A Domain of Herpes Simplex Virus pUL33 Required To Release Monomeric Viral Genomes from Cleaved Concatemeric DNA

ABSTRACT Monomeric herpesvirus DNA is cleaved from concatemers and inserted into preformed capsids through the actions of the viral terminase. The terminase of herpes simplex virus (HSV) is composed of three subunits encoded by UL15, UL28, and UL33. The UL33-encoded protein (pUL33) interacts with pUL28, but its precise role in the DNA cleavage and packaging reaction is unclear. To investigate the function of pUL33, we generated a panel of recombinant viruses with either deletions or substitutions in the most conserved regions of UL33 using a bacterial artificial chromosome system. Deletion of 11 amino acids (residues 50 to 60 or residues 110 to 120) precluded viral replication, whereas the truncation of the last 10 amino acids from the pUL33 C terminus did not affect viral replication or the interaction of pUL33 with pUL28. Mutations that replaced the lysine at codon 110 and the arginine at codon 111 with alanine codons failed to replicate, and the pUL33 mutant interacted with pUL28 less efficiently. Interestingly, genomic termini of the large (L) and small (S) components were detected readily in cells infected with these mutants, indicating that concatemeric DNA was cleaved efficiently. However, the release of monomeric genomes as assessed by pulsed-field gel electrophoresis was greatly diminished, and DNA-containing capsids were not observed. These results suggest that pUL33 is necessary for one of the two viral DNA cleavage events required to release individual genomes from concatemeric viral DNA. IMPORTANCE This paper shows a role for pUL33 in one of the two DNA cleavage events required to release monomeric genomes from concatemeric viral DNA. This is the first time that such a phenotype has been observed and is the first identification of a function of this protein relevant to DNA packaging other than its interaction with other terminase components.

and C capsids, and an inner scaffold layer; A capsids lack the internal layer, and the C capsid contains DNA in place of the inner scaffold. A capsids are believed to be the result of aborted packaging events in which the inner shell was expelled or degraded and DNA was not inserted (10,11).
All herpesviruses encode an enzyme called the terminase; in HSV, the terminase consists of three subunits encoded by the genes U L 15, U L 28, and U L 33 (12)(13)(14)(15)(16)(17)(18)(19). The U L 28 protein (pU L 28) has been shown to bind Pac1 DNA, which is important for the generation of short component termini (20), whereas U L 15 has nonspecific cationdependent nuclease activity in vitro and contains an essential Walker box motif, suggesting an ability to hydrolyze ATP (21). The U L 89-encoded homolog of cytomegalovirus retains the ATPase motif and cation-dependent nuclease activity (22,23). The structures of the C-terminal portions of the HSV-1 U L 15 gene product (pU L 15) and human cytomegalovirus (HCMV) pU L 89 are virtually superimposable and reveal a conserved RNase H-like structure seen in a number of nucleases and bacteriophage terminase subunits responsible for endonucleolytic DNA cleavage (18,21,24).
Cleavage of viral DNA requires both intact capsids and all terminase components (18,(25)(26)(27). Mutations outside the ATP binding and nuclease active sites in pU L 15 can prevent DNA packaging but do not preclude DNA cleavage or the release of intact genomes from concatemeric DNA, indicating that pU L 15 has a packaging function separate from its nuclease activity (28). These observations are consistent with its roles as both a packaging motor and an endonuclease. pU L 28, on the basis of its sequencespecific DNA binding activity, may act to restrict pU L 15's nuclease activity to correct sites at genomic termini.
The predominant model to explain HSV DNA packaging proposed that cleavage at the long terminus is followed by packaging and scanning of DNA until the short terminus is encountered in the concatemer, at which time it is cleaved (29,30). The a sequences that signal cleavage contain subsequences designated DR1-Uc-DR4 m -DR2 n -Ub-DR1, where DR indicates a direct repeat and m and n indicate the number of repeats (31,32). Ub and Uc are two unique sequences. Ub has been designated Pac1, and Uc has been designated Pac2. Although two cleavage events that are required to release genomes from the concatemer occur at different times and require different cis-acting sequences, they cleave DR1 identically, leaving single 3= overhangs with 18 bp of DR1 at the L terminus and a single base pair of DR1 at the S terminus (32). The initial cleavage renders the S terminus on the concatemer nonfunctional because it removes much of DR1. Thus, it is believed that only the L terminus is packaged, whereas the S terminus generated from the first cleavage event is degraded. While most genomes in the concatemer are separated by a single a sequence, some genomic junctions and L component termini contain multiple a sequences that share an intervening DR1 region. During packaging, these tandem a sequences presumably pass by the docked terminase and remain uncleaved. Scanning continues until DR1 in the proper orientation is cleaved to generate the S component terminus of DNA to be packaged and to release the genome from the concatemer. The idea that the S component terminus is packaged last is supported by the observations that (i) the S terminus in packaged DNA is most susceptible to DNase digestion and (ii) the short component of packaged DNA never bears more than one a sequence, suggesting that it is generated when the terminase cleaves the first encountered packaging sequence in the proper orientation after scanning/packaging of the long and short components (7,11,33).
The main goal of the present study was to understand the role of pU L 33 in the packaging reaction. pU L 33 interacts with pU L 28 and enhances the pU L 28-pU L 15 interaction (13). Extensive mutagenic analyses previously identified regions of U L 33 important for DNA packaging and the generation of L component termini (34,35). In extending these analyses, we have identified several novel mutations in a conserved region of pU L 33 with a positive charge that do not prevent the cleavage of concatemeric DNA but preclude the release of monomeric genomes. These data suggest that the pU L 33 component of the terminase is necessary for one of the two DNA cleavage events necessary to release unit-length genomes from concatemeric DNA.

RESULTS
In a previous mutagenesis study, the insertion of amino acids VRPQR at position 111 and AAAAA at position 116 of the 130-codon U L 33 open reading frame precluded the packaging of viral DNA (35). Neither of these insertions precluded interactions with the U L 28 protein (pU L 28), although the insertion at codon 116 diminished this interaction, as assessed by a coimmunoprecipitation assay. Alignment of HSV-1 U L 33 with homologs in other herpesviruses showed that two regions, residues 46 to 77 and 101 to 130, were the most conserved regions of pU L 33. Basic amino acids were highly conserved at positions corresponding to codons 110 to 113 (encoding KRER in HSV-1). These amino acids were followed by invariant phenylalanine and alanine residues at positions 114 and 115, respectively (Fig. 1).
To investigate whether these conserved regions are important for U L 33 function, we generated a series of mutant viruses using a bacterial artificial chromosome (BAC) system and assessed their ability to replicate on CV1 cells and complementing cells expressing U L 33. Consistent with previous work, recombinant viruses with a deletion of residues 50 to 60 or 110 to 120 replicated only in pU L 33-expressing cells (Table 1). In contrast, a mutant virus lacking codons 120 to 130 replicated in noncomplementing cells at levels similar to those of the wild-type virus ( Table 1), suggesting that the last 10 amino acids are dispensable for U L 33 function.
To further investigate the importance of charged residues for pU L 33 functions, we generated a series of mutant viruses in which basic residues were replaced with alanine, using the recombinant bacterial artificial chromosome system. As shown in Table 1, single-alanine-residue substitutions at positions 52, 54, 60, 65, 85, 110, 113, and 120 reduced the replication of the corresponding mutant viruses by 2-to 14-fold, suggesting modest effects on pU L 33 function. In contrast, mutants with multiple amino changes (110-KR-111 changed to 110-AA-111 and 110-KRER-113 changed to 110-AAEA-113, designated KR and KRER, respectively), could be propagated only on cells expressing pU L 33 inasmuch as the ratio of yields from U L 33-expressing cells infected with these viruses to those from CV1 cells was over 10,000-fold (Table 1). These results were similar to those obtained from analyses of the U L 33-null mutant performed at the same time (Table 1). We conclude that there was no detectable replication of the KR or KRER mutation in CV1 cells.
To determine if these lethal substitution mutations interfered with the pU L 28 interaction, cells were mock infected or infected with 5.0 PFU per cell of wild-type HSV-1(F) or U L 33 mutant viruses. Cells were lysed at 18 h postinfection (p.i.), and clarified lysates were reacted separately with antibody against pU L 28 or pU L 33. Antigen-antibody complexes were purified, eluted in SDS-containing buffer, electrophoretically separated on a denaturing SDS-polyacrylamide gel, and transferred onto a nitrocellulose membrane, which was then probed with the same pU L 28-or pU L 33specific antibodies. As shown in Fig. 2, the U L 33 mutant proteins bearing the 110-AA-111 (KR) and 110-AAEA-113 (KRER) mutations were efficiently immunoprecipitated by the U L 33 antibody. Unlike wild-type pU L 33, the mutant proteins failed to coimmunoprecipitate pU L 28. In the reciprocal interaction, pU L 28-specific antibody efficiently immunoprecipitated pU L 33 from cells infected with HSV-1(F), as shown previously (13,15,35). Less pU L 28 was immunoprecipitated with the pU L 28 antibody from lysates of cells infected with the U L 33 mutants, consistent with previous results indicating that an optimal interaction of pU L 33 confers stability to the pU L 28 protein (13). Despite the lower levels of pU L 28 in the lysates of cells infected with the U L 33 mutant viruses, low levels of both mutant U L 33 proteins were coimmunoprecipitated with the pU L 28 antibody. We conclude that the U L 33 mutations diminished the pU L 28-pU L 33 interaction but did not completely eliminate it.
Experiments were then conducted to assess effects of the mutations on the generation of genomic termini. As shown in Fig. 3A, terminase cleavage combined with BamHI cleavage should generate BamHI S fragments of around 2.9 kbp at the L component terminus and a P fragment of 3.45 kbp that reflects the terminus of the S component. Multiple BamHI S fragments were expected because one or more a sequences are present at the L termini of different genomes (Fig. 3A). Both the S and P fragments are covalently linked in the S-P fragment derived from the junction of the long and short components in monomeric DNA and the junctions of tandem genomes within concatemeric DNA. Therefore, to test for terminase cleavage activity, viral DNA was purified from cells infected with wild-type and mutant viruses, digested with BamHI, transferred to a positively charged nylon membrane, and probed with radiolabeled fragments specific for the termini of either the long or short components. The results are shown in Fig. 3B.
BamHI S fragments were readily detectable in cells infected with the wild-type virus (Fig. 3B, lane 1) but were hardly detectable in DNA from cells infected with the U L 33-null virus, indicating that pU L 33 was necessary for optimal terminase cleavage at the L component terminus. In contrast to these results, S fragments from cells infected with the KR or KREK mutant were readily detectable, indicating that the mutations did not block the cleavage that generates the L component terminus. Similarly, BamHI P fragments representing the S component terminus were readily detected in cells infected with both wild-type and U L 33 point mutant viruses, but P fragments were barely detected in U L 33-null-virus-infected cells.
Because genomic DNA within input virions is linear and would be incorporated into preparations of infected cell DNA, we wanted to ensure that the BamHI S and P fragments that we observed were not due to contaminating input virion DNA. Thus, cells were infected with wild-type and mutant viruses in the presence and absence of phosphonoacetic acid (PAA), a potent viral DNA synthesis inhibitor, and viral DNAs were purified, digested with BamHI, and analyzed on Southern blots probed with the radiolabeled HSV-1(F) P fragment, as detailed above. As shown in Fig. 4, P fragments were detected in input viral DNA in all samples from cells treated with PAA. Moreover, the P fragment was present with the S-P junction fragment at a consistent ratio in all PAA-treated samples. This was expected and reflected the presence of exclusively linear DNA in input virions. Also as expected, the amount of viral DNA in samples from cells infected in the presence of PAA was reduced. Most importantly, the ratios of P to S-P fragments were similar in all samples except those from cells infected with the U L 33-null mutant in the absence of PAA. In this sample, the P/S-P ratio was reduced by approximately 55%. These data suggest that while pU L 33 is necessary for the optimal generation of genomic termini, both genomic termini were generated in cells infected with two U L 33 substitution mutants. Deletion of residues 50-60 7 ϫ 10 8 Ͻ10 3 ? Del110-120 Deletion of residues 110-120 1.5 ϫ 10 8 Ͻ10 3 ? Del120-130 Deletion of residues 120-130 5.5 ϫ 10 8 2 ϫ 10 8 ϩ P52A Proline to alanine 4 ϫ 10 8 8 ϫ 10 7 ϩ E54A Glutamic acid to alanine 8 ϫ 10 7 1 ϫ 10 7 ϩ P60A Proline to alanine 7 ϫ 10 7 5 ϫ 10 6 ϩ K65A Lysine to alanine 2 ϫ 10 7 1.2 ϫ 10 7 ϩ K86A Lysine to alanine 2.6 ϫ 10 8 8 ϫ 10 7 ϩ K110A Lysine to alanine 4 ϫ 10 8 1.2 ϫ 10 8 ϩ R113A Arginine to alanine 2.2 ϫ 10 8 4 ϫ 10 7 ϩ R120A Arginine to alanine 9.5 ϫ 10 7 5 ϫ 10 7 ϩ KR Lysine and arginine at positions 110 and 111 to alanine To determine whether the terminase cleavage events in U L 33 mutant viral DNA were sufficient to release genomes from concatemeric viral DNA, we analyzed viral DNA using pulsed-field gel electrophoresis. Cells were infected with HSV-1(F) and U L 33 mutant viruses in the presence and absence of PAA, and agarose plugs containing the infected cell DNA was subjected to electrophoresis on an agarose gel in a pulsed-field apparatus. The separated DNA was then transferred to a nitrocellulose membrane and probed with radiolabeled viral DNA. As shown in Fig. 5, both concatemeric DNA (well DNA) and monomeric DNAs were detected in untreated HSV-1(F)-infected cells, whereas no concatemeric DNA and small amounts of monomeric DNA were detected after infection in the presence of PAA. We attribute the signal in the PAA-treated sample to input virion DNA. Unlike the results with the wild-type virus, the U L 33 mutant viruses produced substantially less monomeric DNA than did the wild-type virus inasmuch as virtually all replicated DNA remained in the well, reflecting its presence within DNA concatemers. Small amounts of monomeric DNA were detected in cells infected with U L 33 mutant viruses in the presence of PAA. Although most of this monomeric DNA signal was attributable to input virion DNA, we could not rule out some generation of monomeric DNA by the U L 33 mutant viruses because a slightly higher monomeric signal was obtained from cells infected in the absence of PAA than in PAA-treated cells. In the absence of PAA, the monomeric mutant DNA bands were less distinct and exhibited broad bands extending below the migration position of monomeric DNA. This observation suggested that monomeric DNA was cleaved nonspecifically or was partially degraded in these samples.
To assess DNA packaging of the mutant viruses, two sets of experiments were conducted. In the first set of these experiments, thin sections of cells infected with wild-type and mutant viruses were examined by electron microscopy. As shown in Fig.  6, HSV-1(F) produced electron-dense capsids containing DNA (type C), capsids with an inner electron-lucent core (type B), and empty capsids (type A). In contrast, while type B capsids were readily detected in cells infected with the U L 33 mutants, neither type A Function of U L 33 in HSV DNA Cleavage/Packaging Journal of Virology nor type C capsids were detected in these cells. These results suggested that the U L 33 mutants exhibited profound defects in DNA packaging.
In the second set of experiments, capsids were purified from cells infected with wild-type or mutant viruses and were separated by rate-zonal centrifugation on continuous sucrose gradients. As shown in Fig. 7, only the wild-type virus produced light-refracting bands at positions consistent with all three capsid types, whereas the U L 33 mutants produced only 1 band consistent with type B capsids that lack DNA. These data further indicated that the U L 33 mutants were defective in DNA packaging.

DISCUSSION
To define the function of pU L 33, we extended mutagenesis studies reported previously, and we report here the generation of new U L 33 mutants using a bacterial artificial chromosome system. The deletion of the conserved regions between either amino acids 50 and 60 or amino acids 110 and 120 of pU L 33 impaired its function, and these mutants were able to replicate only in U L 33-expressing cell lines. Our results are consistent with data from previous work in which pU L 33 mutations with insertions in these regions were unable to complement the growth of a U L 33-null virus in transientpackaging assays (35). The last 10 amino acids of pU L 33 are highly conserved in all alphaherpesviruses but were found to be dispensable for HSV replication.
One goal of this work was to define the region of the pU L 33 interaction with pU L 28. Our previous work showed that pU L 33 bearing a point mutation at position 61 failed to interact with pU L 28 at a nonpermissive temperature (34). Consistent with this work, the deletion of codons 50 to 60 precluded the interaction of pU L 33 with pU L 28 (data not shown), and this mutant virus was unable to replicate in noncomplementing cells

FIG 4 Ratio of terminal to junction BamHI fragments in replicated and input HSV DNA from infected cells. (A) CV1 cells were infected with the indicated viruses at an MOI of 5 PFU per cell in the presence or absence of phosphonoacetic acid (PAA). At 18 h postinfection, viral DNAs were extracted, digested with
BamHI, separated on an agarose gel by electrophoresis, denatured, and transferred onto a nylon membrane. Bound DNAs were probed with the denatured radiolabeled P fragment. (B) The levels of the P or S-P fragments in all samples were quantified by using ImageJ software, and the ratios of P fragments to S-P fragments are plotted on a graph.
( Table 1), suggesting that this region may mediate protein-protein interaction. Surprisingly, mutants with alanine replacements in the conserved residues (52P, 54E, 60P, or 65K) in this region replicated in noncomplementing cells nearly as well as the wild-type virus ( Table 1), suggesting that these point mutations did not dramatically affect pU L 33 function or interactions with pU L 28. However, we have not tested whether these mutations affect pU L 33 function at higher temperatures (e.g., 39°C). Whether this conserved region is sufficient to mediate the interaction of pU L 33 with pU L 28 needs further study. A virus lacking U L 33 codons 110 to 120 was also unable to replicate in noncomplementing cells (Table 1). Attempts to assess pU L 28 interactions with a pU L 33 mutant lacking codons 110 to 120 were unsuccessful because the deletion of this region precluded detection by the pU L 33-specific antibody (data not shown). Therefore, it remains unclear whether the deletion of codons 110 to 120 in pU L 33 affects the interaction with pU L 28.
Previously characterized terminase component mutants either do not cleave DNA or allow both cleavage events but preclude DNA packaging (11,18,26,28,(36)(37)(38)(39)(40). The U L 33 mutants KR and KRER ( Fig. 3 and 4) are unusual because both long and short termini were generated, yet little to no monomeric DNA was released from the concatemer. Because type A capsids that result from aborted packaging events were not detected, it is likely that the U L 33 mutants abort the packaging reaction after DNA cleavage but before the initiation of DNA packaging. Although there are other possibilities, the simplest explanation for the lack of released monomers is a failure of the second DNA cleavage event. Thus, in noncomplementing cells, the short and long termini of these mutants are generated from a single successful DNA cleavage event, but this is not followed by a second coordinated cleavage event that releases genomes from the concatemer. If true, this would suggest that the first DNA cleavage event always occurs at DR1 in the a sequence most distal to U L , leaving an S terminus on the concatemer with an a sequence bearing a single base pair of DR1.
Indirect evidence also favors the possibility that the KR and KRER mutants are defective in the second DNA cleavage event. Specifically, in coimmunoprecipitation experiments, these UL33 mutants were coimmunoprecipitated with pU L 28 by anti-pU L 28 antibodies, but pU L 28 was not pulled down with the pU L 33 mutant by anti-pU L 33 antibodies. These observations suggest that these mutations inhibit the pU L 33- Function of U L 33 in HSV DNA Cleavage/Packaging Journal of Virology pU L 28 interaction but do not completely eliminate it. This imperfect interaction may inhibit proper scanning, which may be required for the coordination of the two cleavage events to release monomeric genomes. The lack of an optimal pU L 33-pU L 28 interaction may also preclude the second DNA cleavage event, which is likely augmented by pU L 28's recognition of Pac1 DNA (20, 41). It is notable that low levels of L termini are detectable in concatemeric DNA (well DNA) from cells infected with the wild-type virus (42,43). Concatemeric DNA has been designated well DNA experimentally because it is large enough to be retained in loading wells even after lengthy pulsed-field gel electrophoresis (44). Remarkably, S termini have not been detected in well DNA (44,45), suggesting that they are absent from concatemeric ends. This observation is surprising because each DNA cleavage event should generate both an S terminus and an L terminus. It is therefore likely that S termini are degraded (when produced from the first terminase DNA cleavage event) or, when produced from the second DNA cleavage event, removed from the concatemer through DNA packaging. Unlike the wild-type virus, the U L 33 KR and KRER mutants described here produce concatemeric DNA almost exclusively, yet both S and L termini were abundant and readily detected. Taken together, these results suggest that pU L 33 may also play a direct or indirect role in the degradation of S termini on the concatemer. Another possibility is that both terminase DNA cleavage events occur in the KR and KRER UL33 mutants, but the monomers are somehow retained with the concatemer in well DNA. We do not favor this possibility because monomers are released when well DNA is digested with SpeI, a restriction enzyme that cuts each genome within the concatemer only once (44). Thus, tandem cleavages a genome apart should be sufficient to release monomeric genomes. It follows that the cleavages that we detected in the KR and KRER mutants are much farther apart than genome length, suggesting a discoordination of the two cleavage events relevant to DNA packaging.
In summary, these data reveal previously unknown functions of pU L 33 and support its role as an important terminase component.

MATERIALS AND METHODS
Cells, viruses, and plasmids. CV1 cells and CV1-derived complementing cells expressing U L 33 (designated CV33) were described previously (13). The wild type F strain of HSV-1 [HSV-1(F)] and the U L 33 deletion virus were described previously (46,47). The HSV-1(F) bacterial artificial chromosome (HSV-BAC) was obtained from Y. Kawaguchi, University of Tokyo (48), and all the recombinant mutants were derived from this HSV-BAC. The pEPKan-S plasmid containing aphA1 (encoding kanamycin resistance) was a gift from Klaus Osterrieder, University of Berlin. Plasmid pCAGGS-nlsCre expressing Cre recombinase was a gift from Michael Kotlikoff, Cornell University. Bacterial Escherichia coli strain GS1783, used to maintain the HSV-BAC, was obtained from Greg Smith, Northwestern University.
Generation of recombinant viruses. All the recombinant viruses were generated with the BAC system as described previously by Tischer at al (49). The primers used to construct recombinant BACs are listed in Table 2. Detailed procedures to generate recombinant BACs were described above, and the expected mutations were confirmed by DNA sequencing. The corresponding recombinant viruses were reconstituted by cotransfecting BAC DNA with the Cre expression plasmid into CV33 cells. Stock viruses of these mutants were prepared in CV33 cells, and the virus titers in CV33 and CV1 cells were determined by using a plaque assay.
Coimmunoprecipitation and Western blotting. CV1 cells were infected with the indicated viruses at a multiplicity of infection (MOI) of 5.0 PFU per cell. Eighteen hours after infection, cells were washed with phosphate-buffered saline (PBS) and lysed in cold radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 1% NP-40, 0.25% sodium deoxycholate, 1 mM EDTA, and a protease inhibitor cocktail). The lysates were clarified by centrifugation at 14,000 rpm for 10 min at 4°C, and the precleared lysates were reacted with anti-pU L 28 or anti-pU L 33 antibodies, followed by the addition of Gamma Bind Sepharose beads for immunoprecipitation. Immune complexes bound on the beads were washed extensively with RIPA buffer, eluted in 2ϫ Laemmli sample buffer (Bio-Rad), separated on an SDS-12% polyacrylamide gel, and transferred onto a nitrocellulose membrane for immunoblotting. Both anti-pU L 28 and anti-pU L 33 antibodies were diluted at 1:100 for immunoprecipitation and at 1:1,000 for immunoblotting. Horseradish peroxidase-conjugated anti-rabbit immunoglobulin G and enhanced chemiluminescence were used for immunodetection.
Southern blotting. CV1 cells were infected with wild-type HSV-1 or U L 33-null mutant viruses at an MOI of 5 PFU per cell in the presence or absence of 300 g/ml of PAA. At 18 h postinfection, viral DNA was extracted as described previously (28). Briefly, cells were lysed in 1% NP-40 and digested with proteinase K, followed by phenol-chloroform extraction and ethanol precipitation. Viral DNAs were digested with BamHI and electrophoretically separated on a 0.8% agarose gel. The separated DNAs were denatured with a denaturation solution (1.5 M NaCl, 0.5 M NaOH), neutralized with neutralization buffer (1 M Tris-HCl [pH 8.0], 1 mM NaCl), and transferred onto a positively charged nylon membrane. Bound DNAs were cross-linked with a UV cross-linker (FB-UVXL-1000; Fisher Scientific) and hybridized with denatured 32 P-labeled BamHI P or S fragments of HSV-1 DNA. The membrane was washed extensively with 1ϫ SSC (1ϫ SSC is 0.15 M NaCl plus 0.015 M sodium citrate), and the positions of the bound probe were determined by fluorography using X-ray film exposed at Ϫ80°C in the presence of an intensifying screen. The film was scanned and processed with Adobe Photoshop software, and the signal intensity was quantified with ImageJ software.
Pulsed-field gel electrophoresis. Pulsed-field gel electrophoresis experiments were carried out as described previously (28). Briefly, about 3.2 ϫ 10 6 CV1 cells in a 60-mm-diameter dish were infected with the indicated viruses at an MOI of 5 PFU per cell in the presence or absence of 300 g/ml PAA for 18 h. Cells were collected in cold PBS and pelleted by spinning them at 4,000 rpm for 5 min in a refrigerated tabletop centrifuge. The pellets were resuspended in 150 l of PBS and mixed with 300 l of 1.2% agarose, and the mixture was poured onto the bottom of a 10-mm-diameter dish to form an agarose block. The agarose block was digested with 100 g/ml proteinase K in digestion buffer ( 8.0], 10 mM EDTA). Roughly equally sized agarose plugs were sliced and loaded into the wells of a 0.8% agarose gel, and the wells were sealed with 0.8% low-melting-point agarose. The gel was run in 0.5ϫ TBE buffer (1ϫ TBE buffer contains 89 mM Tris, 89 mM boric acid, and 2 mM EDTA [pH 8.0]) at 6 V/cm for 16 h at 14°C, with an angle of 120°and a pulse time of 45 to 70 s, with a Bio-Rad CHEF-DR II pulsed-field electrophoresis system. After electrophoresis, the gel was soaked in 0.25 M HCl for 45 min to depurinate the DNAs, and the DNAs were further denatured, neutralized, and transferred onto a positively charged nylon membrane as described above. DNAs were UV cross-linked to the membrane and hybridized with the denatured 32 P-labeled BamHI P fragment of the HSV-1 genome as described above. The bound probe was revealed by exposure of the membrane to X-ray film at Ϫ80°C in the presence of intensifying screens.
Electron microscopy. Electron microscopic examination of infected cells was performed at the Shared Instrument Facility at Louisiana State University. Confluent CV1 cells in T25 flasks were infected with wild-type HSV-1 or U L 33 mutants for 18 h. Medium was removed, and the cells were incubated with a fixative solution (2% formaldehyde and 2.5% glutaraldehyde in 0.1 M phosphate buffer [pH 7.0]) for 10 min and collected by scraping into a centrifuge tube, followed by shaking for 2 h. Cells were pelleted, and the supernatant was discarded. The pellets were mixed with equal volumes of 3% agarose and transferred onto a glass slide before solidification. The agarose cubes were rinsed 5 times (15 min each time) with 0.1 M phosphate buffer (pH 7.4) containing 0.08 M glycine, followed by fixing the cells for 1 h in the dark with 2% osmium tetroxide prepared in 0.1 M phosphate buffer (pH 7.4). After 3 washes with distilled water, samples were dehydrated with a graduated series of ethanol concentrations (50%, 70%, 80%, 90%, and 100%, for 15 min under each concentration). This was followed by infiltration with a 1:1 mixture of ethanol (EtOH) and LR White for 2 h and with 100% LR White for 2 h. Samples were dispersed into the bottom of a Beem capsule, and the resin was polymerized at 65°C for 24 h. Ultrathin sections were cut on a Leica EM UC7 microtome, and thin sections (90 nm thick) were collected on 300-mesh nickel grids. Thin sections were counterstained with 2% uranyl acetate for 20 min and then with lead citrate for 7 min. Stained grids were viewed with a JEOL JEM-1400 transmission electron microscope. Images were captured digitally and processed with Adobe Photoshop software. Letters in boldface type represent introduced mutations, and underlined sequences prime to the 5= or 3= termini of the kanamycin gene of pEPKan-S.
Function of U L 33 in HSV DNA Cleavage/Packaging Journal of Virology