Involvement of the Portal at an Early Step in Herpes Simplex Virus Capsid Assembly

DNA enters the herpes simplex virus capsid by way of a ring-shapedstructure called the portal. Each capsid contains a single portal,located at a unique capsid vertex, that is composed of 12 UL6protein molecules. The position of the portal requires thatcapsid formation take place in such a way that a portal is incorporatedinto one of the 12 capsid vertices and excluded from all otherlocations, including the remaining 11 vertices. Since initiationor nucleation of capsid formation is a unique step in the overallassembly process, involvement of the portal in initiation hasthe potential to cause its incorporation into a unique vertex.In such a mode of assembly, the portal would need to be involvedin initiation but not able to be inserted in subsequent assemblysteps. We have used an in vitro capsid assembly system to testwhether the portal is involved selectively in initiation. Portalincorporation was compared in capsids assembled from reactionsin which (i) portals were present at the beginning of the assemblyprocess and (ii) portals were added after assembly was underway. The results showed that portal-containing capsids wereformed only if portals were present at the outset of assembly.A delay caused formation of capsids lacking portals. The findingsindicate that if portals are present in reaction mixtures, aportal is incorporated during initiation or another early stepin assembly. If no portals are present, assembly is initiatedin another, possibly related, way that does not involve a portal.

INTRODUCTION

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Introduction

Assembly of herpes simplex virus type 1 (HSV-1) can be consideredto begin with formation of the capsid. Capsids are producedin the infected cell nucleus, where they are also packaged withthe double-stranded DNA (dsDNA) genome. Promptly after DNA isintroduced, capsids transit to the cytoplasm, where they areenveloped to form mature virions (5, 21, 23).

The HSV-1 capsid is closely similar in structure to the capsidsof other herpesviruses. It is icosahedral in shape, and itsmajor structural features are 162 capsomers. The capsomers areof three types: (i) hexons that form the capsid faces and edges,(ii) pentons that are located at 11 of the 12 capsid vertices,and (iii) the portal found at one of the 12 vertices. The 150hexons are hexamers of the major capsid protein (UL19), whilethe 11 pentons are UL19 pentamers. The portal is a 12-mer ofUL6, the portal protein. The portal is about the size of a hexonor penton and cylindrical, with an axial channel through whichHSV-1 DNA passes as it is introduced into the capsid (32). Thecapsomers are connected in groups of three by the triplexes,trigonal structures that lie on the outer surface of the capsidfloor (15, 27, 32, 36).

The mechanism of HSV-1 capsid formation has been examined ininfected cells, in insect cells infected with recombinant baculoviruses(rBV) encoding HSV-1 capsid proteins, and in an in vitro assemblysystem (14, 28, 31, 34). Such studies have demonstrated thatassembly of closed icosahedral capsids requires the major capsidprotein, the triplex proteins, and a scaffolding protein. Themajor scaffolding protein, the product of the UL26.5 gene, isrequired for capsid formation, but thereafter it is lost andis not present in the mature capsid or virion.

Capsid assembly was found to proceed by way of a spherical,fragile intermediate called the procapsid. After it is formed,the procapsid undergoes a large-scale energy-independent conformationalchange to create the mature icosahedral capsid. Procapsids havebeen identified during capsid formation in infected cells andas capsids are assembled from purified proteins in vitro (4,12, 13, 18, 22). In vitro studies have demonstrated that procapsidsare assembled by way of angular segments of the closed structure.Called partial procapsids, the angular segments contain themajor capsid, scaffolding, and triplex proteins. They are extendedin small increments to create the closed procapsid. Extensionof the partial procapsid occurs by addition of complexes containingthe major capsid and scaffolding proteins. Like the mature capsid,the procapsid was found to contain a single portal, indicatingthat the portal is incorporated as the capsid is assembled (24).

The location of the portal at a unique site in the mature capsidsuggests it is introduced by an unusual step in the assemblyprocess. Each procapsid must incorporate one portal, but nomore. Such assembly would be expected if the portal were involvedin the initiation of procapsid formation. Since initiation isa singular step in assembly, involvement of the portal couldresult in its incorporation at a unique site. On the other hand,it is clear that capsid assembly can occur in the absence ofthe portal. Formation of capsids lacking portals is observedin cells infected with an HSV-1 mutant lacking the UL6 gene(35) and in similar UL6-negative assembly reactions carriedout in vitro or in rBV-infected insect cells (14, 28, 31). Suchobservations demonstrate that the portal is not required forinitiation of procapsid assembly and raise the possibility thatit may be incorporated at a later step.

Here, we describe the results of experiments performed to testthe idea that the portal is involved in initiation of capsidformation. Using an in vitro capsid assembly system, we comparedthe levels of portal incorporation in reactions where the portalwas added at the outset of the assembly process and after adelay. It was reasoned that if the portal is involved specificallyin initiation, then delay in its addition to reaction mixtureswould permit formation of some capsids lacking portals and thereforedecrease the overall level of portal incorporation.

MATERIALS AND METHODS

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Materials and Methods

Protein purification. Proteins used for in vitro capsid assembly were isolated frominsect cells infected with appropriate rBV. The major capsidprotein (UL19; VP5), scaffolding protein (UL26.5), portal protein(UL6), and an enzymatically inactive form of the protease (UL26/61[30]) were prepared from singly infected cells, while cellscoinfected with rBV encoding UL18 and UL38 were employed fortriplex purification. Earlier publications have described theprocedures used for creation of the rBV, growth and infectionof Spodoptera frugiperda (Sf9) cells, and harvesting of theinfected cells (13, 16, 31).

VP5 was purified to homogeneity using a three-step proceduredescribed previously (13). Preparations used for capsid assemblyhad concentrations in the range of 1.0 to 1.5 mg/ml in phosphate-bufferedsaline (PBS)-10 mM EDTA plus 0.1 volume of a protease inhibitorcocktail (prepared by dissolving 1 tablet of Roche Completeper 5 ml PBS). The scaffolding protein and protease were partiallypurified by ammonium sulfate precipitation of cell lysates asdescribed previously (16). Both were dissolved in PBS-2 mM EDTA-5mM dithiothreitol containing protease inhibitors and used forcapsid assembly on the same day they were prepared. The scaffoldingprotein and protease solutions had concentrations of 1.5 to2.0 mg/ml and $~$ 1 mg/ml, respectively. The scaffolding proteinwas present in solution in the form of 55-nm-diameter scaffoldparticles (11, 16). UL6 was purified to homogeneity by the previouslydescribed method, which yields intact portals in 20 mM Tris-HCl,pH 7.5, containing 1 M arginine and $~$ 20% sucrose (15). UL6 proteinconcentrations were in the range of 0.5 to 1.0 mg/ml. Triplexeswere purified to homogeneity as described previously (16). Triplexsolutions in PBS-10 mM EDTA plus protease inhibitors had concentrationsin the range of 0.5 to 1.0 mg/ml.

In vitro capsid assembly. The compositions of reaction mixtures yielding the highest levelof capsid formation were found to vary with individual preparationsof the reaction components. For each set of experiments, therefore,preliminary tests were carried out to determine the optimalproportions. Representative reaction mixtures contained 30 µlVP5, 10 µl UL26.5, 30 µl triplexes, 2 µl UL26.5/61,2 µl UL6, 60 µl PBS, 16 µl 0.5 M EDTA, 12µl 20 mM dithiothreitol, and 6 µl protease inhibitorcocktail (see above). Optimal capsid formation occurred whenUL26.5/61 was present and VP5 was the limiting component ofthe reaction mixtures. Assembly was allowed to proceed for 20h at 34°C with a 10-µl aliquot withdrawn as necessaryfor electron microscopy. The reaction mixtures were then addedto 400 µl TNE (20 mM Tris-HCl, pH 7.5, 0.5 M NaCl, 1 mMEDTA), and the capsids were purified by centrifugation at 4°Cin 600-µl-capacity tubes (6 by 42 mm) using a BeckmanSW50.1 rotor. Insoluble material was removed by centrifugationat 8,000 rpm for 30 min, after which the supernatant was transferredto a fresh tube and underlaid with 50 µl 35% sucrose inTNE, and the capsids were pelleted by centrifugation for 1 hat 23,000 rpm. The capsids were then suspended in 50 µlTNE, layered on a gradient of 20% to 50% sucrose in TNE, andcentrifuged for 1 h at 23,000 rpm. The gradient was fractionated(13 fractions; $~$ 45 µl each), and the capsid-containingfractions were identified by applying small aliquots (2 µl)to an Immobilon-P polyvinylidine difluoride (PVDF; Millipore,Billerica, MA) sheet and staining them with Ponceau S (16).Capsid fractions identified in this way corresponded to a lightscattering band visible in the gradient. Sodium dodecyl sulfate-polyacrylamidegel electrophoresis (SDS-PAGE) and agarose gel analysis followedby Western immunoblotting were performed with the three peakcapsid-containing fractions.

The amount of UL6 present in capsids assembled in vitro wasdetermined beginning with fractions from the sucrose gradientsused for capsid isolation, as described above. Small aliquots(2 µl) were applied to a PVDF sheet and stained with PonceauS to measure the total amount of protein present. Ponceau stainingwas followed by immunostaining with UL6-specific antibody (describedbelow) to measure the amount of UL6. At both stages, the spotstain was determined quantitatively by scanning on a flatbedscanner, followed by densitometry with Un-Scan-It (Silk Scientific,Orem, UT). The UL6 signal was scaled by the use of HSV-1 B capsids,which were assumed to contain one portal per capsid. The samePVDF sheet was used for analysis of B capsids and all experimentalcapsid preparations. Control experiments showed that capsidsformed normally in the presence of the drug WAY-150138, butportal incorporation was inhibited as described previously (11,16).

Localization of UL6 by immunoelectron microscopy. UL6 was labeled specifically with primary UL6-specific antibody,followed by gold-labeled secondary antibody. The labeling procedurefor capsids formed in vitro was the same as that used previouslyfor capsids isolated from infected cells (15). Primary antibodystaining was with anti-MBP-UL6 (29), which was absorbed with(i) capsids assembled in vitro in the absence of UL6 and (ii)capsids prepared from cells infected with the UL6 deletion strainhr74 (6).

Other methods. Previously described methods were employed for SDS-PAGE, agarosegel electrophoresis, electrophoretic blotting of proteins ontoPVDF sheets, staining of blots with Ponceau S, and quantitativedetermination of protein bands in stained gels and radioautographs(15, 16, 18). Staining of blots with the UL6-specific monoclonalantibody 1C9 was performed as described by Newcomb et al. (16),with 1C9 used at a dilution of 1:10,000. HSV-1 B capsids wereprepared as previously described from Vero cells infected withthe KOS strain of HSV-1 (17). Electron microscopy was performedwith 10-µl aliquots of assembly reaction mixtures. Theywere adsorbed to carbon-Formvar-coated copper electron microscopegrids, stained for 1 min with 1% uranyl acetate, air dried,and photographed in a Philips 400T electron microscope operatedat 80 keV. To count capsids and capsid-related structures, theelectron microscope negatives were digitized and viewed withPhotoshop 5.0.

RESULTS

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Results

In vitro assembly system. In vitro capsid assembly was carried out in reaction mixturescontaining the major capsid, scaffolding, triplex, and portalproteins as described in Materials and Methods. The proportionof reaction components was adjusted by trial and error to optimizethe number of capsids formed. If portals were present, theywere the last component added to the reaction mixtures.

The progress of capsid formation was monitored by electron microscopy.Small samples were removed from reaction mixtures and examinedafter negative staining. Micrographs demonstrated the presenceof assembly intermediates (i.e., partial procapsids) in reactionmixtures after 10 to 15 min of incubation (Fig. 1a, c, and d).Prior to this time, structures observed in reaction mixtureswere too small to be identifiable as capsid precursors. Closedcapsids were first observed after 30 to 60 min of incubation,and they increased in number during 20 h or more of incubation(Fig. 1b). By 20 h, closed capsids predominated over assemblyintermediates in reaction mixtures.

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FIG. 1. Electron micrographs showing the products of in vitro capsid assembly reactions. Samples of reaction mixtures were examined after 15 min (a, c, and d) and 20 h (b) of incubation. Note that partial procapsids predominate in 15-min reactions (arrows in panel a), while completed capsids predominate after 20 h (arrows in panel b). Partial procapsids (arrows) are shown at higher magnification in panels c and d, with more mature capsids close by. Bars = 500 nm (a and b) and 100 nm (c and d).

Control experiments were carried out to test the effect of theportal on capsid assembly. Analysis of micrographs such as thoseshown in Fig. 1 demonstrated little effect of the portal oneither the rate or extent of capsid formation (Fig. 2). Themorphologies of assembly intermediates were the same in thetwo reactions (data not shown). This result is consistent withprevious in vivo and in vitro studies, indicating that capsidformation does not require the presence of the portal (14, 19,28, 31).

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FIG. 2. Time course for appearance of HSV-1 capsids and capsid assembly intermediates during in vitro capsid formation. The total number of completed capsids and capsid assembly intermediates were counted in electron micrographs, such as those shown in Fig. 1. Counts for each time point were made in one x10,000 electron microscope negative. Note that the rate of appearance of assembly products was not affected by the presence of the portal in reaction mixtures.

UL6 incorporation. UL6 incorporation into capsids was tested in standard reactionsas described above and in those in which portal addition wasdelayed for various times. In all cases, the reaction mixtureswere incubated for a total of 20 h. After incubation, the capsidswere isolated by sucrose gradient centrifugation and analyzedby SDS-PAGE, followed by Western immunostaining for UL6. Inparallel studies, capsids isolated by sucrose gradient centrifugationwere also analyzed by agarose gel electrophoresis, followedby UL6 immunostaining.

SDS-PAGE was carried out with the two or three peak capsid-containingfractions isolated from sucrose gradients. After electrophoresis,proteins on the gel were transferred to a PVDF sheet and stainedwith Ponceau S. VP5 staining, as shown in Fig. 3a (top), providesa measure of the total number of capsids analyzed. UL6 immunostainingdemonstrated a high level of UL6 incorporation in complete reactionsbut little in reactions in which portal addition was delayedfor 30 min, 1 h, 2 h, or 4 h (Fig. 3a, bottom). The decreaseswere 7.9-, 12.1-, 6.2-, and 10.6-fold, respectively, for thefour time points.

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FIG. 3. Determination of the UL6 contents in capsids assembled in vitro. Analyses were carried out with capsids formed in incubations in which the time of portal addition varied, as described in the text. After incubation, the capsids were purified by sucrose gradient ultracentrifugation and analyzed in two ways: by SDS-PAGE, followed by Western immunoblotting (a), and by agarose gel electrophoresis, followed by SDS-PAGE and immunoblotting (b). Note that the amount of UL6 incorporated into capsids was greater when UL6 was present at the outset of incubation than when its addition was delayed.

Figure 3b shows the results obtained when capsid-containingsucrose gradient fractions were analyzed by agarose gel electrophoresisfollowed by blotting onto PVDF. Examination of the blot afterPonceau staining demonstrated that capsids migrated as a singleband during electrophoresis (Fig. 3b, top). UL6 staining showeda substantial level of UL6 in capsids isolated from controlincubations but a decreased level when the addition of UL6 wasdelayed (Fig. 3b, bottom). The reductions were 10.0-fold and16.4-fold in incubations where UL6 addition was delayed for15 min and 45 min, respectively.

Amount of UL6 incorporated. If the portal is involved at an early step of procapsid assembly,as suggested by the results described above, then it is furtherexpected that (i) no more than one portal will be able to beincorporated into the capsid and (ii) the portal will be locatedat a vertex in capsids formed in vitro as it is in capsids isolatedfrom infected cells. Tests of both expectations were performedusing capsids assembled in the in vitro system.

The amount of UL6 present was measured beginning with reactionmixtures in which the number of input portals was varied. Afterincubation to allow them to form, capsids were isolated by sucrosegradient ultracentrifugation, and the amount of UL6 was determinedby dot blot analysis. The blots were stained with Ponceau S,followed by immunostaining for UL6 and densitometry as describedin Materials and Methods.

The results (Fig. 4) indicated that the amount of UL6 incorporatedinto capsids increased with increasing input UL6 over the concentrationrange tested (i.e., 0.25 to 2.0 µg/170-µl reactionmixture). A sigmoid relationship was observed with little UL6incorporation observed below $~$ 1.0 µg and evidence of saturationabove $~$ 1.5 µg. The maximum UL6 incorporation correspondedto 10 to 12 UL6 molecules per capsid or approximately 25% ofthe UL6 added to standard incubations. Efforts to add greateramounts of UL6 to reaction mixtures were affected by an inhibitionof capsid formation by the 1 M arginine required to maintainUL6 in solution (15).

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FIG. 4. Effect of input UL6 on the amount of UL6 incorporated into HSV-1 capsids assembled in vitro. Capsids were formed in vitro and analyzed for the presence of UL6 as described in Materials and Methods. The bar indicates the amount of UL6 present in HSV-1 B capsids isolated from infected cells.

Portal location. The location of the portal in the capsid was examined by immunoelectronmicroscopy in a procedure based on the use of a polyclonal antibodyspecific for UL6 (29). Capsids were prepared for immunoelectronmicroscopy by in vitro assembly in reaction mixtures containingportals. They were then purified by sucrose gradient centrifugationand adsorbed onto carbon-Formvar-coated electron microscopegrids. All staining operations were performed with adsorbedcapsids. They were stained with the primary antibody, followedby a secondary, gold-labeled antibody as described in Materialsand Methods.

Electron microscopic examination showed that in capsids containinggold label, the label was confined to a single site that appearedto correspond to a vertex (Fig. 5). Capsids labeled at morethan one site were not observed. The proportion of capsids containinglabel (32% [Table 1]) was in satisfactory agreement with thelabeled fraction observed in capsids isolated from infectedcells, as reported earlier (32% and 41%, respectively, in twoexperiments) (15). In contrast, labeling was 6% in control experimentsperformed with capsids lacking UL6 (Table 1).

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FIG. 5. Electron micrograph showing HSV-1 capsids that were assembled in vitro and stained to identify the location of UL6 protein. The capsids were stained with antibody specific for UL6, followed by a gold-labeled secondary antibody. The gold beads (black dots; $~$ 15 nm in diameter) identify the locations of UL6.

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TABLE 1. In vitro-assembled capsids: immunogold staining for UL6

DISCUSSION

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Discussion

Role of the scaffolding protein in portal incorporation. The scaffolding protein has been the focus of previous effortsto understand the way in which the portal becomes assembledinto the HSV-1 capsid. Intact portals were found to bind UL26.5when the two purified proteins were mixed in vitro (16). Also,studies with deletion mutants identified a region of the scaffoldingprotein required for interaction with the portal (26). Experimentswith one such deletion, UL26.5 ${Delta}$ 143-151, were particularly revealing. ${Delta}$ 143-151 protein failed to bind to the portal, although it supportedthe formation of portalless capsids (which were otherwise morphologicallynormal) in an in vitro assembly system. The results indicatethat the scaffolding protein plays an important role in promotingincorporation of the portal into capsids as they are formed.In particular, the portal is suggested to be donated to thenascent capsid by way of a complex with the scaffolding proteinin a process resembling the way the major capsid protein isdonated (Fig. 6).

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FIG. 6. Schematic diagram illustrating HSV-1 procapsid formation in the absence (a) and presence (b) of the portal. The diagram illustrates the observation that procapsids are assembled normally in the absence of the portal (a). If the portal is present, however, it is suggested to be involved in the initiation of procapsid assembly and to be donated by way of a complex with the scaffolding protein, as shown in panel b.

A role for the scaffolding protein in portal incorporation isalso suggested by results with WAY-150138, a small moleculeinhibitor of HSV-1 replication (11, 16). WAY-150138 was foundto antagonize the in vitro interaction between UL26.5 and theportal. When the drug was present in infected cells, or in invitro assembly reaction mixtures, capsids formed normally butportal incorporation was suppressed (11). The results supportthe view that the portal cannot be incorporated into capsidsif it is prevented from interacting with the scaffolding protein.

Delay in portal addition. The present study extends the previous results by demonstratingthat a delay in addition of the portal to in vitro assemblyreaction mixtures causes a decrease in incorporation of portalsinto product capsids. The decrease in portal incorporation wasobserved despite the fact that few assembly intermediates orcompleted capsids were present in reaction mixtures at the endof the time window (15 to 30 min of incubation) found to bepermissive for portal incorporation. We interpret the findingsto indicate that initiation, and perhaps other early steps inprocapsid formation, takes place during the first 15 to 30 minof incubation in vitro. If the portal is present during thistime, then it becomes incorporated into such early precursorsand is found in product capsids. If not, then assembly takesplace in the absence of the portal as described previously (14).This would be the case, for instance, if nucleation of capsidformation involved a complex of the major capsid, scaffolding,and triplex proteins to which the portal could bind if it werepresent. If not, then assembly would proceed without the portal.Alternatively, initiation of assembly could normally occur ata portal with VP5 or a combination of VP5 with other protein(s)substituting for the portal if it is absent. In either case,subsequent assembly events would alter the initiation complexand thereby prevent the portal from becoming incorporated ata later time.

It is interesting that the degree to which UL6 incorporationwas suppressed by delay in its addition was not dependent onthe length of the delay over the time range examined (i.e.,15 min to 4 h) (Fig. 3). Suppression was $~$ 6-fold to $~$ 16-fold inall cases with no systematic dependence on the extent of delay.The absence of dependence on the time of delay suggests thatthe opportunity for portal incorporation is lost after 15 minof incubation in vitro and is not regained.

If it is true that early assembly intermediates are presentin 15- to 30-min reaction mixtures as postulated above, thenit must be that they are not identifiable as assembly precursorsin electron micrographs, such as Fig. 1 (top). Examination of15- to 30-min reaction mixtures most often revealed a few partialprocapsids and many other smaller structures that did not appearuniform in morphology and did not resemble capsids or partialprocapsids (Fig. 1, top). The postulated early assembly intermediatesmay be among this uncharacterized group of structures.

The amount of UL6 assembled into product capsids was found todepend on the amount added to reaction mixtures. Little UL6was incorporated in reactions containing less than $~$ 0.5 µgUL6, whereas above $~$ 1.5 µg, the amount incorporated correspondedto approximately one portal (i.e., 12 UL6 monomers) per capsid(Fig. 5). Higher levels of UL6 incorporation were not observeddespite the fact that UL6 was present in excess in reactionmixtures. UL6 recovered in product capsids, for example, amountedto 25% or less of the input UL6 in standard reaction mixtures(see Materials and Methods). These results support the viewthat the assembly pathway limits portal incorporation to oneper capsid.

In assembly reactions in which total UL6 incorporation correspondedto less than one portal per capsid (Fig. 4), we assume thatproduct capsids were a mixture of capsids containing one portaland those containing none. The alternative possibility, thatcapsids contain portal fragments, seems less likely becausethe oligomeric state of UL6 as isolated from insect cells hasbeen observed only in the range of 12. For example, values between $~$ 11 and 14 were reported in studies using two different experimentalmethods (15, 32). UL6 11- to 14-mers would need to disassemblefor partial portals to be incorporated into capsids.

Examination of the product capsids by immunoelectron microscopydemonstrated that UL6 was present at a single vertex, as itis in capsids isolated from infected cells (Fig. 5 and Table1). The presence of UL6 at a capsid vertex is consistent withthe view that portal incorporation in vitro is a faithful recapitulationof capsid formation as it occurs in vivo.

Portal assembly in dsDNA bacteriophages. The mechanism of portal assembly into a polyhedral capsid hasbeen studied for many years in dsDNA bacteriophages, such asT4, ${phi}$ 29, P22, and SPP1 (2, 7, 8, 9, 25, 33). As in the case ofHSV-1, bacteriophage portals are (i) cone-shaped structuresformed from 12 or 13 copies of a single polypeptide, the portalprotein; (ii) located at a unique vertex of the capsid polyhedron,where they serve as a channel for DNA to enter and exit; and(iii) present in the procapsid and not able to be added afterthe procapsid is formed (2, 7, 20).

Murialdo and Becker were the first to propose that the portalis involved in initiation of bacteriophage procapsid assembly(10). The proposal was made to account for the location of theportal at a unique location in the capsid structure. Studieswith phage T4 supported a role for the portal in initiationof head formation. The T4 portal protein, gp20, was found associatedwith the scaffolding protein at membrane sites involved in nucleationof procapsid assembly (33). Results with capsid assembly inthe Bacillus subtilis phages SPP1 and ${phi}$ 29 have also supporteda role for the portal in initiation of procapsid formation.In these phages, uniform populations of morphologically normalprocapsids are formed only in the presence of the portal. Inits absence, populations include smaller and malformed structures(2, 3). The results suggest that the portal is involved at anearly stage of procapsid assembly, when the radius of curvatureand triangulation number become fixed.

In contrast, studies with P22 have emphasized the possibilitythat the portal may not be involved in initiation of procapsidassembly. Morphologically normal (T = 7; T, triangulation number)procapsids are formed both in vivo and in vitro in the absenceof the portal protein, indicating that the portal is not obligatorilyinvolved in the initiation process (8, 9). The portal was foundto have little effect on the rate of procapsid formation, andresults with mutants in which portal incorporation was affectedsuggested that the portal could be incorporated at any stageof assembly (1, 8). It is possible, therefore, that virusesdiffer in the manner in which the portal becomes incorporatedinto the nascent procapsid. In some, such as T4, SPP1, and HSV-1,the portal participates in nucleation or other early steps,while in others, such as P22, initiation can be accomplishedwithout involvement of the portal.

In future studies of HSV-1 capsid formation, it will be of interestto determine how portal incorporation is limited to one percapsid. For instance, if the portal is involved in initiationof procapsid formation or is donated at an early assembly step,as suggested here (Fig. 6), then how is it that another portalcannot be donated at a later assembly step? To account for howportal incorporation is limited, we suggest a model in which(i) the scaffolding protein forms a linear, head-to-tail aggregatecontaining many UL26.5 molecules and (ii) the portal binds selectivelyto only one end of the aggregate and not to internal UL26.5molecules. Procapsid assembly would involve a nucleation stepin which a portal binds to one end of the scaffold string, followedby extension steps involving major capsid protein moleculesbound at internal positions, as illustrated schematically inFig. 7. Procapsid assembly in the absence of a portal wouldinvolve binding of a major capsid protein molecule rather thana portal at the end of the scaffold polymer. It is consistentwith the model proposed, which we call the string model, thatUL26.5 is known to form large aggregates called scaffold particlesin insect cells and in cells infected with HSV-1 (11, 16). Itwould be a critical test of the model to determine whether UL26.5forms linear aggregates in scaffold particles or other aggregationstates and how capsids form under conditions where the scaffolddoes not appear to form large aggregates (13).

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FIG. 7. Schematic diagram illustrating the proposed mechanism by which portal incorporation is limited to the initiation step of capsid assembly. The model postulates that (i) the scaffolding protein, UL26.5, is found in the form of a head-to-tail polymer and (ii) the portal attaches only at one end of the polymer, as illustrated in step 1, with major capsid protein molecules bound to all other scaffold molecules. Procapsid formation is suggested to occur by successive steps in which one major capsid protein molecule is delivered to a growing edge of the nascent procapsid (steps 2 and 3). Note that the proposed mechanism restricts portal incorporation to the initiation step.

ACKNOWLEDGMENTS

We thank Greg Singer and Anna Maria Copeland for helpful discussionsduring the course of this investigation and Joel Baines forthe gift of anti-MBP-UL6 antibody.

The work was supported by NIH award AI41466.

FOOTNOTES

* Corresponding author. Mailing address: Department of Microbiology, Box 800734, University of Virginia Health System, 1300 Jefferson Park Ave., Charlottesville, VA 22908. Phone: (434) 924-1814. Fax: (434) 982-1071. E-mail: JCB2G{at}virginia.edu.

REFERENCES

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