Atomic Force Microscopy Investigation of Vaccinia Virus Structure

Vaccinia virus was treated in a controlled manner with variouscombinations of nonionic detergents, reducing agents, and proteolyticenzymes, and successive products of the reactions were visualizedusing atomic force microscopy (AFM). Following removal of theouter lipid/protein membrane, a layer 20 to 40 nm in thicknesswas encountered that was composed of fibrous elements which,under reducing conditions, rapidly decomposed into individualmonomers on the substrate. Beneath this layer was the viruscore and its prominent lateral bodies, which could be dissociatedor degraded with proteases. The core, in addition to the lateralbodies, was composed of a thick, multilayered shell of proteinsof diverse sizes and shapes. The shell, which was readily etchedwith proteases, was thoroughly permeated with pores, or channels.Prolonged exposure to proteases and reductants produced disgorgementof the viral DNA from the remainders of the cores and also leftresidual, flattened, protease-resistant sacs on the imagingsubstrate. The DNA was readily visualized by AFM, which revealedsome regions to be "soldered" by proteins, others to be heavilycomplexed with protein, and yet other parts to apparently existas bundled, naked DNA. Prolonged exposure to proteases deproteinizedthe DNA, leaving masses of extended, free DNA. Estimates ofthe interior core volume suggest moderate but not extreme compactionof the genome.

INTRODUCTION

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INTRODUCTION

Vaccinia virus, the best-characterized member of the Poxviridaefamily, is unusual among most double-stranded DNA viruses inthat both transcription and replication occur in the cytoplasmof the host cell (5, 8, 24). The 191-kbp genome of the Copenhagenstrain contains up to 266 open reading frames encoding proteins>65 amino acids in length (9, 15), with 63 to 80 vacciniavirus gene products being packaged in the virion (4, 53, 57,60). Replication takes place within discrete cytoplasmic viralfactories that are thought to be devoid of cellular organelles(3) but which contain ribosomes (16). Infectious virions canbe isolated in several forms, depending on their degree of maturation,including mature virus (MV), wrapped virus, and enveloped virus(25).

Many general features of vaccinia virion architecture are readilyapparent from a variety of studies (3), notably a genome/enzyme-containingproteinaceous core, proteinaceous lateral bodies (LBs), andsurrounding surface tubular elements and lipid membrane. Nonetheless,despite $~$ 60 years of study by conventional electron microscopy(EM) (11, 14, 15, 45, 48, 54, 58), cryoelectron microscopy (1,9, 54), and more recently cryoelectron tomography (4) freezeetch (31), deep etch (11), and atomic force (22) microscopies,specific controversies remain in issues such as the intrinsicrigidity of the core, the degree of order in genome packaging,the degree and affinity of the association of the genome withprotein in the packaged state, the presence/absence of an "innermembrane" or "palisade" enwrapping the core, and the numberof lipid bilayers that enwrap the intact virion. Structuraldetails of the virion are relevant to several stages of thevaccinia life cycle, including mechanisms of fusion and entry,extrusion of early mRNA from the infecting virion, disassemblyof the infecting virion, genome packaging into nascent virionsduring assembly, and virion maturation/membrane enwrapment.

A persistent drawback with EM images, irrespective of the techniqueapplied, is that they generally provide two-dimensional projectionsof specimens onto a single plane. Three-dimensional informationis usually absent, and approximations can be recovered onlyvia reconstruction. Thus, ambiguities and uncertainties arisein the assignment of features to internal structures and surfaces.In addition, various artifacts may be introduced, particularlyvia the fixing and staining procedures generally employed toenhance contrast. Atomic force microscopy (AFM) complementsEM in a number of ways. Although the resolution of AFM is theoreticallylower than that of EM, it often reveals details of soft, biologicalspecimens, including viruses (20), that cannot otherwise beobtained. The three-dimensionality of specimens can easily bepreserved during imaging, and instead of the specimen beingprojected onto a plane, topographical features of a specimen'ssurface are revealed. This might appear to limit the utilityof the technique for delineating the internal structures ofspecimens such as complex viruses, but these drawbacks can belargely overcome. Viruses, as we have now shown in several investigations(32, 35, 38-40, 42-44, 52), can be broken down or disassembledin controlled ways to reveal, sequentially, internal layers.By chemical and enzymatic dissection of virions accompaniedby AFM visualization of the products, information regardinginternal structures can be revealed. AFM is a single-particleanalytical method that does not rely on a homogeneous population(as would, for example, cryoelectron microscopy reconstruction).It records not only the structures and states common withina population but also, with equivalent accuracy, features foundin just a few individuals. AFM can be applied with equal easeto dried specimens or specimens fully hydrated in physiologicalmedia. AFM records topography irrespective of the electron density.

We have previously established, as a proof of principle, theamenability of vaccinia virus to AFM analysis (22). Here wereport a more in-depth AFM analysis of vaccinia virus that webelieve clarifies some significant structural questions andprovides additional, general details. The images presented hereare, we believe, a significant improvement over those shownin the previous study (22) in terms of resolution and populationcoverage within individual preparations. We have focused inparticular upon the virion core and LBs, along with aspectsof the packaged genome, such as its degree of order and associationwith protein and its appearance when released from virion cores.We focus exclusively on MV released from mammalian cells usingstandard protocols. We have imaged MV in standard buffers akinto those used for storage and infection and have also treatedit with a number of vigorous reagents and reagent combinations.

MATERIALS AND METHODS

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MATERIALS AND METHODS

Sample preparation and AFM analysis. The virus preparation described elsewhere (22) was used throughoutthe current study, at the titer described previously. Intactand treated viruses were imaged on glass coverslips or freshlycleaved mica coated with poly-L-lysine. Genomic DNA was imagedon freshly cleaved, uncoated mica. For virus imaging, 1.5 µlof 50-fold-diluted freshly resuspended MV in 10 mM Tris (pH7.8) was deposited on the substrate and allowed to sedimentfor 30 min at room temperature. For imaging in air, the specimen,on the substrate, was rinsed multiple times with distilled waterand then dried under a stream of N₂ gas. Prior to imaging underliquid, specimens were treated with a solution of 5% glutaraldehydein phosphate-buffered saline (PBS) for 15 min and then rinsedwith distilled water. Fixation with glutaraldehyde has beenshown in previous investigations to not perturb the surfacestructures of virus particles within the resolution of the AFMtechnique (32, 34, 35, 39, 40).

For enzyme digestion experiments, after deposition of the intactspecimen onto the imaging substrate, the substrate was rinsedmultiple times with PBS and excess PBS was wicked away withtorn filter paper. After addition of 15 µl of enzyme solution,the substrate was sealed in a plastic cell (to minimize evaporation),followed by incubation either in an incubator at 37°C orat room temperature. For sequential treatments with differentreagents, the specimen on the substrate was rinsed with PBSafter removal of the first reagent before addition of the second.

Imaging procedures were essentially as previously describedfor other viruses (32, 34, 40, 42, 44) and RNA (33, 37), withuse of a Nanoscope III multimode AFM instrument (Veeco Instruments,Santa Barbara, CA) calibrated to small lateral distances byimaging the 111 face of a thaumatin protein crystal as a standard,based on the known lattice spacings (19, 21). After the samplesubstrates were placed in a 75-µl fluid cell, images wereacquired at 26°C in tapping mode (17, 18) using commerciallyavailable silicon tips (imaging under air) or oxide-sharpenedsilicon nitride tips (imaging under fluid) at a scan frequencyof 1 Hz and an oscillation frequency of 9.2 kHz (under fluid)or 300 kHz (in air). In the AFM images presented here, increasingheight above the substrate is indicated by an increasingly lightercolor. Due to the distortion of lateral distances due to convolutionof the cantilever tip shape and the surface features scanned,quantitative measures of size were based either on heights abovethe substrate or on center-to-center distances between particles.

RESULTS

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RESULTS

The intact virion. The shape, dimensions, and general surface characteristics ofhydrated MV imaged by AFM (Fig. 1a and b) were as previouslyreported (22), with the virus assuming a familiar rectangular("brick") shape upon dehydration (Fig. 1c and d). Particleswere pleomorphic, with an outer surface design varying fromthe more regular, short, haphazardly intersecting "railroadtracks" topography of naturally cell-extruded virus (see reference11and references therein) to a more punctate topography, referredto in the early literature as the "M" or "mulberry" form ofthe virion (7, 25, 47, 58), which may be more diagnostic ofintracellular virus released by cell rupture (10, 11). Parenthetically,the virion surface also closely resembled the external surfacesof various animal cells as imaged by AFM (34, 36, 39, 40), consistentwith a comparable array of membrane proteins anchored in a lipidbilayer, perhaps with an underlying membrane skeleton.

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FIG. 1. Intact vaccinia virions, hydrated and dried. (a and b) AFM images of fully hydrated MV scanned in a fluid cell under PBS. Virion surfaces were very rough, and LBs were invisible or indistinct. (c and d) MV that had been dried on the AFM substrate and scanned in air. LBs in dried particles were prominent and readily visualized.

One LB (or very occasionally both) was discernible attachedto hydrated specimens (Fig. 1 and 2). As observed previously(22), the profile of the LBs visibly associated with every particlebecame greatly accentuated upon virion dehydration (Fig. 1 and2), and a deep trench formed around the visible LBs which clearlydelineated them with respect to the background viral surface.LBs were about one-third the overall dimensions of dried intactMV (albeit the height could not be measured) but less rectangularin shape.

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FIG. 2. Two MV particles in panels a and b were dried in air on the substrate and imaged by AFM. Many dried particles showed a corona of membrane with embedded protein (indicated by arrows). This membrane appears to have withdrawn and disengaged from the particle. The prominence of LBs was also notable.

Virions in both hydrated and dehydrated preparations were frequentlyfound to be aggregated with one another (a well-known propertyof vaccinia virus [17]), albeit isolated virions were also frequentlyobserved. The isolated virions permitted observation of structuralchanges arising from chemical or enzymatic treatment (below).Insofar as individual particles were readily visualized againsta very clean substrate background (Fig. 1), the MV preparationappeared almost completely devoid of large cellular assembliesor subviral materials (such as partially degraded particles,smaller viral fragments, or protein complexes).

In the dehydrated samples, a corona was frequently discernedsurrounding the particle (Fig. 2). We interpreted this as apartial retraction of outer membrane(s) and associated proteinsfrom the upper part of the virion surface toward the imagingsubstrate surface, leading to the exposure of a somewhat smootherunderlying surface that may be diagnostic of a form previouslyreferred to as the "C" form (6, 32). Brief treatment of virionswith nonionic detergent-containing solution (e.g., NP-40 plusdithiothreitol [DTT]) dissolved the membrane quickly but leftthe ring of proteins at the periphery intact.

Interestingly, attempts to induce osmotic phenomena in hydratedparticles by exposing them to pure water had no discernibleeffect on the integrity of virions, indicating a strong insulationfrom ion flux, consistent with the virions' tolerance to low-ionic-strengthbuffers, such as the standard storage buffer (10 mM Tris-HCl).Nonetheless, the severe change in volume upon dehydration/rehydration(Fig. 1) (22) suggests that water can be exchanged.

Chemical and enzymatic dissection of the vaccinia virus virion. Before discussing the treatment of particles with various reagents,we should emphasize that the population of treated particleswas an ensemble of individuals having a variety of structuralstrengths and weaknesses. Not all particles responded to a giventreatment or became degraded in an identical or synchronousmanner. Different particles, exposed to the same reagents, wereobserved in a variety of states of disassembly as a functionof time. Since it seemed unlikely that the glutaraldehyde-fixedparticles were not equivalently exposed to reagents, they werepresumably structurally nonidentical (some being stronger/moreresistant to degradation than others, which were more fragile/easilydisrupted), presumably a result of the harvest of an asynchronousinfection by cell rupture. Therefore, we have exercised cautionin describing degradation pathways, particularly in terms ofthe exposure times necessary to induce structural changes. Ingeneral, we describe the response of the majority of particles.

Isolated STEs had a fibrous rather than tubular appearance. Surface tubular elements (STEs) have been previously observed(42, 45, 50, 51, 56, 58, 59) and are considered to be dissociatedforms of the "short railroad tracks" or punctate spots previouslyobserved associated with extracellular virions or "M"-form virions,respectively (see above) (11). In our hands, STEs dissociatedfrom the virion in the presence of low concentrations of detergentand reducing agent (0.1% NP-40-10 mM DTT) (Fig. 3), revealingtheir structure quite clearly. Thus, although some minor proportion(perhaps 15%) was present as individual globular units, STEsappeared principally as thick fibers with a chain-like appearance(Fig. 3). By AFM, these fibers did not show evidence of a tubular(or otherwise hollow (33) morphology or of helicity. Rather,they appeared more as linear aggregates of one kind (or perhapsjust a few kinds) of protein assembly. We will therefore henceforthrefer to them as surface fibrous elements (SFEs). SFEs had afairly uniform diameter of about 20 nm and lengths mostly inthe range of 100 to 150 nm (though some appeared to be as longas 500 to 600 nm—twice the diameter of the virion). Bycomparison of SFE-positive and SFE-negative virions (Fig. 1c and dversus Fig. 3a and b), AFM results were consistent with thedeep-etch EM (11, 14) observations that when attached to thevirus, SFEs form a dense network or array oriented more or lessparallel to the virion surface ("short railroad tracks"). However,by AFM, some individual SFEs, when packed around the virioncore, appeared laterally twisted and/or bent and sometimes doubledback on themselves (Fig. 1c and d). The observed SFE diameterof 20 nm was consistent with the thickness of the virion "outermembrane" in virion thin sections (6, 9, 12, 21).

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FIG. 3. Particles in the process of SFE shedding. (a) MV were treated for 5 min at 22°C with 0.1% NP-40 and 10 mM DTT, dried in air, and then imaged by AFM. (b) The particles underwent the same treatment, but for 15 min. The SFE layer lying immediately beneath the outer lipid membranes became exposed, with SFEs being rapidly shed onto the imaging substrate as linear aggregates (see the text). Note the substantial increase in density with time of the SFE on the substrate surrounding the particles. SFEs apparently occupy most if not all of the space between the outer membrane and the core. (c and d) Higher-magnification images of shed SFEs. After shedding, SFEs rapidly disassembled into individual globular units.

Upon exposure to higher concentrations of NP-40-DTT (0.5%-50mM, respectively), SFEs appeared to depolymerize/disaggregate,prior to loss from virions (data not shown). Under these conditions,shedding and disaggregation into globular units was so rapidthat it could scarcely be visualized. By comparison, after treatmentin other ways, EM studies have shown partially disaggregatedforms still attached to the virus (see references 14, 15, and20 and references therein). After shedding, the individual globularunits observed by AFM showed remarkable uniformity in size andshape, coating the substrate in the vicinity of the sheddingparticle (Fig. 3a and b). After 60 min of exposure, even atlower concentrations of NP-40-DTT (0.1%-10 mM, respectively),the fibrous layers of most particles were entirely strippedand monomerized and the underlying virion cores were exposed.SFEs appeared to be the principal material filling the spacebetween the virion outer membrane(s) and the virion core andtherefore presumably comprise some of the more abundant of thestructural proteins in the virion, arguably virion protein p4b(33). Other possibilities might be actin or a residual fragmentof the A-type inclusion body protein or inclusion body embeddingfactor VO.

MV cores and sacs. Vaccinia virion cores could be reproducibly generated by 60min of exposure of MV to 0.5% NP-40 plus 50 mM DTT at 37°C.Examples of vaccinia cores are shown in Fig. 4. Those in panelsa and b were dried on the substrate, while those in panels cand d were imaged in their fully hydrated state. Dried coresappeared complex and pleomorphic in their structural detail.Most, however, shared some common features and exhibited similarbehavior upon further exposure to reagents. The typical hydratedcore, as revealed by AFM (Fig. 4c and d), was an oblong bodyof dimensions 220 nm by 200 nm and a height above the substrateof $~$ 120 nm. These dimensions shrank by approximately 15 to 20%when the cores were dried and then imaged in air. However, becauseof their apparent softness, the cores' surface structures wereresolved in considerably greater detail when dry.

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FIG. 4. Longer-duration treatment produced fully stripped virion cores. (a and b) Two examples of dried virion cores prepared by 60-min exposure of MV to 0.5% NP-40-50 mM DTT at 22°C, followed by drying in air. SFEs have been lost, revealing the DNA-containing cores with their associated LBs. In these dried particles, the wall enclosing the nucleic acid appears to be heavily perforated. (c and d) Fully hydrated cores, treated in the same way. Dimensions of the hydrated cores were approximately 220 nm by 200 nm by 120 nm. In panel d, the proteins making up the LBs were particularly well defined.

The surfaces of dried cores appeared as dense networks of proteins,having a variety of shapes and sizes, and were extensively perforatedwith holes or channels. The outer surfaces of hydrated cores,by contrast, generally appeared quite smooth. Cores from mostparticles could be progressively etched by several hours ofexposure to NP-40 and DTT (though a few individuals appearedto degrade much more rapidly). This process was noticeably acceleratedif the incubation contained protease or was followed by proteasetreatment. In the presence of proteinase K, for example (afterfirst washing thoroughly with water to remove NP-40), or inthe presence of trypsin plus 0.5% NP-40, a progressive etchingof the core outer surface could be observed, with the core surfacebecoming rougher in appearance with increased duration of proteolysis(data not shown).

Two interpretations of the "etching" observations were considered:(i) removal of a smoother outer protein layer revealing a rougherinner one or (ii) deepening of preexisting etch pits withina unitary wall. Our observations seemed consistent with thecore wall being identical to the "palisade layer" (also referredto as the "core membrane") identified in EM thin sections, sincethe deeply ridged or etched structure imaged by AFM could beexpected to appear striated in section. Our observations aretherefore consistent with the core wall having a structurallyasymmetric unitary structure, without the need to invoke thegreater structural complexity of a double wall or a membranesurrounding the core. It should be noted, however, that theparticles had been treated with NP-40, which could disrupt anylipid membrane. A multiwalled architecture, however, remainsa possibility.

Cores degraded in the presence of proteinase K finally lostor expelled their internal DNA. Although the remaining proteinsuperstructure of the cores remained largely intact, the coresuperstructure collapsed on the substrate as flat, round "pancakes"of 12 to 15 nm in height (Fig. 5), as one would expect of deflatedballoons or empty sacks ("ghosts" [14]). In Fig. 5d and e, thesurfaces of the ghosts appeared to be very rough in texture,which was apparent whether or not they were hydrated. In theprocess of proteolytic etching of the outer layer of the cores,LBs were also degraded and lost. In some cases we observed whatappeared to be intact LBs separated from cores, suggesting aparticular susceptibility of the LB-core linkage. Most LBs,however, were progressively degraded until only remnants couldbe detected still associated with the cores.

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FIG. 5. (a to c) Virion cores prepared by treatment of MV with 0.5% NP-40 and 50 mM DTT followed by exposure to proteinase K in PBS for 16 h at 37°C, drying in air, and imaging by AFM. Virion DNA has been lost from the cores along with the LBs. Core walls have been substantially degraded and appear as flattened sacs on the imaging substrate. These sacs are 12 to 15 nm in height. Proteins and protein fragments can be seen on the substrate surrounding the sacs. (d and e) Higher-magnification images of the flattened sacs showing their rough and irregular surfaces, presumably maintained by residual protein-protein linkages. Note the absence of any LBs. (f) Residual sac of a core which had not been exposed to proteinase K at all but had nonetheless lost its DNA spontaneously. Its thickness is somewhat greater, 15 to 20 nm. Note the residual remnants of an LB (arrow).

LBs. As illustrated in Fig. 1 and 2, LBs were sometimes apparenteven in fully hydrated, intact MV but became more distinct upondrying. The profound trench developing around the LBs upon dryingshowed them to be distinct entities ("organelles") of the virion.As expected, the LBs were associated with opposing sides ofthe virion core (Fig. 6a and b). In many images, only one lateralsurface of a particle was visible to the AFM tip, and therefore,only one core-associated LB could be verifiably ascertained.In virtually all of those cases in which the orientation ofa particle was such that both LBs would be visible to the AFMtip, two symmetrically placed LBs were in fact observed. Thus,we feel that most particles did indeed possess two LBs. In Fig.4d, the features of a hydrated LB attached to a virion corewere particularly well resolved. The LB was significantly rougherand more irregular in texture than the remainder of the coreand appeared to contain aggregates or assemblies of proteinshaving a range of sizes and shapes (as also indicated in Fig.4c).

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FIG. 6. Additional virion cores. (a) After exposure of MV to 0.5% NP-40 and 50 mM DTT for 60 min at 22°C and imaging under water. Associated with the core were two LBs disposed on opposite sides. Dissociated proteins cluttered the substrate surface. (b) Again, a pair of LBs can be seen associated with some core particles. (c and d) Low- and high-magnification images of a native, untreated MV particle that had been air dried and imaged by AFM. An LB has been lost from the particle (the cause is unknown), and a deep pit remains in the otherwise still-intact virion.

The LBs were firmly attached to (and apparently covalently continuousby other than disulfide bonds with) the core, as indicated bytheir resistance to prolonged treatment with NP-40 plus DTT(though for some cores, an LB appeared to have become completelysheared away even in the absence of added protease, consistentwith some heterogeneity in the preparation of intracellularvaccinia virus). LBs could, however, be consistently degradedby treatment with proteinase K (Fig. 7). As shown in Fig. 6c and d,LBs could occasionally be lost even from intact MV, and theirremoval left a deep pit or indentation in the host particle.It was difficult to approximate the depth of the pit for variousreasons, including the radius of curvature of the AFM tip. However,insofar as the genome did not spill out and DNA (see below)was not imaged within the pit, we do not believe the pit torepresent a perforation in the core wall. The pit was even moreclearly revealed in additional images of cores immediately followingLB removal by proteolysis with trypsin (Fig. 7a and b) (11).While many cores exhibited a profound central depression resultingfrom loss of LBs (as shown in Fig. 7a and b), occasionally trypsintreated cores did not. Interestingly, those that did not appearedto increase in number with time of exposure to protease. Itis possible that cores lacking pits were simply the occasionalproducts of a somewhat arbitrary proteolytic process. On theother hand, we suspected the progressive assumption of a "relaxed"shape from the initially pitted cores. That is, following lossof the LBs, the core wall may be prone to relax or "pop out"into a fuller, more loaf-like shape (as exhibited by the particlesin Fig. 7c and d).

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FIG. 7. Cores produced by treatment of MV with a mixture of 0.5% NP-40 plus 50 mM DTT plus 0.1 mg/ml trypsin at 37°C for 60 min and then imaged by AFM in a fluid cell (thus, the particles represent fully hydrated cores). (a and b) LB loss left a deep, central depression. (c and d) Similarly treated cores, though these had more of a loaf shape and failed to exhibit the pits left by degraded LBs. In panel d, one of the three cores (indicated by an arrow) had lost its DNA and collapsed to a flattened sac, while its two nucleic acid-containing neighbors retained their full shape.

Nucleic acid bundles within the cores. As described above, once the DNA had exited the core, the vacantprotein sac collapsed like a plastic bag or a deflated ballonto the imaging substrate. The genomic DNA, visualized by AFMat various times both within and after leaving the cores andin different states of unraveling, displayed a variety of interestingfeatures. In some cases, after NP-40-DTT treatment, the DNAwithin the virion cores was relatively unperturbed even whenthe surrounding sac was damaged. This is shown in Fig. 8a and b,where the sac was "peeled back" without disturbing its contents.In other instances after prolonged NP-40/DTT treatment, theentire sac was separated from the nucleic acid, leaving theDNA as a condensed packet or bundle with some unraveling atits edges (Fig. 8c). This bundle was reminiscent of the virionnucleoids observed by EM after sodium dodecyl sulfate treatmentof particles (13). The dried nucleic acid bundle had a loaf-likeshape, $~$ 100 to 120 nm in length, reaching up to $~$ 80 nm in height.The observation of this "nucleoid" as an almost entirely intactentity in the absence of the sac implied an absence of stronginternal pressure within the "relaxed" core (after a loss ofLB indentation, albeit there could have been slightly greaterpressure with LBs in place). Although the nucleoid showed noinclination to spontaneously burst or spread, it was able tounravel, starting to assume the more recognizable features ofDNA strands, mostly decorated with proteins (Fig. 8C and 9).

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FIG. 8. Core contents after treatment with 0.5% NP-40 and 50 mM DTT for 20 min at 22°C. (a) The wall/sac of a core has been partially sheared away (arrow), revealing an inner packet of nucleic acid, some of which was apparently associated with protein. (b) A higher-resolution image of the sheared sac and the core contents. Note that the nucleic acid and its associated protein had not burst from the opened core but remained intact. (c) Another unusual observation: the internal contents of a virion core, which has been entirely shorn of its sac, are shown on the AFM substrate. At the top, the nucleic acid with associated protein can be seen initially unraveling and spreading on the substrate around it. The packets of DNA inside the core were present as unique entities that in general unraveled only slowly into recognizable strands of nucleic acid.

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FIG. 9. Nucleoprotein loss from sac. (a) Cores were produced by treatment of MV with 0.5% NP-40 and 50 mM DTT at 22°C, washed with PBS, and subsequently treated with 0.1 mg/ml proteinase K for 30 min at 37°C. The core has begun disgorging its contents via a rupture in the core sac/wall. Spilled nucleic acid appears to be almost entirely associated with protein. (b) Nucleoprotein complex imaged at higher magnification. (c and d) the same nucleoprotein after a 60-min exposure; strands of naked DNA are beginning to appear (indicated by arrows). Note, however, that most of the DNA is still protein coated.

Vaccinia DNA. After protease treatment, the virion DNA was not visualizedas a sac-free packet, as shown in Fig. 8c, but rather as nucleoproteinflowing out of a perforated or protease-damaged core onto thesubstrate surface in the immediate vicinity of the disintegratingcore (Fig. 9). During the time course of protease digestion(which is relatively short), DNA seemed to be most heavily studdedwith protein at the earliest times after its emergence fromthe cores (e.g., see Fig. 9), with some strands being completelycoated with protein (particularly evident in Fig. 9c and d).However, some protein-free strands could also be seen even atearly times. Later in the proteolytic time course, the DNA appearedmore as shown in Fig. 10, with significantly smaller amountsof associated protein. Most of the protein units associatedwith the DNA appeared fairly uniform in size, as though therewas a predominant nucleoprotein unit (consistent with the four-protein"subnucleoid" structure described in reference 12), albeit DNA-freeprotein aggregates of other sizes and shapes were also observed(Fig. 9 and 10). Presumably a variety of proteins (e.g., enzymes)were included in the mass of protein and DNA. After prolongedexposure to proteinase K (for several hours or overnight), proteinwas almost entirely stripped from the DNA, and the DNA appearedultimately as complex networks of folded, tangled, and looped,mostly naked, strands (Fig. 11). Often the most stubbornly remainingproteins appeared to be "soldering" together various strandsand loops in the expelled DNA.

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FIG. 10. Nucleoprotein. (a) A cluster of vaccinia cores at the edge of the image (indicated by arrow) had emptied its nucleic acid entirely, which is seen expanded about it on the AFM substrate. The remains of the sacs are still present. (b to d) Higher-magnification images of various regions of disgorged DNA from the cores in panel a and others (of which there were many). DNA was also frequently exuded from cores after exposure to a mixture of 0.5% NP-40 plus 50 mM DTT plus 0.1 mg/ml proteinase K for 30 min.

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FIG. 11. Naked genomic DNA. (a to d) Images of vaccinia DNA which has emerged from ruptured core particles after exposure to proteinase K for 12 h. The protein was almost entirely degraded, leaving only naked DNA on the AFM substrate.

When condensed in packets, as in the intact virion core, thestrands did not appear to be arranged or packed in an orderedmanner. It seemed as though the DNA or nucleoprotein was simplystuffed inside the sac-like core in a disordered manner, asone might stuff a down parka or sleeping bag inside a nylonsac. Furthermore, when the nucleic acid unraveled at the edgesof the DNA packets, its arrangement appeared arbitrary withno obvious systematic mode of packing. It appeared like a bagfull of wet yarn. We saw no evidence for an ordered, tube-likenucleoprotein structure (27).

Radically ruptured MV. The chemical and enzymatic dissection of vaccinia virions describedabove was a result of slow, reasonably controlled digestionwith specific reagents and may bear some resemblance to whatoccurs during the early stages of vaccinia virus infection.We also identified reagents leading to a more radical rupturekinetic. "Radical rupture" led to the display of nucleic acidon the substrate directly from intact, native virions, evenin the absence of any detergent or protease. For example, exposureto high concentrations of DTT alone (0.25 M) (Fig. 12a and b)or brief exposure to a high pH (pH 12) (Fig. 12c and d) ledsome particles to spontaneously rupture, followed by an apparentlysudden release of nucleic acid to the exterior. When nucleicacid was spilled by a native particle undergoing chemical orphysical "radical rupture," a massive egress of what appearedto be almost entirely naked DNA, with little or no accompanyingprotein, was apparent. Our impression was that only the nakedportions of the DNA exited the particle, with the more protein-richportions perhaps remaining attached to the interior surfaceof the protein sac. This can be seen, to some extent, in Fig.12b and c.

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FIG. 12. (a and b) MV treated with 0.25 M DTT only at 37°C for about 15 min and imaged in air by AFM. (c and d) MV exposed briefly to pH 12. Under either condition, a few MV ruptured precipitously and DNA poured from the damaged virions onto the substrate.

mRNA synthesis. We made extensive efforts to treat virion cores with buffer/reagentmixtures known to support mRNA synthesis in the hope of visualizingnewly made mRNA on the substrate or on the surfaces of particles.In this way we might have characterized the production of earlymRNA from the encapsidated DNA. However, no evidence was everdetected of mRNA emerging from the particles, as might havebeen observable by AFM (33, 37). It must be emphasized, however,that because of protein loss induced by the transcription buffer(which also contained NP-40 and DTT), the substrate around theparticles became very rough and cluttered with shed and damagedproteins. Similarly, the surfaces of the virion cores themselvesbecame very uneven because of their associated protein. Thissurface irregularity may have precluded observation of mRNAby AFM, since a clean, flat surface would normally be required.

Anomalous particles. We made a final, curious observation. In one or two images,we observed what appeared to be a particle that was not recognizableas vaccinia virus. An example can be seen in Fig. 13. It wasnonenveloped, had a diameter of about 160 nm, and exhibitedwhat appeared to be an ordered or partially ordered arrangementof protein clusters, much like capsomers, on its surface, asone might expect of a conventional icosahedral virus. However,we were not able to unambiguously verify the symmetry of theparticles from AFM images. Presumably this was a contaminatingvirus of minor abundance or some aberrant vaccinia virus.

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FIG. 13. Particle X: occasionally, particles like this were observed among untreated MV. These particles were larger in diameter than MV, almost completely spherical (even after drying), and appeared to have an ordered arrangement of protein clusters (or capsomers) on their surfaces.

DISCUSSION

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DISCUSSION

There have been a significant number of structural studies ofvaccinia over the decades, principally via various forms ofEM (see the introduction) but also by AFM (22). This body ofwork is extensive and has recently been reviewed (3). From thecurrent AFM work, we have been able to confirm and lend weightto specific interpretations of EM observations, which, alongwith some additional findings, suggest a consensus model ofvaccinia virion structure. In particular, we have been ableto address SFEs, LBs, the nature of the core wall, and the packagedgenome.

The fibers observed surrounding the vaccinia core (likely consistentwith the "coats" observed in a previous study [22]), which werenamed SFEs, appeared to be composed predominantly of a multimerizedprotein complex, which reverted efficiently to a monomer formupon treatment with NP-40 and DTT. The fibers appeared to beoriented parallel to the virion surface (Fig. 1 and 2), an observationconsistent with both the "short railroad tracks" surface topographydescribed by Heuser (20) and the very notable ropelike helicalsurface fibers of parapoxvirus (46, 48, 49, 55). Although somewhatcytoskeletal in appearance, the mode of depolymerization ofthese SFEs, i.e., primarily under reducing conditions, wouldseem atypical for common cellular cytoskeletal proteins. Overall,our observations would support a view that the inner portionof the virion double outer layer, visible in some EM thin sections,may comprise these fibers in sections covered with a virionouter lipid bilayer, itself studded with proteins.

LBs were clearly imaged by AFM. Other than to say that the LBssurely do exist, we can only speculate as to their compositionand function. The sizes and gross appearances of the two LBsassociated with a core were equivalent (or roughly so). TheLBs appeared similar to large protein complexes found in cellsand may have comparable functions, such as the anchoring ofDNA telomeres, orientation or anchoring of membrane proteinsat the virion surface, or compression of the internal volumeof the core (clamping the core wall down over the genome bytaking up the slack in the core wall). The attachment sitesfor the LBs appeared symmetrically placed, indicating that theydo not adhere arbitrarily to the core but instead have specificattachment or binding sites. This evokes two possible modelsfor LB/virion morphogenesis. In one, the protomers of the coresac are constructed prior to LB attachment, and yet insteadof being randomly distributed, some (at least) occupy discretelocations in the shell to provide the LB anchoring sites. Inan alternative view, the LBs might appear early in the virionmorphogenic pathway, acting as nucleation points for core wallgrowth so that once the LBs are saturated with core wall proteins,the latter would continue to assemble by interacting only withone another. Indeed, the collapsibility of the empty sac comprisingthe core wall (Fig. 5) (14) might be consistent with such amodel. The collapsibility of the core wall would be consistentwith the presence of an apparent external bracing during virionassembly (provided by the vaccinia virus D13 protein's clathrin-likenetworks) (11, 31). The core wall may be built within such atemplate, or mold, within the crescents that are characteristicof the early stages of virion morphogenesis.

With regard to genome packaging, we should first point out thatin a previous AFM study (22), images of degraded vaccinia virusparticles were presented that contained, or so it was believed,segmented tubules of about 16 nm in diameter which appearedon the exterior to have a helical character. These were visualizedin association with thin fibers thought to be extended strandsof DNA. The suggestion was advanced that the DNA might be enclosedin the cores within the tubules and might be superhelicallystressed. These observations and conclusions were almost certainlyincorrect. We have subsequently observed segmented 16-nm-diametertubules, virtually identical to those previously shown, in mediacontaminated with bacteria. We are now convinced that thesetubules were in fact of bacterial origin and were in no waya part of the vaccinia virus structure. We have taken some additionalcare in the experiments reported here to avoid any repetitionof their appearance. Moreover, we have found no evidence inthe current study to suggest that the packaged vaccinia virusgenome is superhelically stressed, at least not in the quiescentstate of the virion.

By contrast, our AFM images of DNA "nucleoid" structures indicatedthat a large portion of the packaged genome may be moderatelytightly packed and perhaps not extensively complexed with protein.With regard to intravirion pressure, if one calculates the volumeoccupied by the DNA in the condensed packets seen in Fig. 8or calculates an approximate volume for the interiors of thehydrated cores, a value of about 4 x 10⁶ nm³ is obtained. Fromthis, along with a genome size of $~$ 191 kbp, a DNA density ofabout 0.05 bp/nm³ may be computed. Similar calculations forbacteriophages lambda (28) and P22 (2), which are fairly representativeof double-stranded DNA phages, give a value of around 0.60 bp/nm³.In the cases of lambda and P22, it is known that the encapsidatedDNA is not accompanied by any protein and that it is packedwith about as high a density as is believed to be theoreticallypossible (28) (about equivalent to that of DNA in a crystal[1]). Comparison of the packing density of the DNA in the vacciniavirus cores with corresponding densities for the bacteriophages(29, 41) clearly shows there to be at least 10 times more spacein the vaccinia virus cores than in the heads of bacteriophagesin proportion to the amount of nucleic acid.

The absence of extreme packaging pressures notwithstanding,our AFM images do suggest that the interior bulk of the vacciniavirus DNA is likely to consist primarily of naked DNA strandsthat are relatively closely packed. This is supported by low-angleX-ray scattering evidence, with $~$ 25-Å spacing being observedfor both vaccinia virus (23) and the bacteriophages (7). A relativelylow packing density for vaccinia virus DNA would be consistentwith the observation that when the core is ruptured by proteases,the DNA does not burst out of the sac but pours out like a thickfluid and unravels gradually. It is interesting to speculatethat stable interstrand connections may have constrained theDNA from radical ejection in terms of flexibility/degrees offreedom. The apparent protein-based "soldering" of DNA strandsand its apparent stability during virion degradation (Fig. 11)may be consistent with this and with the stability of DNA "nucleoids"observed previously under extractive conditions (12, 13) andmay serve to mitigate the internal pressure arising during moderategenome compaction.

In light of the evidence presented here that the packing ofthe DNA chiefly determines the core shape, which in turn isreflected in the virion shape, then ultimately the distributionor packing of the nucleic acid determines virion shape characteristics.In such a case, how is the genome packed that it imposes a rectangularshape on the core? One possibility, consistent with all of theobservations, is that the DNA is simply a bundle of long loops,sometimes referred to as a "climber's rope," equivalent to acoil collapsed about an axis in its plane. Such a folded bundleof rope, soldered predominantly at positions toward its equator,would likely appear relatively disordered when imaged from oneend ("loop-on") or in a partially disassembled state. An attractionof such an arrangement is that it has two distinct directions,one parallel and one perpendicular to the direction of the strandsin the loops, yielding roughly rectilinear and biconcave morphologies,respectively. Moreover, it would provide consistency with priorobservations of an internal tube-like structure (27). It hasbiological virtue too, in that it may be possible to bundleand unbundle in a relatively straightforward and uncomplicatedway when its genetic content is required.

The sac containing the DNA we know to be flexible and able toassume multiple shapes (rectangular and dumbbell shaped in intactcores, loaf shaped in cores lacking LBs, or round and flat followinga loss of DNA). If, as we suspect, there is a change in theoverall shape of partially degraded cores which still containtheir DNA, upon loss of LBs, from particles having profoundcentral depressions to more loaf-like shapes (Fig. 7), thenthere would almost certainly have to be some rearrangement ofthe nucleic acid and its associated proteins within the corethat either produces or accompanies the change in core shape.Conversely, the DNA may be more compressed with the LBs present.

In numerous cases (e.g., see Fig. 9 and 10), the DNA observedpouring from ruptured cores appeared to be partially associatedwith protein in addition to the "soldering sites," with somestrands appearing to be even more fully protein coated. Onepossibility (though not unequivocally demonstrated) is thatwhile much of the nucleic acid may be packaged as bundles ofnaked or "soldered" DNA, portions of the DNA near the insidesurface of the core wall (i.e., the outer surface of the DNAbundle) may be more extensively protein associated. If so, thena possibly related observation might be the action of the vacciniavirion as a transcriptosome early during infection, prior tovirion disassembly. A key criterion might be the rate at whichmRNA could (directionally) exit the bundle given the speed,after the initiation of transcription, at which mRNA startsto be extruded from the virion cores into the external milieu(30). As a result of this efficient extrusion in the contextof a moderately compact genome, mRNA may not be synthesizedthroughout the bulk of the DNA bundle within the sac but onlynear the DNA bundle surface. Channels in the shell may thenbe more immediately available for mRNA escape from the core.

This is speculative, we concede, though it would have a numberof implications for the packaging of the DNA and the internalstructure/protein composition of the core. We will make no effortto address such a model here, other than to point to one obviousimplication. For the purposes of intravirion transcription ofearly genes, the packaging of the DNA could not be an entirelyrandom stuffing of pliable DNA into the sac as some of the AFMimages might suggest. The genome would need to follow a pathin which the early genes interfaced more closely with the interiorof the sac. In this regard, early transcription complexes maybe poised at early promoters during the late gene expressionand/or the packaging phase. Clearly the question of genome packagingrequires further study before clear conclusions can be drawn.Moreover, not having imaged DNA within the core under transcriptionconditions, i.e., in the context of changes known to occur underconditions that would support transcription (18, 26, 29), itis unclear how DNA morphology might be affected.

ACKNOWLEDGMENTS

This research was supported by a grant from the NIH (GM58868-02).

We thank Aaron Greenwood for assistance in preparation of figures.

FOOTNOTES

* Corresponding author. Mailing address: University of California, Irvine, Department of Molecular Biology and Biochemistry, 560 Steinhaus Hall, Irvine, CA 92697-3900. Phone: (949) 824-1931. Fax: (949) 824-1954. E-mail: amcphers{at}uci.edu

Published ahead of print on 28 May 2008.

These two authors contributed equally to this work.

REFERENCES

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