JOURNAL OF VIROLOGY,0022-538X/01/$04.00�0 DOI: 10.1128/JVI.75.21.10505–10510.2001

Nov. 2001, p. 10505–10510 Vol. 75, No. 21

Copyright © 2001, American Society for Microbiology. All Rights Reserved.

Isomerization of a Uniquely Designed Amplicon during HerpesSimplex Virus-Mediated Replication

HUA WANG,1 XINPING FU,1 AND XIAOLIU ZHANG1,2*

Center for Cell and Gene Therapy1 and Departments of Pediatrics and Molecular Virology & Microbiology,2

Baylor College of Medicine, Houston, Texas 77030

Received 18 April 2001/Accepted 22 July 2001

Herpes simplex virus (HSV) type 1 DNA isomerization was studied using a uniquely designed amplicon thatmimics the viral genomic structure. The results revealed that amplicon concatemers frequently containadjacent amplicon units with their segments in opposed orientations. These unusual concatemers weregenerated through homologous recombination, which does not require HSV DNA as the source of homology.

The herpes simplex virus type 1 (HSV-1) genome is a linear,double-stranded DNA molecule of 152 kb. It consists of twocovalently linked segments designated long (L) and short (S).Each segment contains largely unique sequences (UL and US)which are bracketed by inverted repeats. Previous studies haveshown that HSV replicative intermediates are high-molecular-weight molecules in which the genomic termini are fused to-gether in a head-to-tail arrangement (2, 13, 14). These findingshave led to a model in which the linear viral genome circular-izes immediately after infection and replicates unidirectionallyby a rolling-circle mechanism. This mode of replication gener-ates a head-to-tail concatemer that is cleaved into unit-lengthgenomes during packaging (10, 11, 19, 26). Consistent with thismodel, defective HSV-1 genomes are encapsidated as head-to-tail repeats (4, 28), structures that are compatible with arolling-circle mechanism of DNA replication (21, 22).

A prominent feature of HSV DNA replication is the freeinversion of the L and S segments relative to each other,generating four isomeric forms that occur naturally in equimo-lar proportions (5, 11). The molecular mechanism of segmentinversion is poorly understood. The use of alternative cleavagesites during maturation and packaging of concatemeric inter-mediates can account for the generation of only two isomericforms from a single monomeric template (25). The remainingisomers are thought to have been generated by homologousrecombination, and the repeated a sequences appear to play animportant role in this recombination-mediated segment inver-sion (3, 6, 7, 16–18, 24). However, recent studies have provideda different insight into the mechanism of HSV DNA isomer-ization. Analysis of replicative intermediates digested with re-striction enzymes that cleave once in the unique region of thevirus genome has revealed that the concatemers very fre-quently contain adjacent genomic units with L segments indifferent orientations (called concatemers with internal seg-ment isomerization [concatemer-ISI]), from which all four pos-sible HSV isomers can be generated in an equal molar ratiothrough random cleavage and packaging (1, 15, 20, 27). Thisfinding has suggested that HSV genome isomerization is inti-

mately linked to DNA replication, rather than to a late eventassociated with cleavage and packaging.

The mechanism for generating concatemer-ISI is not fullyunderstood. Since conventional rolling-circle replication is un-able to generate such concatemers, they must arise by other,yet-unidentified mechanisms. Further elucidation of the mech-anism(s) requires a more detailed analysis of the viral replica-tive intermediates. However, the large size and complexity ofthe HSV genome make it time-consuming and technically dif-ficult to modify the viral genome in order to facilitate experi-mental designs. For the same reasons, it is also difficult toselect unique restriction enzymes for Southern blot analysis.The HSV amplicon, which has a much smaller genome andcontains only the cis elements required for virus-mediated rep-lication and cleavage-packaging (i.e., the replication origin andthe packaging signal) (8, 9), may be an alternative and simpli-fied tool for this purpose. Upon introduction into cells togetherwith a helper virus, the HSV amplicon can be amplified, pre-sumably by a rolling-circle mechanism using the essential pro-teins provided by the helper virus. The replicated ampliconDNA can be subsequently packaged into viral particles, whichare also provided by the helper virus, in a multiple-copy con-catemeric form.

Earlier studies by us and others showed that neither isomer-ization nor concatemer-ISI could be detected on HSV ampli-cons harboring only a single set of viral cis elements (18, 28).We therefore constructed a new version of the HSV ampliconthat mimics the viral genomic structure; this amplicon has twosets of HSV repeat sequences, each composed of a replicationorigin (oriS) and an a sequence in different locations and inopposite orientations. This amplicon, called pSZ-dPac-EGFP,consequently has its own L and S segments bracketed by theHSV repeats (Fig. 1A), much like the native HSV genome.Purified pSZ-dPac-EGFP DNA was transfected into babyhamster kidney (BHK) cells, which were subsequently infectedwith a helper HSV (wild-type HSV strain 17). The ampliconwas harvested and passaged twice before concatemers wereextracted from packaged viral particles. DNA was digestedwith either ScaI, which has a single recognition site locatedclose to one end of the L segment, or XhoI, which has a singlerecognition site located near one end of the S segment (Fig.1A). If there is no internal segment isomerization and onlyhead-to-tail concatemers are formed, then digestion with ei-

* Corresponding author. Mailing address: Department of Pediatricsand Center for Cell and Gene Therapy, Baylor College of Medicine,One Baylor Plaza, Houston, TX 77030. Phone: (713) 798-1256. Fax:(713) 798-1230. E-mail: xzhang@bcm.tmc.edu.

10505

on July 13, 2018 by guesthttp://jvi.asm

.org/D

ownloaded from

ther of these enzymes will generate only the unit-length 6.6-kbfragment. Alternatively, if the orientation of the L segment isfrequently opposed, ScaI digestion will generate two additionalDNA fragments with sizes of 8.8 and 5.2 kb (Fig. 2A). Accord-ingly, if the orientation of the S segment is also frequentlyopposed, XhoI digestion will produce two additional DNAfragments with sizes of 7.3 and 5.9 kb (Fig. 2B).

Following restriction enzyme digestion, DNA fragmentswere separated by agarose gel electrophoresis, transferred to anylon membrane, and hybridized with a probe (pC) which wasmade from the entire pSZ-dPac-EGFP sequence but lackedthe oriS and a sequences (to limit cross-hybridization withDNA fragments from the helper virus genome). ScaI digestionof the packaged amplicon concatemer generated two strongbands of approximately 8.8 and 5.2 kb in size (designated B1and B3, respectively) in addition to the 6.6-kb unit-length frag-ment (B2) (Fig. 2C, lane 1). A pair of additional bands 5.7 and4.2 kb in size (designated b4 and b5, respectively) were alsoweakly visible; these bands represent two of the terminal frag-ments of the packaged amplicon concatemers.

To add further support to the segment isomerization pred-ication in Fig. 2A, we employed two additional probes, pS1 andpS2, that were made from different regions of the amplicongenome. Fitting the predicted isomerization pattern in Fig. 2A,the results in Fig. 2C (lanes 2 and 3) showed that pS1 hybrid-izes to the unit-length 6.6-kb fragment as well as the smaller5.2-kb fragment, whereas pS2 hybridizes to the unit-lengthfragment and the larger 8.8-kb fragment. Digestion with theXhoI enzyme, which cuts within the S segment, also resulted inthe predicted pattern (shown in Fig. 2B). XhoI digestion gen-erated two fragments (B1 and B3) in addition to the unit-length fragment (B2) when the digested concatemeric DNAwas hybridized with probe pC (Fig. 2D, lane 1). Two weakerterminal fragments (b4 and b5, with sizes of 5.4 and 4.7 kb,respectively) were also visible. The disappearance of band B1

from the hybridization with the subgenomic probe pX1 (Fig.2D, lane 2) and of band B3 from the hybridization with sub-genomic probe pX2 (Fig. 2D, lane 3) confirmed the specificityof each of the bands.

To estimate the percentage of concatemers containing L andS segments in opposing orientations, the intensity of each in-dividual band was quantified by phosphorimager analysis (Im-ageQuant). The results showed that the sum of the intensitiesof B1 and B3 was only slightly less than the intensity of B2 inboth ScaI- and XhoI-digested DNA samples (data not shown,but see lanes 1 of Fig. 2C and D). Collectively, these resultsdemonstrate that during HSV-mediated pSZ-dPac-EGFP rep-lication, a high percentage (30 to 50%) of concatemers containL and S segments in opposite orientations in neighboring am-plicon genomes.

Next, we conducted experiments to determine if the gener-ation of concatemer-ISI is through homologous recombina-tion, as confirmation of this aspect would further support thehypothesis that there is a direct link between the production ofconcatemer-ISI and the generation of equimolar HSV iso-forms. To conduct this experiment, we constructed anotheramplicon plasmid, pW4.2 (Fig. 1B). In addition to a single copyof the oriS and the a sequence of HSV-1, this amplicon con-tains two copies of the ampicillin gene (ampR) arranged indifferent loci of the plasmid and in opposite orientations. Thisarrangement creates the L and S segments of this plasmid,which are bracketed by the ampicillin genes rather than therepeated sequences of HSV. The same Southern blotting strat-egy described above was used to detect the generation ofconcatemer-ISI, with the exception that the amplicon concate-mers were digested with either AlwnI, which cuts the plasmidonce at one end of the L segment, or HindIII, which cuts theplasmid once at one end of the S segment. The predictedisomerization patterns and the DNA fragments and their sizesfollowing AlwnI or HindIII digestion are shown in Fig. 3A. The

FIG. 1. Structures of pSZ-dPac-EGFP and pW4.2. Short and long segments of the plasmids are labeled S and L, respectively. The orientationsof the repeated regions of pSZ-dPac-EGFP including oriS and the a sequence (a-seq) are shown by arrowheads. The names and locations of thefour subgenomic DNA fragments used for making probes are indicated. The orientations of the two ampicillin genes (AmpR) in pW4.2 are alsolabeled. The locations of the unique restriction enzymes used to determine the segment orientation in the concatemers are indicated by individualenzyme names.

10506 NOTES J. VIROL.

on July 13, 2018 by guesthttp://jvi.asm

.org/D

ownloaded from

amplicon concatemers were extracted from stocks which hadbeen passaged either once (Fig. 3B, lanes 1 and 4) or twice(lane 2). AlwnI digestion of the concatemeric DNA generatedthree major DNA bands (B1 to B3) of approximately 10.8, 7.8,and 4.9 kb, respectively (Fig. 3B, lanes 1 and 2). The weaklyhybridizing terminal fragments (b4 and b5, of 5.3 and 5.5 kb,respectively) were also visible. HindIII digestion also gener-ated three strong bands with sizes of approximately 9.3, 7.8,and 6.4 kb (3C, lanes 1 and 2). Quantification of the bandsrepresenting concatemer-ISI (i.e., bands B1 and B3) by phos-phorimager analysis showed that the concatemer-ISI frompW4.2 occurred at a frequency similar to that from pSZ-dPac-EGFP (data not shown). The ratio remained almost un-changed during amplicon passages (compare lanes 1 and 2 inFig. 3B and C). These results indicate that the ampicillin gene

sequence can fully replace the HSV repeated sequences toachieve high-frequency homologous recombination duringHSV-1-mediated DNA replication, leading to the frequentgeneration of concatemer-ISI. These results therefore confirmthat concatemer-ISI generation occurs through homologousrecombination and that it can take place between non-HSVDNA elements.

One possible mechanism to generate concatemer-ISI duringvirus infection is through homologous recombination betweentwo different isoforms of the viral genome. This would requirethe preexistence of at least two different isoforms in the inputvirus. Alternatively, if a single isoform of HSV was used, mul-tiple rounds of viral replication would be required to generatemore than one HSV isoform. However, it is not technicallypossible to perform an experiment with a strict single-round

FIG. 2. Southern blot analysis of pSZ-dPac-EGFP concatemers. (A and B) Schematic representations of pSZ-dPac-EGFP concatemers andpossible orientations of the L (A) and S (B) segments; the orientation of each segment is indicated by an arrow. The repeated regions (oriS andthe a sequence) of each amplicon are represented by filled boxes. The ScaI and XhoI recognition sites are marked along the concatemers, and thesizes of the restriction fragments are noted. The locations of the subgenomic DNA fragments used for making probes are represented by open bars(pS1), hatched bars (pS2), crosshatched bars (pX1), and shaded bars (pX2). (C and D) Southern blot hybridization following ScaI (C) or XhoI (D)digestion. Lanes 1 to 3, digested concatemeric pSZ-dPac-EGFP DNA; lanes 4, undigested DNA; lanes 5, digested purified pSZ-dPac-EGFPplasmid DNA. The marker (lanes M) is the 1-kb ladder (Gibco-BRL). Each lane of the blot was hybridized as follows: in panels C and D, lanes1, 4, and 5 were hybridized with probe pC; in panel C, lanes 2 and 3 were hybridized with pS1 and pS2, respectively; and in panel D, lanes 2 and3 were hybridized with pX1 and pX2, respectively.

VOL. 75, 2001 NOTES 10507

on July 13, 2018 by guesthttp://jvi.asm

.org/D

ownloaded from

infection-replication because each plaque-purified virus wouldundergo many rounds of replication before a sufficiently largestock could be made for experimental characterization. In ad-dition, it is not possible to generate a virus stock containingonly a single HSV isoform, since each new virus stock containsall four possible HSV isoforms in an equimolar ratio. Since thepSZ-dPac-EGFP amplicon generates a similar pattern of con-catemer-ISI, we queried whether a strict single-round DNAamplification of a single isoform could be performed with thisamplicon. To test this, cells were transfected with pSZ-dPac-EGFP and were infected 16 h later with helper virus at 10 PFUper cell, a dose at which the majority of cells will be infected inthe first round and will therefore prevent a subsequent second-round infection. We assumed that under these experimentalconditions, the amplicon would undergo only a single round ofamplification and packaging. Concatemeric DNA extractedfrom virions released from the cells was digested with ScaI andhybridized with probe pC. In this single-step DNA replicationsetting, concatemer-ISI, which was represented by the appear-ance of B1 and B3 bands, was efficiently generated (Fig. 4, lane

1) and largely maintained during subsequent serial passages(Fig. 4, lanes 2, 3, and 4). Phosphorimager quantificationshowed that the ratio of the intensities of bands B1 and B3 tothat of B2 in lane 1 of Fig. 4 was approximately 2:3 and that theratio remained almost unchanged during serial passages of thestock (Fig. 4, lanes 2, 3, and 4). These results therefore suggestthat generation of concatemer-ISI does not require the preex-istence of more than one isomer of pSZ-dPac-EGFP.

Our earlier studies showed that homologous recombinationcould occur between a wild-type HSV genome and a mutantHSV genome that has a single SpeI recognition site deleted(23). However, the relative amount of concatemers generatedfrom recombination between mutant and wild-type HSV wasfar less than that of the concatemer-ISI generated from thewild-type virus genome alone. A possible explanation for thisdiscrepancy could be the branched nature of HSV replicativeintermediates and/or the relatively large sizes of the DNAmolecules that were studied (180 to 260 kb), which may havelimited efficient DNA transfer during Southern blotting pro-cedures. In order to measure the relative amounts of concate-

FIG. 3. Generation of concatemer-ISI is through homologous recombination. (A) Schematic representation of a pW4.2 concatemer andpossible orientations of the L (black arrows) and S segments (gray arrows) along the molecule. The repeated regions (ampicillin gene) of theconstruct are represented by filled boxes. The AlwnI and HindIII sites are marked along the concatemer, and the sizes of the restriction fragmentsare noted. (B and C) Southern blot hybridization following digestion with AlwnI (B), which cuts once within the L segment of pW4.2, or HindIII(C), which cuts once within the S segment. Concatemeric DNA was extracted from amplicon stocks that had been passaged either once (lanes 1and 4) or twice (lane 2). The DNA in lane 3 represents unit-length pW4.2 plasmid DNA. Hybridization was with a probe made from the entirepW4.2 genome but lacking the oriS and a sequence. The marker (lanes M) is the 1-kb ladder.

10508 NOTES J. VIROL.

on July 13, 2018 by guesthttp://jvi.asm

.org/D

ownloaded from

mers more accurately, we carried out an experiment similar tothe ones described above but employed two amplicons whichhad much smaller genomes than the virus. It has been reportedthat when two similar-sized plasmids carrying different markergenes were mixed at a 1:1 ratio for in vitro transfection, themajority of the transfected cells were found to express bothmarker genes (12). We therefore transfected BHK cells with aDNA mixture of two amplicons, pW7-TK and pW7-EGFP, atan equimolar ratio. Plasmid pW7-EGFP was constructed byinserting an enhanced green fluorescent protein (EGFP) genecassette [containing the cytomegalovirus promoter and bovinegrowth hormone poly(A)] into the unique SapI site of pW7-TK, such that pW7-TK has 100% homology to pW7-EGFPoutside the EGFP cassette (Fig. 5A). Sixteen hours after trans-fection with the DNA mixture, the cells were infected with thewild-type helper virus HSV strain 17. Virion DNA extractedfrom virus particles was digested with XhoI, which has a singlerecognition site located in the EGFP cassette region but doesnot cut within pW7-TK itself. The digested DNA was subjectedto gel electrophoresis and Southern blot hybridization with aradioactive probe made from the EGFP gene (this probe willidentify only pW7-EGFP but not pW7-TK unless it has recom-bined with pW7-EGFP). Frequent homologous recombinationbetween these two amplicons during HSV-mediated DNA am-plification should generate a significant number of concatem-ers containing both amplicons interspersed along a single mol-ecule, which upon digestion with XhoI will produce a 19.5-kbDNA fragment in addition to the unit-length 11.7-kb pW7-EGFP fragment. Figure 5B shows that with a normal exposuretime (lanes 1 and 2), only a single band (B2) representing theunit-length pW7-EGFP could be detected. However, after anextremely long exposure (lane 3), a band of approximately 19kb was also visible, although the intensity of this band was onlya small proportion of that of B2. The other two weakly hybrid-izing bands (b3 and b4, of approximately 6 and 14 kb, respec-tively) represent the terminal fragments of concatemers thatcontain the EGFP gene. These results are in agreement with

our previous observations (23) and indicate that random ho-mologous recombination between two different genomes doesoccur but at a frequency too low to contribute significantly tothe observed large amount of concatemer-ISI during HSV-mediated DNA replication.

Based on these observations, we conclude that the genera-tion of concatemer-ISI is a generalized phenomenon that canoccur in the absence of a complete HSV genome. Further-more, concatemer-ISI is the direct result of homologous re-combination, which does not specifically require that the ho-mologous sequence be from HSV DNA. The demonstration ofa similar pattern of ISI during amplicon replication also indi-cates that uniquely designed amplicons such as pSZ-dPac-EGFP may be useful as a simplified model for further inves-tigation into HSV replication mechanisms.

We thank Malcolm K. Brenner for continuous support and C. Mafor technical assistance.

REFERENCES

1. Bataille, D., and A. Epstein. 1994. Herpes simplex virus replicative concate-mers contain L components in inverted orientation. Virology 203:384–388.

2. Ben Porat, T., A. S. Kaplan, B. Stehn, and A. S. Rubensten. 1977. Replica-tion of herpes DNA. II. Sedimentation characteristics of newly synthesizedDNA. Virology 79:292–301.

3. Bruckner, R. C., R. E. Dutch, B. V. Zemelman, E. S. Mocarski, and I. R.Lehman. 1992. Recombination in vitro between herpes simplex virus type 1 asequences. Proc. Natl. Acad. Sci. USA 89:10950–10954.

4. Deiss, L. P., and N. Frenkel. 1986. Herpes simplex virus amplicon: cleavageof concatemeric DNA is linked to packaging and involves amplification of

FIG. 4. Generation of concatemer-ISI during a single-step ampli-con replication. The amplicon was hybridized with probe pC. Lane 1,transfection-infection; lane 2, passage 1; lane 3, passage 2; lane 4,passage 3; lane 5, unit-length pSZ-dPac-EGFP plasmid DNA; lane 6,undigested amplicon concatemer (from the passage 1 sample). Themarker (lane M) is the 1-kb ladder.

FIG. 5. Homologous recombination between different amplicons isa rare event. (A) Schematic representations of pW7-TK and pW7-EGFP. (B) Southern blotting results. Following a mixed transfection ofpW7-TK and pW7-EGFP and subsequent helper HSV infection, con-catemeric DNA was extracted, digested with XhoI, and hybridized witha probe made from the gene encoding EGFP. The DNA marker (laneM) is �-HindIII (Gibco-BRL). Lanes 1 to 3 show different exposures ofthe same Southern blot: lane 1, short exposure; lane 2, medium expo-sure; lane 3, long exposure.

VOL. 75, 2001 NOTES 10509

on July 13, 2018 by guesthttp://jvi.asm

.org/D

ownloaded from

the terminally reiterated a sequence. J. Virol. 57:933–941.5. Delius, H., and J. B. Clements. 1976. A partial denaturation map of herpes

simplex virus type 1 DNA: evidence for inversions of the unique DNAregions. J. Gen. Virol. 33:125–133.

6. Dutch, R. E., R. C. Bruckner, E. S. Mocarski, and I. R. Lehman. 1992.Herpes simplex virus type 1 recombination: role of DNA replication andviral a sequences. J. Virol. 66:277–285.

7. Dutch, R. E., B. V. Zemelman, and I. R. Lehman. 1994. Herpes simplex virustype 1 recombination: the Uc-DR1 region is required for high-level a-se-quence-mediated recombination. J. Virol. 68:3733–3741.

8. Fraefel, C., D. R. Jacoby, and X. O. Breakefield. 2000. Herpes simplex virustype 1-based amplicon vector systems. Adv. Virus Res. 55:425–451.

9. Frenkel, N. 1981. Defective interfering herpesviruses, p. 91–120. In A. J.Nahmias, W. R. Dowdle, and R. S. Schinazy (ed.), The human herpesvi-ruses—an interdisciplinary perspective. Elsevier-North Holland Inc., NewYork, N.Y.

10. Garber, D. A., S. M. Beverley, and D. M. Coen. 1993. Demonstration ofcircularization of herpes simplex virus DNA following infection using pulsedfield gel electrophoresis. Virology 197:459–462.

11. Hayward, G. S., N. Frenkel, and B. Roizman. 1975. Anatomy of herpessimplex virus DNA: strain differences and heterogeneity in the locations ofrestriction endonuclease cleavage sites. Proc. Natl. Acad. Sci. USA 72:1768–1772.

12. Horbinski, C., M. K. Stachowiak, D. Higgins, and S. G. Finnegan. 2001.Polyethyleneimine-mediated transfection of cultured postmitotic neuronsfrom rat sympathetic ganglia and adult human retina. BMC Neurosci. 2:2.

13. Jacob, R. J., L. S. Morse, and B. Roizman. 1979. Anatomy of herpes simplexvirus DNA. XIII. Accumulation of head to tail concatemers in nuclei ofinfected cells and their role in the generation of the four isomeric arrange-ments of viral DNA. J. Virol. 29:448–457.

14. Jacob, R. J., and B. Roizman. 1977. Anatomy of herpes simplex virus DNA.VIII. Properties of the replicating DNA. J. Virol. 23:394–411.

15. McVoy, M. A., and S. P. Adler. 1994. Human cytomegalovirus DNA repli-cates after early circularization by concatemer formation, and inversionoccurs within the concatemer. J. Virol. 68:1040–1051.

16. Mocarski, E. S., L. E. Post, and B. Roizman. 1980. Molecular engineering ofthe herpes simplex virus genome: insertion of a second L-S junction into thegenome causes additional genome inversions. Cell 22:243–255.

17. Mocarski, E. S., and B. Roizman. 1981. Site-specific inversion sequence of

the herpes simplex virus genome: domain and structural features. Proc. Natl.Acad. Sci. USA 78:7047–7051.

18. Mocarski, E. S., and B. Roizman. 1982. Herpesvirus-dependent amplifica-tion and inversion of cell-associated viral thymidine kinase gene flanked byviral a sequences and linked to an origin of viral DNA replication. Proc. Natl.Acad. Sci. USA 79:5626–5630.

19. Poffenberger, K. L., and B. Roizman. 1985. Studies on non-inverting genomeof a viable herpes simplex virus 1. Presence of head to tail linkages inpackaged genomes and requirements for circularization after infection. J. Vi-rol. 53:589–595.

20. Severini, A., A. R. Morgan, D. R. Tovell, and D. L. Tyrrell. 1994. Study of thestructure of replicative intermediates of HSV-1 DNA by pulsed-field gelelectrophoresis. Virology 200:428–435.

21. Skaliter, R., and I. R. Lehman. 1994. Rolling circle DNA replication in vitroby a complex of herpes simplex virus type 1-encoded enzymes. Proc. Natl.Acad. Sci. USA 91:10665–10669.

22. Skaliter, R., A. M. Makhov, J. D. Griffith, and I. R. Lehman. 1996. Rolling-circle DNA replication by extracts of herpes simplex virus type 1-infectedhuman cells. J. Virol. 70:1132–1136.

23. Slobedman, B., X. Zhang, and A. Simmons. 1999. Herpes simplex virusgenome isomerization: origins of adjacent long segments in concatemericviral DNA. J. Virol. 73:810–813.

24. Smiley, J. R., B. S. Fong, and W. C. Leung. 1981. Construction of a double-jointed herpes simplex viral DNA molecule: inverted repeats are requiredfor segment inversion, and direct repeats promote deletions. Virology 113:345–362.

25. Varmuza, S. L., and J. R. Smiley. 1985. Signals for site-specific cleavage ofHSV DNA: maturation involves two separate cleavage events at sites distalto the recognition sequences. Cell 41:793–802.

26. Vlazny, D., A. Kwong, and N. Frenkel. 1982. Site-specific cleavage/packagingof herpes simplex virus DNA and the selective maturation of nucleocapsidscontaining full length viral DNA. Proc. Natl. Acad. Sci. USA 79:1423–1427.

27. Zhang, X., S. Efstathiou, and A. Simmons. 1994. Identification of novelherpes simplex virus replicative intermediates by field inversion gel electro-phoresis: implications for viral DNA amplification strategies. Virology 202:530–539.

28. Zhang, X., H. O’Shea, C. Entwisle, M. Boursnell, S. Efstathiou, and S.Inglis. 1998. An efficient selection system for packaging herpes simplex virusamplicons. J. Gen Virol. 79:125–131.

10510 NOTES J. VIROL.

on July 13, 2018 by guesthttp://jvi.asm

.org/D

ownloaded from