Fusion of the influenza A H1N1 virus envelope with the endosomal membrane at low pH allows the intracellular delivery of the viral genome and plays an essential role in the infection process. Low pH induces an irreversible modification of the virus envelope, which has so far resisted 3D structural analysis, partly due to the virus pleiomorphy. This study showed that atomic force microscopy (AFM) in physiological buffer could be used to image the structural details of the virus envelope, both at neutral pH and after a low-pH treatment. At low and intermediate magnification, AFM of control virions confirmed both the pleiomorphy and the existence of zones devoid of glycoprotein spikes at the virus surface, as established by electron microscopy (EM). At higher magnification, the unique vertical resolution of the AFM in 3D topography demonstrated the lateral heterogeneity in spike distribution and strongly suggested that, at least locally, the spikes can be organized in an irregular honeycomb pattern. The surface honeycomb pattern was more easily detected due to an increase in spike height following low-pH treatment at low temperature, which probably prevented disruption of the organization. This enhanced contrast associated with low-pH treatment emphasized differences in the glycoprotein distribution between virions. It was concluded that, together with EM approaches, AFM may help to establish a correlation between surface structure and influenza virus infectivity/pathogenicity.


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