Structure of Intracellular Mature Vaccinia Virus Visualized by In Situ Atomic Force Microscopy

Abstract

Vaccinia virus, the basis of the smallpox vaccine, is one of the largest viruses to replicate in humans. We have used in situ atomic force microscopy (AFM) to directly visualize fully hydrated, intact intracellular mature vaccinia virus (IMV) virions and chemical and enzymatic treatment products thereof. The latter included virion cores, core-enveloping coats, and core substructures. The isolated coats appeared to be composed of a highly cross-linked protein array. AFM imaging of core substructures indicated association of the linear viral DNA genome with a segmented protein sheath forming an extended ϳ16-nm-diameter filament with helical surface topography; enclosure of this filament within a 30-to 40-nm-diameter tubule which also shows helical topography; and enclosure of the folded, condensed 30-to 40-nm-diameter tubule within the core by a wall covered with peg-like projections. Proteins observed attached to the 30-to 40-nm-diameter tubules may mediate folding and/or compaction of the tubules and/or represent vestiges of the core wall and/or pegs. An accessory "satellite domain" was observed protruding from the intact core. This corresponded in size to isolated 70-to 100-nm-diameter particles that were imaged independently and might represent detached accessory domains. AFM imaging of intact virions indicated that IMV underwent a reversible shrinkage upon dehydration (as much as 2.2-to 2.5-fold in the height dimension), accompanied by topological and topographical changes, including protrusion of the satellite domain. As shown here, the chemical and enzymatic dissection of large, asymmetrical virus particles in combination with in situ AFM provides an informative complement to other structure determination techniques.

Atomic Force Microscopy Investigation of Viruses

“…This approach is particularly effective with large, complex viruses such as vaccinia virus (25,26) or mimivirus (9). With these large assemblies, ordered and disordered protein shells, lipid membranes, and the nucleic acid within can be revealed and analyzed.…”

“…The best example of a complete dissection of a complex virus using AFM is that of vaccinia virus, a pox virus of about 300-nm diameter that is delimited by a lipid membrane (25,26). It contains a double-stranded DNA genome bounded by several protein shells.…”

“…The latter reduces disulfide bonds and liberates polypeptides otherwise bonded to one another. Disulfide bond reduction appears to be particularly important in large, complex viruses where such covalent linkages cross-link coat proteins and stabilize capsids (25,39) (see Note 10).…”

“…The first AFM studies of noncrystalline viruses were retroviruses on cell surfaces (20)(21)(22), again, principally because they were immobilized by their association with cell surfaces. Single particles of larger viruses, and helical viruses, were eventually visualized by AFM, and these included tobacco mosaic virus, cauliflower mosaic virus, Tipula iridescent virus (15,23), herpes simplex virus (24), vaccinia virus (25,26), and mimivirus (9). Although virus crystals were investigated using both contact and tapping mode, noncrystalline specimens were imaged exclusively with tapping mode, in both air and buffer.…”

Atomic force microscopy investigation of a chlorella virus, PBCV-1

“…The methods employed for AFM visualization of PBCV-1 are essentially the same as previously described for the analyses of HIV (Kuznetsov et al, 2003), MuLV (Kuznetsov et al, 2004), and vaccinia virus (Malkin et al, 2003). After appropriate dilution into fresh buVer, virions were spread on poly-Llysine coated glass coverslips.…”