Proc. Nati. Acad. Sci. USAVol. 82, pp. 694-698, February 1985Biochemistry

The herpes simplex virus amplicon: Analyses of cis-actingreplication functions

(oriin/vectors/defective genomes/cdeavage/Dackaings)

RICHARD R. SPAETE AND NIZA FRENKELDepartment of Molecular Genetics and Cell Biology, The University of Chicago, 910 East 58th Street, Chicago, IL 60637

Communicated by Bernard Roizman, September 26, 1984

ABSTRACT Previous studies have shown that defectivevirus rectors (amplicons) derived from herpes simplex virusescould be efficiently propagated in virus stocks in the presenceof trans-acting helper virus functions. The present study estab-lished that two separate cis-acting functions-a DNA replica-tion origin and a cleavage/packaging signal-are required foramplicon propagation. Using deleted derivatives of cloned am-plicons, we mapped one of the viral DNA replication origins(ori-2 or OriL) at coordinate 0.422 of the standard HSV-1genome and at an equivalent position within the HSV-2genome.

Herpes simplex viruses 1 and 2 (HSV-1 and HSV-2) containlinear, double-stranded DNA molecules -150 kilobases (kb)in size. The viral genome consists of two components, L andS, bounded by inverted repeats-the ab and b'a' sequencesbounding L, and the a'c' and ca sequences bounding S. TheL and S components invert relative to each other (reviewedin ref. 1).

Serial, undiluted propagation of standard HSV was shownto result in the accumulation of two classes of defective virusDNA molecules, composed of head-to-tail reiterations oflimited DNA sequences (reviewed in ref. 2). Repeat units ofthe class I defective HSV genomes contained sequencesfrom the S component of standard HSV DNA, whereas re-peat units of the class II defective genomes contained se-quences from the middle of the L component linked to the acinverted repeat sequences. The two classes thus shared theac sequences but differed with respect to the remaining re-peat unit sequences. On the basis of these structural fea-tures, it was suggested (reviewed in ref. 2) that two separatecis-acting functions were required for viral DNA propaga-tion. The first constituted a cleavage/packaging signal resid-ing within the ac sequences. The second corresponded to areplication origin-ori-i (oris), residing in S and initiating thereplication of the class I defective genomes, and ori-2 (oriL),residing in L and initiating the replication of class II defec-tive HSV genomes.

It has been shown (3-6) that cotransfection of cells withhelper virus DNA and monomeric repeat units of defectivegenomes (amplicons) resulted in the generation of packaged,full-sized DNA molecules consisting of head-to-tail reitera-tions of the seed amplicon sequences. This transfection ap-proach provided a functional assay for the fine mapping ofcis-acting functions required for the propagation of viralDNA. In the present study we show that two separate cis-acting functions (a cleavage/packaging signal and a replica-tion origin) were required for amplicon propagation in virusstocks. Furthermore, using the amplicon propagation assay,we have finely mapped the replication origin contained inclass II defective genomes (ori-2 or oriL). In an independent

study, Stow et al. (7) have reported recently that two cis-acting functions were required for the propagation of ori-I(oris)-containing amplicons.A preliminary report of the studies described in this paper

has been presented.t

MATERIALS AND METHODSCells and Viruses: DNA Transfections and Derivation of Vi-

rus Series. The sources of cells and viruses, the proceduresfor preparation of labeled infected cell and bacterial plasmidDNAs, and the procedures for restriction enzyme and blothybridization analyses have been described (4). Rabbit skincells were cotransfected with helper virus DNA and test am-plicons, and transfection-derived virus stocks were seriallypropagated in HEp-2 cells at 1:4 dilutions. Amplicons andtransfection-derived virus stocks were designated by nomen-clature described earlier (4).

Construction of the pac-2, pP2-111, p-112, and pP2-204Plasmids. The pac-2 clone was derived by cloning a 1275-base-pair (bp) fragment spanning the S-L junction (bac) ofHSV-1 (F) DNA into the EcoRI site of pKC7. The 1275-bpfragment originally was cloned and sequenced by Mocarskiand Roizman (8) and was obtained from them. The plasmidpP2-111 was derived by inserting the BamHI V fragment (ob-tained from defective genomes propagated from pP2-102)into the Bgl II site of pac-2. pP2-204 and p-112 were con-structed by (i) insertion of a Kpn I linker (Chiron, Emery-ville, CA) into the Pvu II site ofpBR322 and by (it) ligation ofthe Kpn I-Pst I fragment spanning nucleotides 2065-3608 ofthe Kpn I/pBR322 derivative to the Pst I-Kpn I fragmentsderived from pP2-111 and from a similar plasmid containingthe BamHI V fragment in the reverse orientation. These fu-sions generated pP2-204 and p-112, respectively.

RESULTSTwo Separate Regions Are Required for the Propagation of

Class II Amplicons. As previously described (4), the ampli-con pP2-102 was derived by cloning a 3.9-kb repeat unit ofHSV-1 (Patton) class II defective genomes into the Bgl II siteof pKC7. The majority of the HSV DNA sequences in pP2-102 corresponded to the UL sequences within map coordi-nates 0.407-0.429 of standard HSV DNA. A stretch of a andadjacent c sequences resided within the Bgl II-Xho I frag-ment denoted by ac* in Fig. 1. Bacterial cloning of the 3.9-kbrepeat unit resulted in small deletions ('100 bp in pP2-102)within the Kpn I-BamHI segment shown as the stippled areain Fig. 1. pP2-201 contained the same (-3.8 kb) HSV DNAinsert in the reverse orientation. The cotransfection of cellswith helper virus DNA and the pP2-102 or pP2-201 ampli-

Abbreviations: HSV, herpes simplex virus; bp, base pair(s); kb, ki-lobase(s).tSpaete, R. R., Deiss, L. P. & Frenkel, N., Sixth Cold Spring Har-bor Meeting on Herpesviruses, Aug. 31-Sept. 5, 1982, Cold SpringHarbor, NY, p. 229.

694

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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FIG. 1. Structure of pP2-102, pP2-201, and derivative con-

structs. pP2-102 and pP2-201 were derived by inserting a 3.9-kb re-

peat unit of HSV-1 (Patton) defective genomes into the Bgl II site ofpKC7 (4). The HSV DNA sequences are denoted by the double-lineportions of each construct. The Cla I site shown in parentheses ismethylated in plasmid DNA propagated in dam' bacteria. pP2-111,p-112, and pP2-204 were derived as described. pP2-104, p-106, andp-108 were derived after partial Xho I digestion of pP2-102. p-203was derived after a complete digestion of pP2-201 with Xho I.

Shown are locations of sites for Bgl II (Bg), BamHI (Ba), Xho I (X),Kpn I (K), HindIll (H), Cla I (Cl), Sal I (S), and EcoRI (E). Bg/Badenotes a fusion of the Bgl II and BamH1 sites. K(P) denotes inser-tion of a Kpn I linker into the Pvu II site.

cons typically resulted in generation of full-sized concate-meric defective genomes, which could be propagated furtherin derivative virus stocks (4).The Xho I sites in pP2-102 define three segments (denoted

A, B, and C in Fig. 1) within the HSV DNA Bgl II insert. Toidentify the DNA sequences required for defective genomepropagation, we tested the ability of plasmids containing thesegments A, C, A + C, and A + B (Fig. 1) to become propa-gated in transfection-derived virus stocks. Replicate sets ofrabbit skin cell cultures were cotransfected with helper virusDNA, and each of the test plasmids and the resultant virusstocks were serially propagated. Restriction enzyme digestsof 32P-labeled DNAs prepared from cells infected with thethird passages (P3) of representative series are shown in Fig.2. Also shown are the hybridization patterns of a 32P-labeledpKC7 DNA probe to a blot containing Bgl II digests of theinfected cell DNAs. The results of these and additional anal-yses (data not shown) revealed that sequences containedwithin the Xho I A and C segments were simultaneously re-

quired for defective genome propagation. Thus, test plas-mids containing all three Xho I segments (A + B + C) or thecombination of segments A + C were efficiently propagated,whereas plasmids containing segment A, segment C, or seg-ments A + B were not authentically propagated in the trans-fection-derived virus stocks.DNA prepared from the series derived from a cotransfec-

tion receiving HSV-1 (Justin) helper DNA and the p-106 testplasmid (termed as the J-106 series, Fig. 2, lanes 4 and 12)contained a 3.8-kb Bgi II species that hybridized the pKC7probe. However, this species differed in size from the 6.1-kbseed p-106 plasmid. To further analyze its structure, the 3.8-

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FIG. 2. Tests for propagation of the Xho I derivative clones intransfection-derived virus stocks. (Left) Bgl 11-digested 32P-labeledDNAs from cells infected with HSV-1 (Justin) standard virus (lane1) or from cells infected with the third passages of the series shown(lanes 2-6). Each stock in the series was derived from a cotransfec-tion of 25-cm2 rabbit skin cells with 0.5 Mg of helper virus DNA and0.1 pg of test plasmid. The Xho I segments present in the test plas-mids are summarized above the figure. (Center) Hybridization of a32P-labeled pKC7 probe to blots containing BgI 11-digested test plas-mid DNA (sublanes p in lanes 7-16) or DNA from cells infected withpassage 3 of the transfection-derived virus (sublanes V). (Right) Hy-bridization of 32P-labeled HSV-1 (Justin) DNA (lane 17) or the 3.8-kb Bgl II fragment from passage 5 of J-106 virus (lane 18) to blotscontaining the separated Kpn I fragments of standard HSV-1 (Justin)DNA. The map shows the arrangement of the relevant Kpn I frag-ments (exhibiting homology to the 3.8-kb species) within the stan-dard HSV genome. The probe also hybridized to partially digestedbands as indicated next to lane 18.

kb DNA fragment was eluted from an agarose gel, labeled invitro with [a-32P]dCTP, and used to probe nitrocelluloseblots containing the Kpn I fragments of standard HSV-1(Justin) DNA. This hybridization (Fig. 2, lanes 17 and 18)revealed that additional sequences arising from the Kpn Ifragments V, A', and X of the helper virus DNA were incor-porated into the defective genome repeat units. These se-quences included the sequences present in the Xho I C seg-ment of pP2-102, as well as adjacent helper virus DNA se-quences that were not present in the input p-106 clone. Thus,the p-106 test plasmid that contained the Xho I segments A +B was propagated in virus stocks only after incorporation ofC segment sequences, most likely by recombination withhelper virus DNA. This observation further supports theconclusion that cis-acting functions residing in segments Aand C were simultaneously required for the successful prop-agation of the class II HSV amplicon.The UL Sequences of pP2-102 Contain a Replication Origin.

The p-203 plasmid contained the C segment sequences re-quired for amplicon propagation but lacked the required Asegment sequences. As described above, p-203 could not bepropagated in virus stocks. To test whether p-203 was never-theless replicated within the transfected cells (i.e., containeda replication origin), a rabbit skin cell culture was cotrans-fected with the p-203 plasmid and HSV-1 (Justin) helper vi-rus DNA. Total cell DNA prepared from the transfected cul-ture at 4 days post-transfection was digested with restrictionenzymes. The resultant fragments were transferred to nitro-cellulose blots and hybridized with 32P-labeled pKC7 DNA.

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The restriction enzymes Dpn I and Cla I were chosen forthese digests so as to permit the distinction between replicat-ed and unreplicated input plasmid DNA (9). Specifically, theDpn I enzyme recognizes only the methylated mG-A-T-C se-quence and, therefore, cleaves input plasmid DNA propagat-ed in dam' bacteria (which methylate the site). However,Dpn I does not cleave DNA molecules that have been repli-cated in animal cells (lacking the ability to methylate thesite). Furthermore, the Cla I enzyme recognizes only the un-methylated sequence A-T-C-G-A-T but fails to cleave itsmethylated (A-T-C-mG-A-T) counterpart. One of the twoCla I sites of pP2-102 [denoted as (Cl) in Fig. 1] was found tobe cleaved by Cla I only after propagation in animal cells orin dam- bacteria but not after the propagation of pP2-102 indam' bacteria (Fig. 3, lanes 1-4). Based on these consider-ations, DNA extracted from the transfected cells was doublycleaved with (i) Dpn IlBgl II, (ii) Dpn I/Cla I, and (iii) Dpn Iwith limiting amounts of Cla I. Input p-203 plasmid controlsprepared from dam' bacteria also were included in thesetests.The results of these analyses (Fig. 3) can be summarized

as follows: (i) As expected, the cleavage of input p-203 withDpn I (lane 5) yielded small fragments. (ii) Digestion of the J-203 DNA with Dpn I/Bgl II (lane 7) yielded the monomericfragment, showing that substantial proportions of the J-203DNA sequences homologous to the pKC7 probe were resist-ant to Dpn I and, thus, arose by replication in the animalcells. As with all clones carrying segment C, the J-203 repeatunits migrated in the gel somewhat slower than their p-203plasmid progenitors due to the apparent repair of the smallpP2-102 deletion, resulting in the increase in the repeat unitsize from 6.8 to 6.9 kb (unpublished data). (iii) Digestion ofthe p-203 plasmid with Cla I (lane 6) yielded a 6.8-kb frag-ment corresponding to the p-203 monomer. (iv) The diges-tion of J-203 DNA with the Dpn I/Cla I enzymes (lane 8)yielded two fragments (4.5 and 2.4 kb) due to the "unmask-ing" of the methylated Cla I site after replication in animalcells. Furthermore, a series of high molecular weight bands,homologous to the pKC7 probe, were apparent in the Dpn

pP2- 102Clal

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I/Cla I-partial digest (lane 9), revealing the concatemericstructure of the p-203 progeny DNA.Thus, the results of these experiments showed that the p-

203 plasmid was capable of replication in the presence ofhelper virus DNA, yielding high molecular weight DNA mol-ecules that were devoid of dam-type (G-A-T-C) methylation.Therefore, we conclude that the Xho I C segment of pP2-102contained a replication origin. Moreover, because as shownabove no progeny-defective genomes were observed in virusstocks propagated from the J-203 transfections, it could beconcluded that the cis-acting function residing in segment Aof the pP2-102 amplicon (and absent from p-203) was re-quired for the propagation of the replicated viral DNA frompassage to passage-a conclusion consistent with the sug-gested role of this sequence during concatemeric cleavageand packaging of viral DNA (2, 7, 10).

Further Mapping of the Set of Sequences Required for Am-plicon Propagation. To further map the replication origin re-siding within pP2-102, three additional plasmids were testedfor the ability to become propagated in transfection-derivedvirus stocks. All three plasmids contained as a source for thecleavage/packaging signal a 1275-bp fragment cloned by Mo-carski and Roizman (8) from an S-L junction (bac) of stan-dard HSV-1 (F) DNA. In addition, the test plasmid pP2-111contained the 2.2-kb BamHI fragment corresponding to theBamHI V fragment of standard virus DNA. The plasmidpP2-204 contained the 660-bp right-hand BamHI-Kpn I por-tion of the cloned BamHI V fragment, whereas the plasmidp-112 contained the remaining 1500 bp. Both pP2-111 and

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FIG. 3. Tests for replication of the p-203 plasmid. (Left) Ethidi-um bromide-stained gels (lanes 1 and 2) containing Cla I-digestedpP2-102 DNA propagated in dam' and dam- bacteria. (Center)Autoradiograms of Cla I digests of 32P-labeled pP2-102 ampliconpropagated in dam' bacteria (p) (lane 3) and of "P-labeled DNAfrom cells infected with the JP2-102 series (V) (lane 4). Note the"unmasking" of the second Cla I site in the amplicon sequencesfollowing propagation in animal cells. (Right) Autoradiograms ofblots that were probed with 3"P-labeled pKC7 DNA. The blots con-tained Dpn I (lane 5)- and Cla I (lane 6)-digested p-203 plasmid fromdam' bacteria or digested fragments of DNA prepared from a trans-fected culture (J-203) receiving 0.5 ,g of helper and 0.1 ug of p-203DNA. Digestion was with Dpn I/Bgl II (lane 7), Dpn I/Cla I (lane 8),and Dpn I with a limited amount of Cla I (lane 9). Both short andlong autoradiographic exposures of lanes 8 and 9 are shown.

FIG. 4. Further localization of the ori-2 sequences. (Left) Sche-matic representation of standard virus DNA, the 3.9-kb Patton re-peat unit, and the UL sequences present in the test constructs pP2-104, pP2-111, p-112, and pP2-204. Plus and minus denote the abilityor lack of ability of each construct to become propagated in transfec-tion-derived virus stocks. The dotted area denotes the BamHI-KpnI segment, which contains ori-2. (Right) Restriction enzyme patternsof 32P-labeled test plasmid DNA (lanes p), DNA from cells infectedwith standard Justin virus (lanes J), and DNA from cells infectedwith the third passage of the corresponding derivative series (lanesV). pac denotes a series propagated from cells cotransfected withhelper virus DNA and the pac-2 plasmid containing the cleavage/packaging signal. All series were derived from transfections receiv-ing 0.5 ug of HSV-1 (Justin) helper virus DNA and 0.1 ,g of plasmidDNA. Note that pP2-204 does not contain Xho I sites (lane 9, bandR) and, therefore, when propagated in the JP2-204 series, yields con-catameric defective genomes that are resistant to Xho I digestion(lanes 10, band R). The p-112 construct contains a single Xho I siteand, therefore, could be expected to generate defective genomesthat would yield a 5.4-kb fragment upon Xho I digestion. Note theabsence of this band in the digested J-112 DNA (lane 11).

I

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Proc. NatL Acad Sci. USA 82 (1985) 697

Baml~ v-- ;-BgIIlJ JT2ITT2 i iT2 TT220111021 201 102

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O fragment of standard HSV-2 (333) DNA. (Left) Schematic dia-gram depicting the structure of the pT2-102 and pT2-201 amplicons,which were constructed by insertion of the Bgl II 0 fragment ofHSV-2 strain 333 DNA into pac-2 in the two possible orientations.(Right) BamHI and BgI II digestions of 32P-labeled DNA preparedfrom cells infected with the third passages of the series are shown.Numbers denote fragment sizes (kb). To conform with our nomen-clature for amplicons and derivative virus stocks (4), we have used Tas the initial letter designating the strain 333. Accordingly, JT2-201aand JT2-201b represent series derived from two duplicate transfec-tions receiving HSV-1 (Justin) helper virus DNA and the pT2-201amplicon, whereas TT2-102a is a series derived from a transfectionreceiving HSV-2 (333) helper DNA and the pT2-102 amplicon. J rep-resents DNA from cells infected with plaque-purified HSV-1 (Jus-tin).

pP2-204 could be efficiently propagated in the transfection-derived virus stocks (Fig. 4). In contrast, p-112 failed to yielddetectable progeny defective virus genomes. These resultsmap the UL replication origin of HSV-1 DNA (ori-2) withinthe 660-bp segment delimited by the Kpn I and BamHI sitesof the pP2-102 amplicon, or the corresponding map coordi-nates 0.418-0.423 of standard HSV DNA.The Mapping of oni-2 of HSV-2 DNA. On the assumption

that the replication origins of HSV-1 and HSV-2 DNAsmight be colinear, we cloned the Bgl II 0 fragment spanningcoordinates 0.408-0.440 of HSV-2 (333) DNA into the plas-mid pac-2, containing the cleavage/packaging bac sequence.The resultant clones (differing in orientation of the Bgl II 0

insert) were designated pT2-102 and pT2-201. As with thecloning of the HSV-1 origin region, the bacterial cloning ofthe HSV-2 Bgl II 0 fragment resulted in small deletions(mapped within the Sal I fragment spanning positions 4.35-6.03 of pT2-102; see Fig. 5). The two clones were tested fortheir ability to serve as seed amplicons during cotransfec-tions with HSV-1 (Justin) and HSV-2 (333) helper virusDNAs. Virus stocks derived from these transfections con-

tained large quantities of derivative defective genomes (Fig.5). Thus, HSV-2 DNA contained a replication origin withinthe Bgl II 0 fragment. Furthermore, based on the location ofdeletion-prone sequences, the L replication origin of HSV-2strain 333 most likely resided within the 1670-bp Sal I frag-ment spanning coordinates 4.35-6.03 of the pT2-102 ampli-con.

DISCUSSION

The presence of several replication origins within HSV DNAwas first suggested by Friedmann et al. (11) on the basis ofelectron microscopy studies of replicating viral DNA mole-cules. The exact location of these functional replication ori-gins has been established further in studies designed to testthe ability of defined HSV DNA sequences to become prop-agated in virus stocks in the presence of helper virus. Thus,our own studies using class I defective virus genomes (3) andthe more recent fine mapping studies by Stow and McMona-gle (12) and Mocarski and Roizman (13) using cloned seg-ments of standard virus DNA have mapped two identicalreplication origins within the inverted repeat sequences ofthe S component of standard virus DNA. The experimentsdescribed in this communication have localized the UL ori-gin, initiating the replication of the class II defective ge-nomes, to a 760-bp fragment extending from the Kpn I site atcoordinate 0.417 to the BamHI site at coordinate 0.423 (atthe right-hand portion of BamHI V fragment) of standardHSV DNA.The origin sequences within this fragment can be further

defined by considering the location of the sequences that areuniformly deleted upon bacterial cloning. Specifically, se-quencing analyses of pP2-102 (unpublished results) have re-vealed that the right hand boundary of the pP2-102 deletionis located 120 bp from the BamHI site. Because defectivegenomes arising from replication of pP2-102 and derivativeplasmids invariably restored the deleted sequences (this pa-per, ref. 14, and unpublished data), it is most reasonable tosuggest that the deletion-prone sequences reside within thereplication origin itself. Hence ori-2 of HSV-1 could bemapped within the BamHI V segment, approximately 100 bpto the left of the BamHI site, coordinate 0.422 of HSV-1DNA. In line with this conclusion are recent studies by Grayand Kaerner (15) and by Weller et al. ,: documenting thepresence of large palindromic sequences within this regionof standard HSV-1 DNA.That HSV-2 DNA contains a similar distribution of two

replication origins in the inverted repeat sequences of the Scomponent has been suggested on the basis of the findingthat HSV-2 class I defective genomes consist of repeat unitssimilar to their HSV-1 counterparts (2). Although (to the bestof our knowledge) class II defective genomes have not beendocumented in serially passaged HSV-2 stocks, the presentset of data clearly shows that HSV-2 DNA contains a thirdreplication origin in the L component, at an equivalent posi-tion to ori-2 of HSV-1 DNA.The other set of sequences required for the successful

propagation of the recombinant amplicons in virions wasshown to reside in the Xho I A segment containing the acsequences. As elaborated elsewhere (ref. 14; unpublisheddata), the signal for cleavage of concatameric DNA and thepackaging of viral DNA into nucleocapsids resided in its en-tirety within the sequence a. The ability of both HSV-1 (Jus-tin) and HSV-2 (333) to support the replication and propaga-tion of pT2-102 containing the S-L junction of HSV-1showed that the cleavage/packaging signals residing withinthe a sequences of HSV-1 and HSV-2 DNAs were function-ally conserved.

Finally, we have presented in this paper the results of anassay designed to test for the presence of a replication originwithin a given set of viral DNA sequences. This assay,which was modeled after earlier experiments in the simianvirus 40 system (e.g., ref. 9), used the lack of significant dammethylation in animal cells to distinguish input unreplicated

tWeller, S., Spadiro, A., Murray, A. & Schaffer, P., Ninth Interna-tional Herpesvirus Workshop, Aug. 24-29, 1984, Seattle, WA, p.103.

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plasmid DNA from DNA that had undergone replication af-ter transfection. The choice to use the Dpn I and Cla I meth-ylation assays was based upon initial control experiments in-volving the pKC7 bacterial plasmid (devoid of HSV DNAsequences). In these experiments, substantial amounts ofpKC7 plasmid DNA remained in the transfected cells 4 dayspost-transfection as seen from blot-hybridization tests (datanot shown). The residual DNA was similar to the input plas-mid DNA inasmuch as it was sensitive to Dpn I and exhibit-ed similar mobility in agarose gels. The use of methylation-specific enzymes in our tests has provided an adequate solu-tion to the input plasmid background problem and extendedthe previously established observations concerning the lackof dam methylation to the HSV-infected cell.

We thank Ms. Glynis McCray and Ms. Malera Traylor for theirskillful technical help, Drs. E. Mocarski and B. Roizman for theplasmid pRB373, and Chiron Corporation for their gift of the Kpn Ilinker. These studies were supported by U.S. Public Health ServiceResearch Grants AI-15488 and CA 19264 from the National CancerInstitute and by National Science Foundation Grant PCM 8118303.R.R.S. was a predoctoral fellow supported by Public Health ServiceTraining Grant CA 09241.

1. Roizman, B. (1979) Cell 16, 481-494.2. Frenkel, N. (1981) in The Human Herpesviruses: An Interdis-

ciplinary Perspective, eds. Nahmias, A. J., Dowdle, W. R. &Schinazy, R. S. (Elsevier/North-Holland, New York), pp. 91-120.

3. Vlazny, D. A. & Frenkel, N. (1981) Proc. Natl. Acad. Sci.USA 78, 742-746.

4. Spaete, R. R. & Frenkel, N. (1982) Cell 30, 295-304.5. Stow, N. D. (1982) EMBO J. 1, 863-867.6. Barnett, J. W., Eppstein, D. A. & Chan, H. W. (1983) J. Virol.

48, 384-395.7. Stow, N. D., McMonagle, E. C. & Davison, A. J. (1983) Nu-

cleic Acids Res. 11, 8205-8220.8. Mocarski, E. S. & Roizman, B. (1981) Proc. Natl. Acad. Sci.

USA 78, 7047-7051.9. Bergsma, D. J., Olive, D. M., Harzell, S. W. & Subramanian,

K. N. (1982) Proc. Natl. Acad. Sci. USA 79, 381-385.10. Vlazny, D. A., Kwong, A. & Frenkel, N. (1982) Proc. Natl.

Acad. Sci. USA 79, 1423-1427.11. Friedmann, A., Schlomai, J. & Becker, Y. (1977) J. Gen.

Virol. 34, 507-522.12. Stow, N. D. & McMonagle, E. C. (1983) Virology 130, 427-

438.13. Mocarski, E. S. & Roizman, B. (1982) Proc. Nail. Acad. Sci.

USA 79, 5626-5630.14. Frenkel, N., Deiss, L. P. & Spaete, R. R. (1984) in UCLA

Symposia on Molecular and Cellular Biology, New Series, ed.Rapp, F. (Liss, New York), Vol. 21, 289-299.

15. Gray, C. P. & Kaerner, H. C. (1985) J. Gen. Virol., in press.

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