Delocalization of vaccinia virus components observed by atomic force and fluorescence microscopy

Better understanding of viral genomes is emerging as an urgent need as these genomes evolve and pandemic fears surface and for better understanding of viral infection processes. To address this need, we report a method to visualize intact, viral DNA and its interaction with viral proteins with the use of the atomic force microscope (AFM) in conjunction with fluorescence microscopy. Through a series of multifaceted experiments, we were able to visualize time-dependent progressive stages of proteolytic digestion and disassembly of extracellular enveloped vaccinia virus particles. After a 1-h treatment, the viral particles were partially digested and the viral cores showed slight disassociation in the AFM as evidenced by height analysis of individual virions. Most of the components of the virions were still intact. Further verification with florescence microscopy with nucleophilic and lipophilic stains demonstrated that viral DNA was, indeed still, co-localized within the viral core. However, with prolonged treatment with proteinase K and sodium dodecylsulfate, the AFM revealed that the viral core completely collapsed onto the substrate and had delocalized from the enclosed DNA. This process was again verified using fluorescence microscopy, the viral DNA was observed to be completely released from the viral core, in globular condensed form. These studies suggest that AFM imaging and fluorescence microscopy verification with stains specific for different constituents of viral particles is a valuable method to study the structural and mechano elastic properties of virus morphology and interactions of viral nucleoproteins with its DNA core.