro. Influenza virus particles range in length by three orders of magnitude (~55 nm to ~30 μm). All influenza particles package at most a single genome, but the total number of the surface-exposed viral glycoproteins, hemagglutinin (HA) and neuraminidase (NA), and the HA/NA ratio scale with particle length. HA is the cell-entry protein of influenza, which mediates fusion between the viral and the endosomal membranes by the combined action of 3-5 active HA neighbors (fusion cluster). Here we identify influenza filaments as viral persisters increasing the probability of fusion-cluster formation and cell entry under HA-directed selective pressure. We fractionated viruses to enrich for spherical or filamentous particles and measured the single-particle kinetics of membrane fusion. As a surrogate for HA-directed selective pressure, we used a Fab fragment of a broadly neutralizing antibody that inactivates bound HA. In its presence, filamentous particles fuse more rapidly and more efficiently than do spherical ones. We show that the infectious advantage of filaments derives from their enhanced fusion efficiency rather than from rate effects. Filaments also offer universal protection from extreme HA inactivation. Our results show how the virus can adapt to any condition limiting HA function, and suggest targeting viral filaments as a strategy to prolong vaccine effectiveness or to thwart viral pandemic adaptation.