Quantification of the DNA Cleavage and Packaging Proteins UL15 and UL28 in A and B Capsids of Herpes Simplex Virus Type 1

The proteins produced by the herpes simplex virus type 1 (HSV-1)genes U_L15 and U_L28 are believed to form part of the terminaseenzyme, a protein complex essential for the cleavage of newlysynthesized, concatameric herpesvirus DNA and the packagingof the resultant genome lengths into preformed capsids. Thiswork describes the purification of recombinant forms of pU_L15and pU_L28, which allowed the calculation of the average numberof copies of each protein in A and B capsids and in capsidslacking the putative portal encoded by U_L6. On average, 1.0(±0.29 [standard deviation]) copies of pU_L15 and 2.4(±0.97) copies of pU_L28 were present in B capsids, 1.2(±0.72) copies of pU_L15 and 1.5 (±0.86) copiesof pU_L28 were found in mutant capsids lacking the putative portalprotein pU_L6, and approximately 12.0 (±5.63) copies ofpU_L15 and 0.6 (±0.32) copies of pU_L28 were present ineach A capsid. These results suggest that the packaging machineis partly comprised of approximately 12 copies of pU_L15, asfound in A capsids, with wild-type B and mutant U_L6(-) capsidscontaining an incomplete complement of cleavage and packagingproteins. These results are consistent with observations thatB capsids form by default in the absence of packaging machineryin vitro and in vivo. In contrast, A capsids may be the resultof initiated but aborted attempts at DNA packaging, resultingin the retention of at least part of the DNA packaging machinery.

INTRODUCTION

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Introduction

During herpesvirus replication, branched concatemers of viralDNA are produced in the nucleus of an infected cell (47). Individualviral genomes are cleaved from the concatemer, and each is packagedinto a preformed capsid (reviewed in references 9 and 26). Thisassembly step involves extensive interactions between the capsid,DNA, and the packaging machinery.

Four forms of herpesvirus intranuclear capsid have been described(22, 37). Procapsids, believed to be the precursors of all capsidtypes, are spherical structures containing an inner shell orscaffold consisting largely of the protein VP22a. During thepackaging reaction, VP22a is cleaved by the U_L26 protease, releasingit from the inside surface of the outer shell (19, 33, 45, 59).This dissociation of the scaffold coincides with a dramaticconformational change in the outer shell, which eventually formsa stable icosahedron (24, 57). Each procapsid is thereby convertedinto one of three angular capsid types. Type A capsids containonly the icosahedral shell, B capsids contain cleaved scaffoldmaterial within the outer shell, and C capsids contain packagedDNA in place of the cleaved scaffold (22).

All three mature forms of capsids contain outer shells consistingof 150 hexons and 12 pentons forming a T=16 structure (62).The pentons and hexons are made up of five and six copies, respectively,of VP5, the product of U_L19; thus, each capsid contains 960copies of VP5. Hexons and pentons are linked by triplexes whichconsist of one copy of VP19C (encoded by U_L38) and two copiesof VP23 (encoded by U_L18) (14, 17, 22, 39, 66). Only C capsidsexit the cell nucleus to become enveloped virions (46). Oneinterpretation from previous studies of the different capsidtypes (35, 37, 52, 57) is that A capsids result from packagingreactions in which the DNA is inserted but not retained, whereasB capsids represent a mismatch in timing such that the formationof the impervious icosahedral shell precludes exit of the cleavedscaffold proteins.

Many DNA viruses use similar mechanisms to cleave genome-lengthDNA from concatemers and package it into preformed capsids.A model deduced from studies of double-stranded DNA bacteriophagespredicts that in cells infected with herpesviruses, the newlysynthesized viral DNA is transported to the empty procapsidby the terminase, an enzyme that specifically binds genomicends and cleaves the DNA. The terminase, with bound DNA, isbelieved to attach to the portal protein which comprises a uniquepore at one of the five fold axes of the capsid. The DNA isthen translocated through the portal into the capsid using theATPase and helicase activities of the terminase (reviewed inreference 12).

Six genes are known to be essential for the cleavage and packagingof the herpesvirus genome: U_L6, U_L15, U_L17, U_L28, U_L32, andU_L33. Deletion of any of these six genes precludes cleavageof viral DNA and results in the accumulation of mutant B capsidsin the nuclei of infected cells (2, 3, 5, 6, 15, 31, 32, 41,43, 49, 52, 55, 56, 60, 64).

There is increasing indirect evidence that the product of U_L6encodes the portal protein, and the U_L15 and U_L28 proteins compriseterminase subunits. The U_L6 gene product (pU_L6) has been localizedto one vertex of the herpesvirus B capsid, where 14.8 ±2.6 copies (mean ± standard deviation) of the proteinwere calculated to be present (38). Moreover, when purifiedfrom recombinant baculovirus-infected insect cells and solubilizedin 1 M arginine, pU_L6 forms rings with a mass correspondingto an oligomeric state of 12 (38), consistent with previouslydescribed bacteriophage portals or connector proteins (34, 53,58). The U_L15 gene shares limited homology with a nucleotidebinding motif found in a number of bacteriophage terminase components,including gp17, the large subunit of the T4 bacteriophage (16,36). In addition, both pU_L15 and gp17 are susceptible to N-terminalcleavage (10, 48, 50). The U_L15 protein is found in wild-typeB capsids in three forms—a full-length 83-kDa proteinand two N-terminally cleaved forms that migrate at 80 and 79kDa. However, only the full-length form is detected in cellsinfected with viral mutants incompetent for DNA cleavage andpackaging, suggesting that the N-terminal cleavage of pU_L15is linked to the cleavage and packaging process. The U_L28 protein,when overexpressed and purified from Escherichia coli cells,has been shown to bind specifically to the pac1 sequence inherpes simplex virus type 1 (HSV-1) DNA, which is essentialfor the generation of correct genomic termini (4, 25). In addition,several studies have shown that pU_L15 and pU_L28 interact invitro and in infected and uninfected cells (1, 11, 29, 30).

The association of pU_L6, pU_L15, and pU_L28 with different typesof capsids has supported the putative functions described above.The U_L6 protein has been detected in procapsids, and similaramounts are present in B capsids and C capsids (41, 51, 65),indicating that it is an integral component of the capsid shell.The putative terminase components pU_L15 and pU_L28 are also presentin procapsids (51); however, smaller amounts of these two proteinshave been detected in C capsids than in B capsids (48, 54, 65).The reduction in the amount of pU_L15 and pU_L28 in C capsidsis consistent with the behavior exhibited by bacteriophage terminasesubunits that disengage from the capsid after packaging is complete.The amount of pU_L15 has been reported to be smaller in capsidslacking pU_L6 or pU_L28 than in wild-type B capsids (50, 65),and recombinant forms of pU_L15 and pU_L28 have been shown tointeract independently with each other and with pU_L6 in vitro(61), suggesting that the ability of the U_L15 protein to bindto B capsids is mediated through interactions with the U_L6 andU_L28 proteins.

In these studies, a stoichiometric approach was used to investigatethe structure of the HSV packaging machine and to clarify theassociation of the putative terminase subunits with the presumedportal vertex. Histidine-tagged U_L15 and U_L28 proteins werepurified and used as standards to calculate the number of copiesof the U_L15 and U_L28 proteins in wild-type A and B capsids andin capsids lacking the U_L6-encoded portal.

MATERIALS AND METHODS

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Materials and Methods

Purification of the U_L28 protein. The U_L28 open reading frame (ORF), excluding the stop codonand including a C-terminal six-histidine tag, was cloned intothe pBakPAK8 vector (Clontech). Using the manufacturer's protocol,the DNA was used to generate a recombinant baculovirus thatcontained tagged U_L28 under the control of the baculovirus polyhedronpromoter. SF-21 insect cells were infected with the recombinantbaculovirus at a multiplicity of infection of 5, and the cellswere harvested 44 h later, pelleted by centrifugation, and storedat -20°C until use.

Approximately 3 x 10⁷ cells were left on ice for 15 min to thawand then resuspended in lysis buffer (50 mM Tris HCl [pH 8],50 mM KCl, 10% glycerol, 5 mM ß-mercaptoethanol) andEDTA-free Complete protease inhibitors (Roche Applied Science,Indianapolis, Ind.). The cells were lysed by Dounce homogenization,and the insoluble material was pelleted by centrifugation at10,000 rpm in a JA-14 rotor for 20 min at 4°C. The pelletwas resuspended in lysis buffer plus 6 M guanidine hydrochloride(GuHCl) and mixed slowly with a magnetic stirrer at 4°Cfor 90 min. After clarification at 12,000 rpm for 15 min ina JA-14 rotor at 4°C, the supernatant was collected andadded to preequilibrated Ni-nitrilotriacetic acid beads (Qiagen)and the sample was rotated slowly at 4°C for 1 h. The beadswere then washed extensively in lysis buffer and 6 M GuHCl.The GuHCl was then diluted by the addition of lysis buffer,and the beads with bound U_L28 protein were washed sequentiallyin 20 mM imidazole and 50 mM imidazole and finally eluted threetimes with 0.75 ml of lysis buffer plus 1 M imidazole. The eluateswere dialyzed extensively against storage buffer containing50 mM Tris HCl (pH 8.0), 200 mM KCl, and protease inhibitorsand stored at 4°C. The concentration of protein was calculatedusing a bicinchoninic acid (BCA) kit (Pierce Biotechnology Inc.,Rockford, Ill.) and confirmed by electrophoretic separationon a denaturing polyacrylamide gel containing known amountsof bovine serum albumin (BSA) followed by fixation, stainingwith Coomassie blue, and densitometry.

Purification of the U_L15 protein. Plasmid pJB278 was cut with EcoRV and HindIII, allowing theisolation of a 2.2-kbp fragment containing the entire U_L15 ORFtagged with six-histidine codons at the 3' end. A kanamycin-resistantvector, pET30b (Novagen), was cut with NdeI and EcoRV and ligatedto remove extraneous sequences in the vector. A blunt end wasintroduced into the multiple cloning site of this altered vectorby cutting with BamHI and filling in with T4 polymerase. Thevector was then cut with HindIII, and the 2.2-kbp EcoRV/HindIIIfragment from plasmid pJB278 was inserted such that expressionof the tagged U_L15 ORF was driven by the T7 promoter withinthe vector. The plasmid was designated pJB279. Plasmid DNA wastransfected into BL-21+ ${lambda}$ DE3 codon plus bacteria (Stratagene)and grown in Luria-Bertani medium supplemented with kanamycinto an optical density of 0.6, and protein production was inducedby adding 5 mM isopropyl-ß-D-thiogalactoside (IPTG)and incubating for 2 h at 37°C. The cells were then harvestedand frozen at -80°C. Cells from a 500-ml culture were resuspendedin 100 ml of lysis buffer (50 mM Tris [pH 7.9 to 8], 50 mM NaCl,10% glycerol, and 5 mM ß-mercaptoethanol) and EDTA-freeComplete protease inhibitors, Dounce homogenized, and left onice for 45 min. Insoluble material was pelleted by centrifugationat 8,000 rpm in a JA-14 rotor for 15 min at 4°C. The pelletwas resuspended in 50 ml of lysis buffer, left on ice for 15min, and centrifuged for 20 min in a JA-14 rotor at 10,000 rpmat 4°C. The pellet was resuspended in 50 ml of denaturationbuffer (lysis buffer supplemented with 6 M GuHCl) and stirredat 4°C for 90 min. The solution was then centrifuged at12,000 rpm for 15 min in a JA-14 rotor at 4°C, and the subsequentsupernatant was passed through a 0.8-µm filter. The filtratewas added to preequilibrated Ni-nitrilotriacetic acid beads,and the sample was rotated slowly at 4°C for 1 h. The beadswere washed three times in denaturation buffer with 1% Tween20 and increasing amounts of imidazole, to a final concentrationof 20 mM, to remove contaminants and then renatured by gradualdilution of the GuHCl. Protein was eluted from the beads bytwo separate elution steps using lysis buffer plus 1 M imidazoleand dialysis against 50 mM Tris (pH 7.9 to 8), 100 mM NaCl,50% glycerol, 1 mM dithiothreitol, 0.25 mM EDTA, 0.5% Tween20, and 5 mM L-arginine. Proteins were stored at -20°C.

Viruses and cells. The G33 cell line is derived from Vero cells and contains HSV-1DNA from U_L6 to U_L8 (41). G5 cells were transformed from Verocells and contain HSV-1 DNA from U_L16 to U_L21 (18). Vero, G33,and G5 cell lines were maintained in Dulbecco's modified Eagle'smedium supplemented with 10% newborn calf serum and penicillinand streptomycin. The HSV-1(F) virus has been previously described(20), and the titer was determined on Vero cell monolayers.Virus CosU_L6- was derived from HSV-1 strain 17 (HSV-1[17]) andcontains a 4-bp insertion at a site corresponding to amino acidresidue 381 (41). It was grown and the titer was determinedon G33 cells. The K23Z virus contains a lacZ cassette in theU_L18 ORF (18) and was grown and titers were determined on G5cells. All CosU_L6- and K23Z viral stocks were tested for revertantvirus by titration on nonrescuing Vero cells. Only stocks withtiters on rescuing cells greater than 10,000-fold over thoseobtained on Vero cells were used for further studies.

Capsid purification and analysis. Capsids were purified as described previously with some modifications(42). Ten 175-cm² flasks of confluent Vero cells were infectedat a multiplicity of infection of 5, followed by a 16-h incubationat 37°C. The cells were then lysed in 1% Triton X and sonicatedfor 40 s at moderate power. After clarification for 15 min at8,000 rpm at 4°C (7,227 x g) in a Sorvall Legend RT centrifuge,Heraeus rotor no. 3334, the supernatant was pelleted througha 6-ml 35% (wt/vol) sucrose cushion in a Beckman SW28.1 ultracentrifugerotor for 1.5 h at 20,000 rpm. The pellet was then sonicatedbriefly and loaded on a 20-to-50% (wt/vol) continuous sucrosegradient and centrifuged for 1 h at 23,000 rpm in an SW41 rotor.The light-scattering bands were collected with a Pasteur pipetteand pelleted at 30,000 rpm for 2 h in an SW41 rotor. Examinationof the pelleted material by electron microscopy identified significantmaterial inconsistent with capsid morphology (results not shown),so the resuspended material was further purified by centrifugationthrough a second continuous sucrose gradient. The capsids werecollected again, pelleted, and resuspended in 50 µl ofwater (DNase-, RNase-, and protease-free; Acros) and storedat 4°C. Examination of the material by electron microscopyshowed abundant capsids with minimal background material. Allcapsids were used within 24 h of purification. Where mentioned,the second sucrose gradient was fractioned using a fractioningdevice (Haake Buchler) beginning at the top of the gradient.

Protein quantification. Capsids were resuspended in a buffer containing sodium dodecylsulfate and ß-mercaptoethanol, boiled for 3 min, andloaded on two denaturing 8% polyacrylamide gels that were runin parallel. BSA standards were included in one gel to allowcalculation of the number of capsids present in the sample (seeResults). The other gel contained either purified pU_L15 or pU_L28to allow calculation of the amount of this protein in the capsidsample. The BSA-containing gel was stained with Coomassie blueand digitally scanned, and the band intensity was determinedusing Scion Image densitometry software for Windows. Initially,both the VP5 and VP19C protein bands were used to estimate thenumber of capsids present, and the data were compared. Similarresults were obtained from both calculations, and the VP5 proteinwas used for all further calculations. The proteins on the secondgel were transferred to either polyvinylidene difluoride (PVDF)or nitrocellulose paper and immunoblotted as previously described(8). Bands were visualized using either ECF (Amersham Bioscience)or ECL+ (Amersham Bioscience) development reagents, and chemiluminescenceintensities were quantified using a Molecular Dynamics StormImager and associated ImageQuant software. The 83,000, 80,000,and 79,000 M_r pU_L15 bands were included in the quantification.

The data were analyzed by a two-tailed t test, using Statistix7 (Analytical Software, Tallahassee, Fla.).

Antibodies. The U_L15 protein was identified using either pU_L15-specificrabbit polyclonal antisera, generated by immunization with anaffinity-purified bacterial fusion protein (U_L15-MBP) containingthe malE gene product fused to the protein encoded by the majorityof U_L15 exon II (7), or rabbit polyclonal antisera recognizingthe N-terminal 35 amino acids of pU_L15 (50). The rabbit polyclonalantisera directed against the U_L28 protein has been describedpreviously (11).

RESULTS

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Results

Purification of pU_L15 and pU_L28. The capsid shell components are present in more than 300 copiesper capsid; however, the much lower copy numbers of pU_L15 andpU_L28 have precluded direct methods to quantify these proteinsin samples of purified capsids. For example, bands specificfor pU_L15 and pU_L28 are not readily distinguishable from ICP5degradation products in profiles of Coomassie-stained electrophoreticallyseparated capsid proteins (data not shown). Calculation of theamounts of these proteins in capsids therefore required moreindirect methods. The strategy was to generate standard curvesof purified pU_L15 and pU_L28 by subjecting known amounts of purifiedproteins to immunoblot analyses with specific antibodies. Thus,the intensity of a given band on an immunoblot would correspondto a known amount of purified protein, and the amounts in unknownsamples (such as electrophoretically separated capsid proteins)could be calculated.

To purify pU_L15, the U_L15 ORF under the control of the T7 promoterwas cloned in frame with a six-histidine tag such that the tagwas inserted at the C terminus. Protein production was inducedin E. coli by addition of IPTG to the culture medium, and inducedprotein was solubilized in GuHCl. The protein was then purifiedby affinity chromatography using Ni⁺ beads, followed by dilutionof the GuHCl and eventual elution in imidazole (see Materialsand Methods for details.) A representative purification is shownin Fig. 1A. Approximately 10 µg of protein was purifiedfrom 500 ml of an induced culture.

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FIG. 1. Scanned digital image of Coomassie-stained gels showing protein profiles from relevant steps of the purification of U_L15 (A) and U_L28 (B) proteins. (A) Lane 1, initial cell lysate; lane 2, supernatant after lysis; lane 3, supernatant from wash of insoluble material; lane 4, eluted protein after dialysis. (B) Lane 1, initial cell lysate; lane 2, supernatant after lysis; lane 3, Ni-nitrilotriacetic acid agarose beads after incubation with the insoluble, denatured protein fraction and extensive washing; lane 4, supernatant from wash of agarose beads; lane 5, eluted protein after dialysis. Molecular mass standards are indicated on the left in kilodaltons.

A similar strategy was used to purify the pU_L28 protein, exceptthat the C-terminally histidine-tagged protein was expressedin insect cells by infection with a recombinant baculovirusexpressing the tagged protein. The recombinant protein was thenpurified by solubilization of inclusion bodies with GuHCl followedby affinity chromography on Ni⁺-containing beads and elutionin imidazole. A representative purification is shown in Fig.1B. The procedure yielded approximately 120 µg of purifiedprotein per 2 x 10⁷ insect cells.

Quantification of U_L15 and U_L28 in HSV-1(F) B capsids. As viewed by examination of each capsid purification step byelectron microscopy, it was necessary to use two successivesucrose gradients to generate highly purified B capsids. Thenumber of capsids present in a given sample was then determinedby calculating the amount of VP5 protein on a Coomassie-stainedgel, using a standard curve of known amounts of BSA protein(Fig. 2A). The amount of U_L15 or U_L28 protein in a known amountof capsids was calculated from immunoblots by comparing theintensity of the band in a sample of the capsids to a standardcurve of purified pU_L15 or pU_L28 (Fig. 2B and C).

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FIG. 2. Data used for calculating the average number of copies of pU_L15 or pU_L28 in capsids. (A) Coomassie-stained gel with B capsids (lanes 1 and 2) and BSA standards (lanes 3 to 8). The amount of BSA (in nanograms) loaded is indicated at the bottom of lanes 3 to 8. The R² value of the graph produced by analysis of the data was 0.98, and the estimated amount of VP5 (arrow) is indicated in italics below lanes 1 and 2. The positions of molecular weight standards are indicated on the left. (B) Immunoblot of B capsids (lanes 1 and 2) and purified U_L15 protein standards (lanes 3 to 8) probed with antisera directed against pU_L15. The amount of pU_L15 (in nanograms) is indicated below each lane. (C) Immunoblot probed with antisera directed against pU_L28. B capsids are in lanes 1 and 2, and purified U_L28 protein standards are in lanes 3 to 9. The amount of pU_L28 (in nanograms) is indicated below each lane.

To ensure that pU_L15 and pU_L28 were associated with capsidsand that background levels within the gradients did not affectthe calculations, a representative second gradient was fractionatedand each fraction was split between three separate gels thatwere then probed with antibodies directed against either VP5,pU_L28, or pU_L15 (Fig. 3). All three proteins were present onlyin fractions 5 and 6, the fractions corresponding to the light-scatteringB capsid band seen on the sucrose gradient. As a further control,a capsid purification procedure was also carried out using lysatesfrom cells infected with K23Z in parallel with HSV-1(F)-infectedcells. The K23Z virus has a lacZ construct replacing most ofthe U_L18 gene and is therefore unable to assemble capsids (18).No light-scattering bands were visible in the first K23Z sucrosegradient; however, the area corresponding to the A and B capsidband on the HSV-1(F) gradient was collected and run on a secondsucrose gradient. This gradient was fractionated, the fractionswere separated on a denaturing sodium dodecyl sulfate-polyacrylamidegel, and the proteins were transferred to PVDF. The membraneswere then probed with antibodies directed against either pU_L15or pU_L28, and the blot was intentionally overdeveloped (Fig.4). No pU_L15 or pU_L28 was detected in any fraction; therefore,background levels of immunoreactivity as a consequence of pU_L15or pU_L28 within the gradients would not be expected to contributeto immunoreactivity within capsid-containing fractions. Parenthetically,no U_L15 or VP5 was detected when a nuclear lysate of K23Z-infectedVero cells was run on a single sucrose gradient, fractionated,and examined by immunoblotting (data not shown).

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FIG. 3. Immunoblots of B capsids purified through two sequential 20- to-50% sucrose gradients. The 14-ml gradient was fractionated from the top (fraction one), and the proteins present in each fraction were separated on an 8% denaturing polyacrylamide gel before being transferred to a PVDF membrane. The membrane was then probed with antisera against U_L28, U_L15, or VP5 and developed using the ECL+ method (see Materials and Methods). The image was generated using a Molecular Dynamics Storm PhosphorImager with chemiluminescence detection capability.

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FIG. 4. Nuclear lysate of Vero cells infected with the U_L18 deletion virus K23Z, purified on two sequential 20- to-50% sucrose gradients. The 14-ml gradient was fractionated from top (lane 1) to bottom (lane 11), and the proteins present in each fraction were separated on an 8% denaturing polyacrylamide gel before being transferred to nitrocellulose membrane. The membrane was then probed with antisera against U_L15 (A) or U_L28 (C) and developed using the ECL+ method (see Materials and Methods). Lane 12 contains purified protein as a positive control (arrow). As a further control, lysates of HSV-1(F)-infected, K23Z-infected, or mock-infected cells were electrophoretically separated and reacted with antisera against U_L15 (B) or U_L28 (D) to confirm expression of the proteins in the cells infected with mutant virus.

Once it was confirmed that all of the pU_L15 and pU_L28 immunoreactivitywas associated with capsids, the number of copies of each ofthe proteins present in HSV-1(F) B capsids was determined. Anew preparation of capsids was used for each calculation. Assumingequal distribution of the proteins among all B capsids, eachcapsid was calculated to contain 1.0 copies of pU_L15 and 2.4copies of pU_L28 (Table 1). Statistical analysis of these datarevealed that each B capsid contained significantly more pU_L28than pU_L15 (P = 0.03) (Table 1).

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TABLE 1. Summary of the amounts of pU_L15 and pU_L28 present in B, U_L6(-), and A capsids^f

The amount of pU_L15 and pU_L28 in pU_L6(-) capsids is similar to the amount present in HSV-1(F) B capsids. To determine whether the presence or absence of the putativeportal encoded by pU_L6 influenced the capsid association ofpU_L15 and pU_L28, the number of copies of pU_L15 and pU_L28 inpU_L6(-) capsids was calculated using the same method as describedabove. An average of 1.5 copies of pU_L28 and 1.2 copies of pU_L15were present in pU_L6(-) capsids. No statistical difference wasdetected when comparing the amount of either pU_L15 or pU_L28in U_L6(-) and wild-type B capsids (P > 0.1) (Table 1).

There is significantly more pU_L15 and less pU_L28 in A capsids than in B capsids. The association of any of the six cleavage and packaging proteinswith wild-type A capsids has not been reported previously. TypeA capsids were purified from approximately 4 x 10⁸ HSV-1(F)-infectedcells, associated proteins were electrophoretically separatedon an 8% polyacrylamide gel, and the gel was stained with Coomassiebrilliant blue. Lanes containing denatured A capsid proteinscontained markedly reduced levels of scaffold proteins (VP22a)relative to other capsid proteins, confirming that the purifiedA capsids did not contain substantial numbers of B capsids (Fig.5).

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FIG. 5. Coomassie-stained gel of A and B capsids purified as described in Materials and Methods. The positions of the capsid shell proteins are indicated on the right. Note the very small amount of VP22a present in the A capsid sample compared to that in B capsids.

The number of copies of pU_L15 and pU_L28 per A capsid was thendetermined. An average of 12.0 copies of pU_L15 per capsid wasdetected, significantly more than amounts detected in wild-typeB capsids and pU_L6(-) capsids (P = 0.08 for both comparisons)(Table 1). In contrast, the level of pU_L28 in A capsid preparationswas near the lowest limit of detection, as assessed by comparisonwith the standard curve. Consequently, immunoreactivity thatcorresponded to an intensity within the standard curve was onlyobtained on two occasions. For these experiments, an averageof 0.6 copies of pU_L28 per capsid was calculated, which wassignificantly less than the amount of pU_L28 present in B capsids(P = 0.06). Since three further experiments failed to revealsufficient pU_L28 to allow calculation from comparison with thestandard curve, the 0.6 copies calculated likely representsthe higher end of the range of levels of pU_L28 in A capsids.

Cleaved pU_L15 is present in A capsids. The U_L15 protein is proteolytically cleaved at the N terminusin close association with the maturation of genomic DNA. Thecleavage shortens the 83-kDa full-length U_L15 protein to approximately80,000 and 79,000 M_r (48, 50). To determine whether the pU_L15was proteolytically cleaved in A capsids, A, B, and pU_L6(-)capsids were purified on two successive sucrose gradients asdetailed above. Capsid-associated proteins were electrophoreticallyseparated on 8% polyacrylamide gels, transferred to a PVDF membrane,and probed with antisera directed against either the entireexon II of the U_L15 protein or the N-terminal 35 amino acidsof the protein. Figure 6 shows that both the full-length andcleaved form of pU_L15 were present in A capsids and wild-typeB capsids but, as has been reported previously, only the full-lengthform was present in pU_L6(-) capsids (48). A sample from thesame A capsid preparation was separated on another gel and stainedwith Coomassie blue to confirm the lack of scaffold proteinVP22a and, hence, the absence of contamination by B capsids(data not shown).

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FIG. 6. Digital image of immunoblot probed with antisera directed against the portion of the U_L15 protein encoded by the entire exon II of the gene (A) or the N-terminal 35 amino acids (B). Lanes 1 and 2, A capsids; lanes 3, 4, and 5, B capsids; lanes 6 and 7, U_L6(-) capsids. All capsids were purified through two successive sucrose gradients (see Materials and Methods). The 83,000 and 80,000 apparent M_r forms of pU_L15 are indicated with arrows.

DISCUSSION

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Discussion

Purified recombinant U_L15 and U_L28 proteins were used as standardsto calculate an average of 1.0 (±0.29) copies of pU_L15and 2.4 (±0.97) copies of pU_L28 per B capsid, substantiallyless than the 15 copies of pU_L6 (38) or 42 copies of pU_L25 (40)previously reported in B capsids. Similar amounts of pU_L15 andpU_L28 were detected in capsids lacking the putative portal proteinpU_L6 (Table 1). In contrast, highly purified A capsids werefound to contain an average of 12.0 (±5.6) copies ofthe U_L15 protein per capsid, a significantly larger amount thanwas found in either wild-type B capsids or pU_L6(-) capsids.Only 0.6 (±0.32) copies of pU_L28 were detected per Acapsid on average, significantly less than the amount of pU_L28present in wild-type B capsids. Extensive efforts were madeto quantify pU_L15 and pU_L28 in C capsids, but the instabilityof these capsids in successive sucrose gradients and the relativelylow levels of pU_L15 and pU_L28 within them precluded accuratecalculations.

In many double-stranded DNA viruses, including bacteriophages ${lambda}$ (63) and ${phi}$ 29 (28), the portal vertex of the capsid representsthe docking site for the terminase enzyme. When extrapolatedto the HSV system, a prediction of this hypothesis is that pU_L6(-)capsids should lack the putative terminase components pU_L15and pU_L28. There have been previous reports of smaller amountsof pU_L15 in U_L6(-) capsids than in wild-type B capsids (65),and our preliminary data seemed to support this observation(50). However, once quantitative analyses were applied, no significantdifference was detected between the amounts of pU_L15 or pU_L28in the U_L6(-) capsids and those in wild-type B capsids (P >0.1) (Table 1). Thus, small amounts of both pU_L15 and pU_L28are able to associate with B-like capsids in the presence orabsence of the U_L6-encoded portal protein. This discrepancybetween present and previous studies in estimating the relativeamounts of pU_L15 in B and U_L6(-) capsids may stem from the factthat in this report capsids were purified through two, ratherthan one, sucrose gradients and extensive efforts were takento separate A capsids from B capsids.

Type A capsids are thought to be the result of an aborted cleavageand packaging reaction and are widely assumed to have at leastengaged the packaging machinery (37, 52, 57). The presence ofthe N-terminally cleaved form of pU_L15 in A capsids (Fig. 6)is consistent with this hypothesis. It is therefore possiblethat A capsids bear 12 copies of pU_L15 as a remnant of the functionalDNA packaging complex docked with the capsid. If these subunitswere to form a complex in conjunction with the portal vertex,such a structure would be reminiscent of the proposed DNA translocationmachinery assembled by the bacteriophages ${phi}$ 29 and SPP1 (23, 27,53). In these models the oligomeric ATPase subunit of the terminaseis present at the portal vertex, possibly in 6- or 12-fold symmetry,and participates in the packaging of the DNA into the capsid.However, it must also be acknowledged that the method describedhere for estimating the copy number of both pU_L15 and pU_L28in A and B capsids is indirect and therefore provides more accuraterelative, rather than absolute, data.

The consistently small amounts of pU_L15 present in both wild-typeB capsids and pU_L6(-) capsids do not exclude the possibilitythat the terminase docks with the capsid via interaction withthe portal protein. Rather, the data suggest that neither Bcapsids nor U_L6(-) capsids contain the fully assembled pU_L15structure, therefore adding to the increasing amount of indirectevidence suggesting that B capsids are dead-end by-productsrather than intermediates in capsid development. For example,several lines of evidence indicate that proteolytic cleavageand expulsion of the scaffold protein are tightly linked withentry of viral DNA (13, 21, 35, 44). This implies that the relativelystable B capsids, with a rigid, icosahedral shell and cleavedbut retained scaffold protein, are unlikely to be able to packageDNA. Further, there is in vitro evidence that, over time, procapsidsautomatically progress to B-like capsids in the absence of anypackaging machinery (37), suggesting that B capsids form bydefault if the cleavage and packaging reaction is delayed. Thus,while wild-type B capsids may be relevant for structural studiesof the portal vertex, they are unlikely to be useful for investigationsinto the portal vertex-terminase complex.

The disparity between the stoichiometry of pU_L15 and pU_L28 inA and B capsids was unexpected and may indicate that the interactionbetween these proteins changes during DNA packaging. Thus, differentstages of the packaging reaction may require different stoichiometriesof, and interactions between, terminase subunits as recentlysuggested for bacteriophage T4 (10). An alternative explanationis that many copies of pU_L28 may also comprise part of the cleavageand packaging machinery but that these are lost either duringthe capsid purification procedure or during A capsid morphogenesis,where a greater affinity for DNA than for capsids or pU_L15 (4)causes the protein to be lost when the DNA with which it isassociated aberrantly exits the capsid.

Both pU_L15 and pU_L28 have been detected in the procapsid (51),a precursor to A, B, and C capsids, suggesting that the cleavageand packaging machinery associates with capsids at an earlystage. Quantification of the amounts of pU_L15 and pU_L28 in procapsidswould likely shed further light on the overall structure ofthe packaging machine and allude to the possible functions ofthese proteins.

ACKNOWLEDGMENTS

These studies were supported by R01 grants GM50740 and AI52341from the National Institutes of Health.

We gratefully acknowledge the technical assistance of JarekOkulicz-Kozaryn, Elizabeth Wills for electron microscopy, andHollis Erb for statistical advice. We also thank P. Desai andS. Person for the K23Z virus, A. Patel for the U_L6 deletionvirus, and G. Cohen and R. Eisenberg for the VP5 antibody.

FOOTNOTES

* Corresponding author. Mailing address: C5169 Veterinary Medical Center, Cornell University, Ithaca, NY 14853. Phone: (607) 253-3391. Fax: (607) 253-3384. E-mail: jdb11{at}cornell.edu.

REFERENCES

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