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The cleavage recognition signal is contained within sequences surrounding an a-a junction in herpes simplex virus DNA
VIROLOGY
167,25-30
(1988)
The Cleavage Recognition Signal Is Contained within Sequences Surrounding an a-a Junction in Herpes Simplex Virus DNA M. NASSERI Department
of Microbiology
and Immunology, Received
AND
Stanford
E. S. MOCARSKI’ University
April 25, 1988; accepted
School
of Medicine,
Stanford,
California
94305
July 11, 1988
Herpesvirus genome maturation involves site-specific cleavage of viral DNA concatemers and encapsidation of unitlength molecules, processes that are apparently coupled. Here, applying a transfection-infection approach, we have investigated the arrangement of the DNA sequence elements involved in cleavage and shown that specific cleavage occurs independently of DNA replication. We show that the &-acting signal for cleavage is located within a 179-bp fragment from across an a-a junction formed as part of the genome maturation process of herpes simplex virus 1. Plasmids carrying the 179-bp fragment are cleaved at the appropriate site even though they are unable to replicate in HSV-infected cells. When linked to an origin, the same 179-bp a-a fragment will replicate and package into progeny virus as a defective genome. Two highly conserved homologies, pacl andpac2, that have been observed in all herpesviruses examined, including cytomegalovirus, Epstein-Barr virus, varicella-zoster virus, and herpes simplex virus 2 as well as the herpes simplex virus 1 genome, are contained within the 179-bp fragment. This suggests that a common 0 1988 Academic PESS. inc. mechanism is utilized for genome maturation in the herpesvirus group.
Smiley, 1985; Deiss and Frenkel, 1986) and genome maturation (Vlazny and Frenkel, 1981; Vlazny et al., 1982; Spaete and Frenkel, 1982; Varmuza and Smiley, 1985; Deiss and Frenkel, 1986; Deiss et al., 1986). In different strains of HSV-1, the a sequence ranges in size from 220 to 500 bp but retains considerable sequence identity, including two quasi-unique elements (U, and U,) and several direct repeats (DR). The a sequence of HSV-1 (F) can be depicted DRl-U,--DR2,DR4,-U,--DRl (Fig. 1) (Mocarski and Roizman, 1982). DRl is a 20-bp repeat that is shared between adjacent copies of the a sequence when it is present in tandem arrays and is the sequence within which cleavage occurs to generate genomic termini (Mocarski and Roizman, 1982). Three observations have suggested that the process of genome maturation in HSV-1 is complex. (i) The cleavage site is within DRl; however, this sequence is not required for genome maturation (Varmuza and Smiley, 1985; Mocarski et a/., 1985). (ii) Sequences within both the&, and U, elements appear to be critical for the cleavage/packaging process (Varmuza and Smiley, 1985; Deiss and Frenkel, 1986; Deiss eta/., 1986). (iii) Single a sequences predominate in replicating concatemeric DNA, although mature progeny molecules have at least one a sequence at each end (Locker and Frenkel, 1979; Mocarski and Roizman, 1981). This situation has led to speculation that there are two separate cleavage reactions (Varmuza and Smiley, 1985) or that a transpositional event obligatorily duplicates the a sequence during cleavage (Deiss and Frenkel, 1986).
INTRODUCTION Herpesviruses are large, enveloped viruses with double-stranded linear DNA genomes ranging in size from 120 to 240 kilobase pairs (kbp) (reviewed by Roizman and Batterson, 1985). Different members of the family vary widely in the occurrence of direct and inverted repeats within the viral genome and in the process of inversion that leads multiple sequence isomers of theviral genome. The herpes simplexvirus (HSV) genome is a 150-kbp molecule composed of two covalently linked components, L (long) and S (short), each consisting of unique sequences (U, and U,) bracketed by inverted repeats such that the genome structure may be depicted ab-UL--b’a’c’-Us-ca (Fig. 1). The a sequence is present as a direct repeat at the genomic termini and as an inverted repeat (a’) at the L-S junction (Mocarski and Roizman, 1981; Davison and Wilkie, 198 1; Mocarski and Roizman, 1982). The HSV genome undergoes inversion, mediated by the a sequence (Mocarski and Roizman, 1982) such that virion DNA consists of four sequence isomers differing only in their relative orientation (Roizman, 1979). The a sequence is present in a single copy at the S terminus of the viral genome and in one to several tandem copies at the L terminus and the L-S junction (Mocarski and Roizman, 1982). This sequence carries signals for activities besides inversion, including transcription (Chou and Roizman, 1986) localized self-amplification (Varmuza and ’ To whom
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for reprints
should
be addressed. 25
0042-6822188
$3.00
Copyright Q 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.
26
NASSERI AND MOCARSKI
Given the evidence that the genomic termini contain single-base 3’ extensions that can be generated only by cleavage and, when aligned, the termini re-form a complete DRl (Mocarski and Roizman, 1982) the last step in the maturation process would be predicted to be a specific cleavage between adjacent a sequences no matter what intermediate steps are needed to create the adjacent a sequences. The genomes of all herpesviruses carry signals located near the genomic termini that are required in cis for packaging progeny DNA into capsids (Vlazny and Frenkel, 1981; Spaete and Frenkel, 1982, 1985; Stow et al., 1983; Spaete and Mocarski, 1985). The signals controlling herpesvirus genome cleavage appear to be structurally conserved. The presence of so-called pat 1 and pad homologies (Deiss eta/., 1986) near the ends of a wide variety of herpesviruses (Spaete and Mocarski, 1985; Albrecht eta/., 1985; Davison, 1984; Bankier et al., 1985; Mocarski et al., 1987; Marks and Spector, 1988; Hammerschmidt et a/., 1988) strongly suggests their functional role in the cleavage/packaging process, a role that is supported by the demonstration (Spaete and Mocarski, 1985) of functional conservation of the packaging signal in two divergent herpesviruses: HSV and cytomegalovirus (CMV). We set out to create a herpesvirus cleavage site using sequences carried in the HSV-1 a sequence. The single-base 3’ extensions on the termini of HSV DNA (Mocarski and Roizman, 1982) suggested that the last step in the genome maturation process must be a cleavage event. Cleaved termini can be juxtaposed to re-form an a-a junction identical to that already known to exist in virion DNA (Mocarski and Roizman, 1981). This work, as well as more recent studies (Varmuza and Smiley, 1985; Deiss and Frenkel, 1986; Deiss et a/., 1986), predicted that signals carried in a single copy of the a sequence would need to be permuted in the process of genome maturation to generate the appropriate sequence arrangement for cleavage. The arrangement we chose to test carries both pacl and pac2 elements and is in an arrangement that is natural for certain herpesviruses such as varicella-zoster virus (VZV), herpesvirus saimiri, herpesvirus tupaia, bovine herpesvirus 1, and murine CMV (Davison, 1984; Bankier et a/., 1985; Albrecht et a/., 1985, Hammerschmidt et a/., 1988; Marks and Spector, 1988). MATERIALS
AND METHODS
Virus and cells HSV-1 (F) was propagated in Vero cells as described previously (Mocarski eta/., 1980). For analysis of cleavage, cells (2 x 1 06) were transfected with 40 pg of plasmid DNA through electroporation (Neumann et a/.,
1982) and 4 hr later the surviving cells (w-40-60%) were infected with 10 plaque-forming units (PFU) of HSV-1 (F) per cell and maintained for a total of 24 hr. The ability of plasmids to propagate was assayed in cells transfected with 5 pg of test plasmid DNA employing DEAE-Dextran (Hall et al., 1983). The cells were infected 2 hr later with 10 PFU of HSV-1 per cell, the cell homogenate was prepared by freeze/thaw 18 hr postinfection, and the resultant virus stock was employed in four consecutive undiluted passages. Plasmid
constructs
A 179-bp Mn/l fragment spanning an a-a junction (consisting of 22 bp of DR4 (DR4 is 37 bp), U, (58 bp), DRl (20 bp), lJb (64 bp), and one complete DR2 (12 bp) plus 3 bp of a second DR2) was isolated from pRB602 (Mocarski and Roizman, 1981) and cloned into the Smal site of pGEM-1 (Promega Biotech, Inc.) to generate pON 1 14. A 169-bp Mnll fragment consisting of DR4, U,, and DRl derived from an a sequence (as above) plus a 69-bp c repeat sequences was also isolated from pRB602 (Mocarski and Roizman, 1981) and cloned into the same vector to generate pON 1 15. Plasmids pON 120 and pON 121 contained a 300-bp HSV DNA replication origin (or&) (Elias et a/., 1986) in addition to the 179- or 169-bp fragments, respectively. These plasmids were made by ligating BarnHI-digested pON114 or pON115 with pONlO3 (Elias et al., 1986) which was cut with Bg/ll and BarnHI. The resultant clones carried two indirect copies of the pBR322 replicon and the /3-lactamase gene separating regions carrying either the oris sequence or the a sequence fragment. The structure of the plasmids was verified by restriction enzyme digestion. DNA methods Infected cells were washed with PBS and total cellular DNA was prepared as described (Spaete and Frenkel, 1982). One-fifth of the DNA from 2 X 1O6 cells was digested with Bgll @g/l cuts once in pGEM-1) or left undigested and then electrophoresed on a 0.7% agarose gel and analyzed by Southern blot hybridization (Southern, 1975) using a 32P-labeled pBR322 probe. For end-labeling the cleavage product, linearized DNA was electroeluted from the agarose gel and labeled with [32P]cordycepin 5’-triphosphate (3000 Gil mmol, Amersham) and terminal deoxynucleotidyl transferase (BRL) as recommended by the manufacturer. After labeled DNA was phenol-extracted, passed through Sephadex G-50, and ethanol-precipitated, the resuspended DNA was digested with HindIll and EcoRl (sites flanking the Smal site in pGEM-l), electropho-
HSV
HSV-1
CLEAVAGE/PACKAGING
SIGNAL
27
(F)
L
ab
bWa%
S
ca
m_______________-------______________-------%. ______________---------r---+-
DRI-U~(DR2#DR4);Uc-
pRB602
DRI -Ub*(DR2j9$)R4);Uc- D;;
944 bp
pON114,
pON120
(DR4)&
DRId+j(DR2),
179 bp
pON115,
pON121
(DR4#
DRI
169 bp
FIG. 1. Schematic representation of the and a’c’and ca, flanking the S component within the L-S junction and the sequences pON 115. and pON 121 contained different of DR4 (DR4 is 37 bp), U, (58 bp), DRl (20
resed on a 6% polyacrylamide autoradiography.
150-kbp HSV genome showing terminal and internal repeats, ab and b’a’, flanking the L component, (Roizman and Batterson, 1985). The expanded region shows the arrangement of a sequence elements carried on plasmid clones used in these studies. As indicated, plasmid clones pON 114, pON 120, elements derived from an a-a junction sequence (Mocarski and Roizman, 1981) consisting of 22 bp bp). Ub (64 bp), and DR2 (12 bp), as described under Materials and Methods.
gel, and subjected
to
of tandemly repeated a sequences contained a recognition signal for HSV cleavage enzymes and, with respect to the cleavage, the 179-bp signal apparently
RESULTS Cleavage at an a-a junction replication
independent
of DNA
Plasmid clone pONl14 carried a 179-bp fragment (Fig. 1) that spanned the junction of two tandemly repeated HSV-1 a sequences [containing UC-DRl-lJb (Mocarski and Roizman, 1981)] was tested in a transient cleavage assay (Fig. 2). Following electroporation of the test plasmid into Vero cells and subsequent infection with HSV for 18 hr, total cellular DNA was prepared, digested with f3g/l (for which there is a single site in the,test plasmid), and analyzed by Southern blot hybridization using vector (pBR322) DNA as a probe. A specific cleavage within the 179-bp fragment would generate two fragments (1.3 and 1.8 kbp) and two bands corresponding to these fragments were produced (Fig. 2). A plasmid clone (pON1 15) carrying a 169-bp a sequence fragment that contained UC-DRl identical to pON 114, but lacked Ub, was prepared in the same vector and remained uncleaved when tested in the transient assay (Fig. 2). Furthermore, cleavage activity specific for pON 114 was not detected following transfection of uninfected Vero cells (data not shown). A plasmid carrying two complete copies of the a sequence, pRB602 (Mocarski and Roizman, 1981) was also tested in the transient cleavage assay and similar to pON 1 14, only a minor portion of the plasmid was cleaved (data not shown). In the plasmids tested, cleavage occurred at a-a junctions, not at either a-c or b-a junctions. Taken together, these findings demonstrated that the 179-bp fragment from the junction
FIG. 2. Specific cleavage within the 179-bp fragment. Southern blot of undigested (U) and Bgll-digested DNA from cells transfected with pON 114 or pON 115 and infected (for 18 hr) with HSV-1 (F) hybridized with a 32P-labeled pBR322 DNA probe. Cleavage could not be detected in the cells transfected with pON 115 even after 20-fold longer exposure. On the right, the arrows indicate the 1.3- and 1.8kbp cleavage products and the hash marks indicate the position of X DNA HindIll fragments (23, 9.4, 6.5, 4.3, 2.3, and 2.0 kbp). Plasmid forms I, II, and III DNAs are indicated on the left.
28
NASSERI
AND
MOCARSKI
sult that suggested that the 133-bp band (the L-terminus analog) was potentially blocked.
Propagation and oris
FIG. 3. Autoradiograph of a polyacrylamide gel showing “P-endlabeled cleavage products. Linearized plasmid as in Fig. 2, track U, was eluted from the agarose gel and end-labeled with [w~‘P]cordycepin 5’triphosphate and terminal deoxynucleotidyl transferase. The labeled DNA was purified as described under Materials and Methods and analyzed on 6% polyacrylamide after digestion with Hindlll and EcoRI. Irrelevant control plasmid DNAs were included to monitor for complete digestion. The size markers (M) of relevant Hpall fragments of pBR322, labeled with [a-32P]dCTP and Klenow fragment (Boehringer). are indicated in bp.
contains the same recognition signals present on the two complete a sequences. In order to further investigate the specificity of cleavage within the 179-bp fragment, we analyzed the product of the transient cleavage assay using 3’-[(r-3zP]cordycepin 5’-triphosphate along with terminal deoxynucleotidyl transferase. The DNA from Vero cells transfected with pON1 14 and infected with virus was prepared and the cleavage product (linearized plasmid) was electroeluted from an agarose gel and 3’-end-labeled. Following digestion with HindIll and EcoRI, the end-labeled products were analyzed by polyacrylamide gel electrophoresis. Specific cleavage would generate two fragments (91 and 133 bp) representing the S- and L-terminal analogs, respectively. Two bands with the expected sizes were generated (Fig. 3), indicating that the cleavage occurred at a site analogous to an a-a junction in the viral genome. Even though restriction enzyme digestion was complete, the incorporation of radiolabel into the two ends was not equivalent, a re-
of plasmids
carrying a cleavage signal
The a sequence domains involved in genome maturation have been mapped within the Ub and U, elements (Varmuza and Smiley, 1985; Deiss and Frenkel, 1986; Deiss et a/., 1986). The appropriate arrangement of these elements for cleavage is U,-DRl-U,,, which is permuted relative to their arrangement in a single a sequence. It has been reported that propagation of plasmids with a single intact a sequence (or certain deletion mutants) is invariably accompanied by a sequence amplification or recombination of the a sequence with the helper virus (Deiss and Frenkel, 1986; Deiss et al., 1986). Based on those reports as well as on our observations on the sequence requirements for cleavage, we constructed a plasmid (pON 120) carrying the 179-bp fragment along with a DNA replication origin (HSV-1 oris) (Elias eta/., 1986) and tested it for propagation as a defective genome. The plasmid was transfected into Vero cells and the cells were subsequently infected with helper virus. Infected cell DNA was extracted at 24 hr postinfection and analyzed by Southern blot hybridization using vector (pBR322) DNA as a probe. Plasmid replication was demonstrated by the presence of high-molecular-weight DNA that consisted of head-to-tail concatemers of the input plasmid. We tested the ability of the concatemeric plasmid to be packaged by preparing virus stocks which were employed in four consecutive undiluted passages. Cell DNA from passage four (P4) was prepared for analysis. The presence of multimeric plasmid DNA in these cells was demonstrated by hybridization of vector DNA to high-molecular-weight material which upon digestion with HindIll produced two expected bands, 3.1 and 2.5 kbp (Fig. 4) and, thus, the 179-bp fragment carried all functions necessary for packaging as well as cleavage. As a control, the 169-bp a sequence fragment containing only UC-DRl elements was cloned along with oris in the same vector and the resultant construct (pON121) did not propagate to any detectable level in four passages. Furthermore, pON121 did not undergo recombination with the helper virus, a phenomenon that has been reported to enhance propagation in similar constructs (Deiss et al., 1986). Finally, we also examined the 179-bp fragment for recombination with helper virus by reisolating BarnHI-digested and religated material from P4 DNA by transformation back into Escherichia co/i. No evidence for size changes in the 179-bp fragment was observed in 48 independent clones (data not shown). Taken together, these experi-
HSV
Hind III
CLEAVAGE/PACKAGING
U
FIG. 4. Southern blot analysis of and HindIll-digested and undigested (U) P4 cell DNA to investigate the propagation of pON120 in presence of helper virus. Nitrocellulose filters were hybridized with 3zP-labeled vector (pBR322) DNA. No bands can be detected in experiments with pON121 even after 20-fold longer exposure. As a marker, pON120 DNA was digested with HindIll and electrophoresed in parallel with P4 DNA. The hash marks on the right (shown separately for U and HindIll tracks) indicate phage X DNA fragments as in Fig. 2.
ments demonstrated that the 179-bp fragment contains all necessary signals for HSV-1 genome maturation.
DISCUSSION We have shown that a 179-bp fragment from the junction of two tandem a sequences carries all sequence elements necessary for cleavage recognition and encapsidation. The arrangement of a sequence elements on the test plasmid is permuted relative to their normal arrangement; however, reasonable evidence has been previously presented to predict that this is the arrangement which must be attained for genome maturation to occur (Mocarski and Roizman, 1982; Varmuza and Smiley, 1985; Deiss et al., 1986). The 179-bp sequence tested here included all sequence elements required for cleavage because the degree of cleavage of two complete tandem a sequences assayed in parallel with the shorter fragment was quantitatively similar to that obtained by the shorter fragment. Sequence elements which have been shown to be es-
29
SIGNAL
sential for encapsidation (Varmuza and Smiley, 1985; Deiss et a/., 1986) are contained entirely within the 179-bp fragment. It would therefore appear that in standard virus the cleavage signal is present only in those junctions bearing two or more tandem a sequences and the cleavage of the junctions containing a single a sequence would occur only if the proper signal is formed by either amplification (Varmuza and Smiley, 1985) or transposition (Deiss et a/., 1986). This would allow for the generation of a single a sequence at each terminus during maturation process despite the fact that majority of the junctions within replicative concatemers contain a single a sequence. The two quasi-unique elements, Ub and U,, which carry two highly conserved sequence blocks, pat-1 and pat-2, are present in all herpesviruses examined including CMV, EBV, VZV, different HSV strains, and a number of animal herpesviruses (Deiss et al., 1986; Mocarski et al., 1985, 1987; van den Berg et a/., 1984; Spaete and Mocarski, 1985; Albrecht et al., 1985; Davison, 1984; Bankier et al., 1985; Marks and Spector, 1988; Hammerschmidt et al., 1988). Based on our findings we speculate that a sequence arrangement similar to that of the 179 bp contains the cleavage recognition and genome maturation signals in other herpesviruses. This speculation is supported by two observations: (i) the cytomegalovirus a sequence functions as a cleavage-packaging signal in an HSV-dependent assay (Spaete and Mocarski, 1985) and (ii) plasmids carrying inserts that join both ends of the pseudorabies virus genome contain the packaging signal (Wu et a/., 1986). It should be noted that the pat- 1 and pac2 homologies in VZV, whose genome is arranged similar to pseudorabies, are present at opposite ends of the genome (Davison, 1984). The proposed models of herpesvirus maturation predict that newly replicated concatemers interact with a packaging complex that would traverse the genome, cleaving DNA at specific sites while it is packaged into capsids (Varmuza and Smiley, 1985; Deiss and Frenkel, 1986; Deiss et al., 1986). Our results show that cleavage is independent of replication or concatemerization. Based on this information, we propose that a sequence amplification or transposition occurs as the initial step in maturation that is essential to create the proper arrangement of a sequence elements that are then the substrate for cleavage.
ACKNOWLEDGEMENT This work was supported by a Public Health National Institutes of Health (Al2021 1).
Service
grant from the
REFERENCES ALBRECHT, M., DARAI, G., and FLUGEL, R. M. (1985). Analysis of the genomic termini of tupaia herpesvirus DNA by restriction mapping and nucleotide sequencing. J. Viral. 56, 466-474.
30
NASSERI
BANKIER, A. T., DIETRICH, W., BAER, R., BARRELL, B. G., COLBERE GARAPIN, F., FLECKENSTEIN, B., and BODEMER, W. (1985). Terminal repetitive sequences in herpesvirus saimiri virion DNA. 1. Viral. 55, 133139. BEN-P• RAT, T. (1982). In “Organization and Replication of Viral DNA” (A. S. Kaplan, Ed.), pp. 147-l 72. CRC Press, Boca Raton, FL. CHOU, J., and ROIZMAN, B. (1985). lsomerization of herpes simplex virus 1 genome: Identification of the c&acting and recombination sites within the domain of the a sequence. Ce//41, 803-811, CHOU, J., and ROIZMAN, B. (1986). The terminal a sequence of the herpes simplex virus genome contains a promoter of a gene located in the repeat sequences in the L component. J. Viral. 57, 629-637. DAVISON, A. 1. (1984). Structure of the genome termini of varicellazoster virus. J. Gen. Viral. 65, 1969-l 977. DAVISON, A. J., and WILKIE, N. M. (1981). Nucleotide sequences of the joint between the L and S segments of herpes simplex virus types 1 and 2. J. Gen. Viral. 55, 3 15-331. DEISS, L. P., CHOU, J., and FRENKEL, N. (1986). Functional domains within the a sequence involved in the cleavage-packaging of herpes simplex virus DNA. J. l&o/. 59, 605-618. DEISS, L. P., and FRENKEL, N. (1986). Herpes simplex virus amplicon: Cleavage of concatemeric DNA is linked to packaging and involves amplification of the terminally reiterated a sequence. J. Viral. 57, 933-941. ELIAS, P., O’DONNELL, M. E., MOCARSKI, E. S., and LEHMAN, I. R. (1986). A DNA binding protein specific for an origin of replication of herpes simplex virus type 1. froc. Nat/. Acad. Sci. USA 83, 63226326. HALL, C. V., JACOB, P. E.. RINGOLD, G. M., and LEE, F. (1983). Expression and regulation of Escherichia co/i /acZ gene fusions in mammalian cells. J. Mol. Appl. Genet. 2, 101-l 09. HAMMERSCHMIDT, W., LUDWIG, H., and BUDK, H. J. (1988). Specificity of cleavage in replicative-form DNA of bovine herpesvirus 1. 1. Viral. 62, 1355-l 363. LOCKER, H., and FRENKEL, N. (1979). Baml, Kpnl. and Sal1 restriction enzyme maps of the DNAs of herpes simplex virus strains Justin and F: Occurrence of heterogeneities in defined regions of the viral DNA. J. Virol. 32,429-441. MARKS, J. R., and SPECTOR, D. H. (1988). Replication of the murine cytomegalovirus genome: Structure and role of the termini in the generation and cleavage of concatenates. Virology 162,98-l 07. MOCARSKI, E. S., DEISS, L. P.. and FRENKEL. N. (1985). Nucleotide sequence and structural features of a novel Us-a junction present in a defective herpes simplex virus genome. J. Viral. 55, 140-l 46. MOCARSKI, E. S., Lu, A. C., and SPAETE, R. R. (1987). Structure and variability of the a sequence in the genome of human cytomegalovirus (Towne strain). 1. Gen. Viral. 68, 2223-2230. MOCARSKI, E. S., POST, L. E., and ROIZMAN, B. (1980). Molecular engineering of the herpes simplex virus genome: Insertion of a second
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MOCARSKI L-S junction into the genome causes additional genome inversions. Cell22, 243-255. MOCARSKI, E. S., and ROIZMAN, B. (1981). Site-specific inversion sequence of the herpes simplex virus genome: Domain and structural features. Proc. Nat/. Acad. Sci. USA 78,7047-7051. MOCARSKI, E. S., and ROIZMAN, B. (1982). Herpesvirus-dependent amplification and inversion of cell-associated viral thymidine kinase gene flanked by viral a sequences and linked to an origin of viral DNA replication. Proc. Nat/. Acad. Sci. USA 79, 5626-5630. NEUMANN, E., SCHAEFER-RIDDER, M., WANG, Y.. and HOFSCHNEIDER, P. H. (1982). Gene transfer into mouse lyoma cells by electroporation in high electric fields. EMBOJ. 1, 841-845. ROIZMAN, B. (1979). The structure and isomerization of herpes simplex virus genomes. Cell 16,48 l-494. ROIZMAN, B., and BATTERSON, W. (1985). In “Virology (B. N. Fields, Ed.), pp. 497-529. Raven Press, New York. SOUTHERN, E. M. (1975). Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol 98, 503-517. SPAETE, R. R., and FRENKEL, N. (1982). The herpes simplex virus amplicon: A new eucaryotic defective-virus cloning-amplifying vector. Cell30, 295-304. SPAETE, R. R., and FRENKEL, N. (1985). The herpes simplex virus amplicon: analyses of cis-acting replication functions. Proc. Nat/. Acad. Sci. USA 82,694-698. SPAETE, R. R., and MOCARSKI, E. S. (1985). The a sequence of the cytomegalovirus genome functions as a cleavage/packaging signal for herpes simplex virus defective genomes. 1. Viral. 54, 817824. STOW, N. D., MCMONAGLE, E. C., and DAVISON, A. 1. (1983). Fragmentsfrom both termini of the herpes simplexvirustype 1 genome contain signals required for the encapsidation of viral DNA. Nucleic Acids Res. 11, 8205-8220. VAN DEN BERG, F. M., VAN OOYEN, A. J. J., VOLKERS, H., and WALBOOMERS, J. M. M. (1984). Heterogeneity in subregions of the terminal repeats of herpes simplex type 2 DNA. lnfervirology 21,96-l 03. VARMUZA, S. L.. and SMILEY. J. R. (1985). Signals for site-specific cleavage of HSV DNA: Maturation involves two separate cleavage events at sites distal to the recognition sequences. Cell 41, 793802. VLA~NY, D. A., and FRENKEL, N. (1981). Replication of herpes simplex virus DNA: Localization of replication recognition signals within defective virus genomes. Proc. Natl. Acad. Sci. USA 78,742-746. VLA~NY, 0. A., KWONG, A., and FRENKEL, N. (1982). Site-specific cleavage/packaging of herpes simplexvirus DNA and the selective maturation of nucleocapsids containing full-length viral DNA. Proc. Natl. Acad. Sci. USA 79, 1423-l 427. Wu, C. A., HARPER, L., and BEN PORAT, T. (1986). cis functions involved in replication and cleavage-encapsidation of pseudorabies virus. J. Viral. 59, 318-327.
167,25-30
(1988)
The Cleavage Recognition Signal Is Contained within Sequences Surrounding an a-a Junction in Herpes Simplex Virus DNA M. NASSERI Department
of Microbiology
and Immunology, Received
AND
Stanford
E. S. MOCARSKI’ University
April 25, 1988; accepted
School
of Medicine,
Stanford,
California
94305
July 11, 1988
Herpesvirus genome maturation involves site-specific cleavage of viral DNA concatemers and encapsidation of unitlength molecules, processes that are apparently coupled. Here, applying a transfection-infection approach, we have investigated the arrangement of the DNA sequence elements involved in cleavage and shown that specific cleavage occurs independently of DNA replication. We show that the &-acting signal for cleavage is located within a 179-bp fragment from across an a-a junction formed as part of the genome maturation process of herpes simplex virus 1. Plasmids carrying the 179-bp fragment are cleaved at the appropriate site even though they are unable to replicate in HSV-infected cells. When linked to an origin, the same 179-bp a-a fragment will replicate and package into progeny virus as a defective genome. Two highly conserved homologies, pacl andpac2, that have been observed in all herpesviruses examined, including cytomegalovirus, Epstein-Barr virus, varicella-zoster virus, and herpes simplex virus 2 as well as the herpes simplex virus 1 genome, are contained within the 179-bp fragment. This suggests that a common 0 1988 Academic PESS. inc. mechanism is utilized for genome maturation in the herpesvirus group.
Smiley, 1985; Deiss and Frenkel, 1986) and genome maturation (Vlazny and Frenkel, 1981; Vlazny et al., 1982; Spaete and Frenkel, 1982; Varmuza and Smiley, 1985; Deiss and Frenkel, 1986; Deiss et al., 1986). In different strains of HSV-1, the a sequence ranges in size from 220 to 500 bp but retains considerable sequence identity, including two quasi-unique elements (U, and U,) and several direct repeats (DR). The a sequence of HSV-1 (F) can be depicted DRl-U,--DR2,DR4,-U,--DRl (Fig. 1) (Mocarski and Roizman, 1982). DRl is a 20-bp repeat that is shared between adjacent copies of the a sequence when it is present in tandem arrays and is the sequence within which cleavage occurs to generate genomic termini (Mocarski and Roizman, 1982). Three observations have suggested that the process of genome maturation in HSV-1 is complex. (i) The cleavage site is within DRl; however, this sequence is not required for genome maturation (Varmuza and Smiley, 1985; Mocarski et a/., 1985). (ii) Sequences within both the&, and U, elements appear to be critical for the cleavage/packaging process (Varmuza and Smiley, 1985; Deiss and Frenkel, 1986; Deiss eta/., 1986). (iii) Single a sequences predominate in replicating concatemeric DNA, although mature progeny molecules have at least one a sequence at each end (Locker and Frenkel, 1979; Mocarski and Roizman, 1981). This situation has led to speculation that there are two separate cleavage reactions (Varmuza and Smiley, 1985) or that a transpositional event obligatorily duplicates the a sequence during cleavage (Deiss and Frenkel, 1986).
INTRODUCTION Herpesviruses are large, enveloped viruses with double-stranded linear DNA genomes ranging in size from 120 to 240 kilobase pairs (kbp) (reviewed by Roizman and Batterson, 1985). Different members of the family vary widely in the occurrence of direct and inverted repeats within the viral genome and in the process of inversion that leads multiple sequence isomers of theviral genome. The herpes simplexvirus (HSV) genome is a 150-kbp molecule composed of two covalently linked components, L (long) and S (short), each consisting of unique sequences (U, and U,) bracketed by inverted repeats such that the genome structure may be depicted ab-UL--b’a’c’-Us-ca (Fig. 1). The a sequence is present as a direct repeat at the genomic termini and as an inverted repeat (a’) at the L-S junction (Mocarski and Roizman, 1981; Davison and Wilkie, 198 1; Mocarski and Roizman, 1982). The HSV genome undergoes inversion, mediated by the a sequence (Mocarski and Roizman, 1982) such that virion DNA consists of four sequence isomers differing only in their relative orientation (Roizman, 1979). The a sequence is present in a single copy at the S terminus of the viral genome and in one to several tandem copies at the L terminus and the L-S junction (Mocarski and Roizman, 1982). This sequence carries signals for activities besides inversion, including transcription (Chou and Roizman, 1986) localized self-amplification (Varmuza and ’ To whom
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for reprints
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be addressed. 25
0042-6822188
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26
NASSERI AND MOCARSKI
Given the evidence that the genomic termini contain single-base 3’ extensions that can be generated only by cleavage and, when aligned, the termini re-form a complete DRl (Mocarski and Roizman, 1982) the last step in the maturation process would be predicted to be a specific cleavage between adjacent a sequences no matter what intermediate steps are needed to create the adjacent a sequences. The genomes of all herpesviruses carry signals located near the genomic termini that are required in cis for packaging progeny DNA into capsids (Vlazny and Frenkel, 1981; Spaete and Frenkel, 1982, 1985; Stow et al., 1983; Spaete and Mocarski, 1985). The signals controlling herpesvirus genome cleavage appear to be structurally conserved. The presence of so-called pat 1 and pad homologies (Deiss eta/., 1986) near the ends of a wide variety of herpesviruses (Spaete and Mocarski, 1985; Albrecht eta/., 1985; Davison, 1984; Bankier et al., 1985; Mocarski et al., 1987; Marks and Spector, 1988; Hammerschmidt et a/., 1988) strongly suggests their functional role in the cleavage/packaging process, a role that is supported by the demonstration (Spaete and Mocarski, 1985) of functional conservation of the packaging signal in two divergent herpesviruses: HSV and cytomegalovirus (CMV). We set out to create a herpesvirus cleavage site using sequences carried in the HSV-1 a sequence. The single-base 3’ extensions on the termini of HSV DNA (Mocarski and Roizman, 1982) suggested that the last step in the genome maturation process must be a cleavage event. Cleaved termini can be juxtaposed to re-form an a-a junction identical to that already known to exist in virion DNA (Mocarski and Roizman, 1981). This work, as well as more recent studies (Varmuza and Smiley, 1985; Deiss and Frenkel, 1986; Deiss et a/., 1986), predicted that signals carried in a single copy of the a sequence would need to be permuted in the process of genome maturation to generate the appropriate sequence arrangement for cleavage. The arrangement we chose to test carries both pacl and pac2 elements and is in an arrangement that is natural for certain herpesviruses such as varicella-zoster virus (VZV), herpesvirus saimiri, herpesvirus tupaia, bovine herpesvirus 1, and murine CMV (Davison, 1984; Bankier et a/., 1985; Albrecht et a/., 1985, Hammerschmidt et a/., 1988; Marks and Spector, 1988). MATERIALS
AND METHODS
Virus and cells HSV-1 (F) was propagated in Vero cells as described previously (Mocarski eta/., 1980). For analysis of cleavage, cells (2 x 1 06) were transfected with 40 pg of plasmid DNA through electroporation (Neumann et a/.,
1982) and 4 hr later the surviving cells (w-40-60%) were infected with 10 plaque-forming units (PFU) of HSV-1 (F) per cell and maintained for a total of 24 hr. The ability of plasmids to propagate was assayed in cells transfected with 5 pg of test plasmid DNA employing DEAE-Dextran (Hall et al., 1983). The cells were infected 2 hr later with 10 PFU of HSV-1 per cell, the cell homogenate was prepared by freeze/thaw 18 hr postinfection, and the resultant virus stock was employed in four consecutive undiluted passages. Plasmid
constructs
A 179-bp Mn/l fragment spanning an a-a junction (consisting of 22 bp of DR4 (DR4 is 37 bp), U, (58 bp), DRl (20 bp), lJb (64 bp), and one complete DR2 (12 bp) plus 3 bp of a second DR2) was isolated from pRB602 (Mocarski and Roizman, 1981) and cloned into the Smal site of pGEM-1 (Promega Biotech, Inc.) to generate pON 1 14. A 169-bp Mnll fragment consisting of DR4, U,, and DRl derived from an a sequence (as above) plus a 69-bp c repeat sequences was also isolated from pRB602 (Mocarski and Roizman, 1981) and cloned into the same vector to generate pON 1 15. Plasmids pON 120 and pON 121 contained a 300-bp HSV DNA replication origin (or&) (Elias et a/., 1986) in addition to the 179- or 169-bp fragments, respectively. These plasmids were made by ligating BarnHI-digested pON114 or pON115 with pONlO3 (Elias et al., 1986) which was cut with Bg/ll and BarnHI. The resultant clones carried two indirect copies of the pBR322 replicon and the /3-lactamase gene separating regions carrying either the oris sequence or the a sequence fragment. The structure of the plasmids was verified by restriction enzyme digestion. DNA methods Infected cells were washed with PBS and total cellular DNA was prepared as described (Spaete and Frenkel, 1982). One-fifth of the DNA from 2 X 1O6 cells was digested with Bgll @g/l cuts once in pGEM-1) or left undigested and then electrophoresed on a 0.7% agarose gel and analyzed by Southern blot hybridization (Southern, 1975) using a 32P-labeled pBR322 probe. For end-labeling the cleavage product, linearized DNA was electroeluted from the agarose gel and labeled with [32P]cordycepin 5’-triphosphate (3000 Gil mmol, Amersham) and terminal deoxynucleotidyl transferase (BRL) as recommended by the manufacturer. After labeled DNA was phenol-extracted, passed through Sephadex G-50, and ethanol-precipitated, the resuspended DNA was digested with HindIll and EcoRl (sites flanking the Smal site in pGEM-l), electropho-
HSV
HSV-1
CLEAVAGE/PACKAGING
SIGNAL
27
(F)
L
ab
bWa%
S
ca
m_______________-------______________-------%. ______________---------r---+-
DRI-U~(DR2#DR4);Uc-
pRB602
DRI -Ub*(DR2j9$)R4);Uc- D;;
944 bp
pON114,
pON120
(DR4)&
DRId+j(DR2),
179 bp
pON115,
pON121
(DR4#
DRI
169 bp
FIG. 1. Schematic representation of the and a’c’and ca, flanking the S component within the L-S junction and the sequences pON 115. and pON 121 contained different of DR4 (DR4 is 37 bp), U, (58 bp), DRl (20
resed on a 6% polyacrylamide autoradiography.
150-kbp HSV genome showing terminal and internal repeats, ab and b’a’, flanking the L component, (Roizman and Batterson, 1985). The expanded region shows the arrangement of a sequence elements carried on plasmid clones used in these studies. As indicated, plasmid clones pON 114, pON 120, elements derived from an a-a junction sequence (Mocarski and Roizman, 1981) consisting of 22 bp bp). Ub (64 bp), and DR2 (12 bp), as described under Materials and Methods.
gel, and subjected
to
of tandemly repeated a sequences contained a recognition signal for HSV cleavage enzymes and, with respect to the cleavage, the 179-bp signal apparently
RESULTS Cleavage at an a-a junction replication
independent
of DNA
Plasmid clone pONl14 carried a 179-bp fragment (Fig. 1) that spanned the junction of two tandemly repeated HSV-1 a sequences [containing UC-DRl-lJb (Mocarski and Roizman, 1981)] was tested in a transient cleavage assay (Fig. 2). Following electroporation of the test plasmid into Vero cells and subsequent infection with HSV for 18 hr, total cellular DNA was prepared, digested with f3g/l (for which there is a single site in the,test plasmid), and analyzed by Southern blot hybridization using vector (pBR322) DNA as a probe. A specific cleavage within the 179-bp fragment would generate two fragments (1.3 and 1.8 kbp) and two bands corresponding to these fragments were produced (Fig. 2). A plasmid clone (pON1 15) carrying a 169-bp a sequence fragment that contained UC-DRl identical to pON 114, but lacked Ub, was prepared in the same vector and remained uncleaved when tested in the transient assay (Fig. 2). Furthermore, cleavage activity specific for pON 114 was not detected following transfection of uninfected Vero cells (data not shown). A plasmid carrying two complete copies of the a sequence, pRB602 (Mocarski and Roizman, 1981) was also tested in the transient cleavage assay and similar to pON 1 14, only a minor portion of the plasmid was cleaved (data not shown). In the plasmids tested, cleavage occurred at a-a junctions, not at either a-c or b-a junctions. Taken together, these findings demonstrated that the 179-bp fragment from the junction
FIG. 2. Specific cleavage within the 179-bp fragment. Southern blot of undigested (U) and Bgll-digested DNA from cells transfected with pON 114 or pON 115 and infected (for 18 hr) with HSV-1 (F) hybridized with a 32P-labeled pBR322 DNA probe. Cleavage could not be detected in the cells transfected with pON 115 even after 20-fold longer exposure. On the right, the arrows indicate the 1.3- and 1.8kbp cleavage products and the hash marks indicate the position of X DNA HindIll fragments (23, 9.4, 6.5, 4.3, 2.3, and 2.0 kbp). Plasmid forms I, II, and III DNAs are indicated on the left.
28
NASSERI
AND
MOCARSKI
sult that suggested that the 133-bp band (the L-terminus analog) was potentially blocked.
Propagation and oris
FIG. 3. Autoradiograph of a polyacrylamide gel showing “P-endlabeled cleavage products. Linearized plasmid as in Fig. 2, track U, was eluted from the agarose gel and end-labeled with [w~‘P]cordycepin 5’triphosphate and terminal deoxynucleotidyl transferase. The labeled DNA was purified as described under Materials and Methods and analyzed on 6% polyacrylamide after digestion with Hindlll and EcoRI. Irrelevant control plasmid DNAs were included to monitor for complete digestion. The size markers (M) of relevant Hpall fragments of pBR322, labeled with [a-32P]dCTP and Klenow fragment (Boehringer). are indicated in bp.
contains the same recognition signals present on the two complete a sequences. In order to further investigate the specificity of cleavage within the 179-bp fragment, we analyzed the product of the transient cleavage assay using 3’-[(r-3zP]cordycepin 5’-triphosphate along with terminal deoxynucleotidyl transferase. The DNA from Vero cells transfected with pON1 14 and infected with virus was prepared and the cleavage product (linearized plasmid) was electroeluted from an agarose gel and 3’-end-labeled. Following digestion with HindIll and EcoRI, the end-labeled products were analyzed by polyacrylamide gel electrophoresis. Specific cleavage would generate two fragments (91 and 133 bp) representing the S- and L-terminal analogs, respectively. Two bands with the expected sizes were generated (Fig. 3), indicating that the cleavage occurred at a site analogous to an a-a junction in the viral genome. Even though restriction enzyme digestion was complete, the incorporation of radiolabel into the two ends was not equivalent, a re-
of plasmids
carrying a cleavage signal
The a sequence domains involved in genome maturation have been mapped within the Ub and U, elements (Varmuza and Smiley, 1985; Deiss and Frenkel, 1986; Deiss et a/., 1986). The appropriate arrangement of these elements for cleavage is U,-DRl-U,,, which is permuted relative to their arrangement in a single a sequence. It has been reported that propagation of plasmids with a single intact a sequence (or certain deletion mutants) is invariably accompanied by a sequence amplification or recombination of the a sequence with the helper virus (Deiss and Frenkel, 1986; Deiss et al., 1986). Based on those reports as well as on our observations on the sequence requirements for cleavage, we constructed a plasmid (pON 120) carrying the 179-bp fragment along with a DNA replication origin (HSV-1 oris) (Elias eta/., 1986) and tested it for propagation as a defective genome. The plasmid was transfected into Vero cells and the cells were subsequently infected with helper virus. Infected cell DNA was extracted at 24 hr postinfection and analyzed by Southern blot hybridization using vector (pBR322) DNA as a probe. Plasmid replication was demonstrated by the presence of high-molecular-weight DNA that consisted of head-to-tail concatemers of the input plasmid. We tested the ability of the concatemeric plasmid to be packaged by preparing virus stocks which were employed in four consecutive undiluted passages. Cell DNA from passage four (P4) was prepared for analysis. The presence of multimeric plasmid DNA in these cells was demonstrated by hybridization of vector DNA to high-molecular-weight material which upon digestion with HindIll produced two expected bands, 3.1 and 2.5 kbp (Fig. 4) and, thus, the 179-bp fragment carried all functions necessary for packaging as well as cleavage. As a control, the 169-bp a sequence fragment containing only UC-DRl elements was cloned along with oris in the same vector and the resultant construct (pON121) did not propagate to any detectable level in four passages. Furthermore, pON121 did not undergo recombination with the helper virus, a phenomenon that has been reported to enhance propagation in similar constructs (Deiss et al., 1986). Finally, we also examined the 179-bp fragment for recombination with helper virus by reisolating BarnHI-digested and religated material from P4 DNA by transformation back into Escherichia co/i. No evidence for size changes in the 179-bp fragment was observed in 48 independent clones (data not shown). Taken together, these experi-
HSV
Hind III
CLEAVAGE/PACKAGING
U
FIG. 4. Southern blot analysis of and HindIll-digested and undigested (U) P4 cell DNA to investigate the propagation of pON120 in presence of helper virus. Nitrocellulose filters were hybridized with 3zP-labeled vector (pBR322) DNA. No bands can be detected in experiments with pON121 even after 20-fold longer exposure. As a marker, pON120 DNA was digested with HindIll and electrophoresed in parallel with P4 DNA. The hash marks on the right (shown separately for U and HindIll tracks) indicate phage X DNA fragments as in Fig. 2.
ments demonstrated that the 179-bp fragment contains all necessary signals for HSV-1 genome maturation.
DISCUSSION We have shown that a 179-bp fragment from the junction of two tandem a sequences carries all sequence elements necessary for cleavage recognition and encapsidation. The arrangement of a sequence elements on the test plasmid is permuted relative to their normal arrangement; however, reasonable evidence has been previously presented to predict that this is the arrangement which must be attained for genome maturation to occur (Mocarski and Roizman, 1982; Varmuza and Smiley, 1985; Deiss et al., 1986). The 179-bp sequence tested here included all sequence elements required for cleavage because the degree of cleavage of two complete tandem a sequences assayed in parallel with the shorter fragment was quantitatively similar to that obtained by the shorter fragment. Sequence elements which have been shown to be es-
29
SIGNAL
sential for encapsidation (Varmuza and Smiley, 1985; Deiss et a/., 1986) are contained entirely within the 179-bp fragment. It would therefore appear that in standard virus the cleavage signal is present only in those junctions bearing two or more tandem a sequences and the cleavage of the junctions containing a single a sequence would occur only if the proper signal is formed by either amplification (Varmuza and Smiley, 1985) or transposition (Deiss et a/., 1986). This would allow for the generation of a single a sequence at each terminus during maturation process despite the fact that majority of the junctions within replicative concatemers contain a single a sequence. The two quasi-unique elements, Ub and U,, which carry two highly conserved sequence blocks, pat-1 and pat-2, are present in all herpesviruses examined including CMV, EBV, VZV, different HSV strains, and a number of animal herpesviruses (Deiss et al., 1986; Mocarski et al., 1985, 1987; van den Berg et a/., 1984; Spaete and Mocarski, 1985; Albrecht et al., 1985; Davison, 1984; Bankier et al., 1985; Marks and Spector, 1988; Hammerschmidt et al., 1988). Based on our findings we speculate that a sequence arrangement similar to that of the 179 bp contains the cleavage recognition and genome maturation signals in other herpesviruses. This speculation is supported by two observations: (i) the cytomegalovirus a sequence functions as a cleavage-packaging signal in an HSV-dependent assay (Spaete and Mocarski, 1985) and (ii) plasmids carrying inserts that join both ends of the pseudorabies virus genome contain the packaging signal (Wu et a/., 1986). It should be noted that the pat- 1 and pac2 homologies in VZV, whose genome is arranged similar to pseudorabies, are present at opposite ends of the genome (Davison, 1984). The proposed models of herpesvirus maturation predict that newly replicated concatemers interact with a packaging complex that would traverse the genome, cleaving DNA at specific sites while it is packaged into capsids (Varmuza and Smiley, 1985; Deiss and Frenkel, 1986; Deiss et al., 1986). Our results show that cleavage is independent of replication or concatemerization. Based on this information, we propose that a sequence amplification or transposition occurs as the initial step in maturation that is essential to create the proper arrangement of a sequence elements that are then the substrate for cleavage.
ACKNOWLEDGEMENT This work was supported by a Public Health National Institutes of Health (Al2021 1).
Service
grant from the
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BANKIER, A. T., DIETRICH, W., BAER, R., BARRELL, B. G., COLBERE GARAPIN, F., FLECKENSTEIN, B., and BODEMER, W. (1985). Terminal repetitive sequences in herpesvirus saimiri virion DNA. 1. Viral. 55, 133139. BEN-P• RAT, T. (1982). In “Organization and Replication of Viral DNA” (A. S. Kaplan, Ed.), pp. 147-l 72. CRC Press, Boca Raton, FL. CHOU, J., and ROIZMAN, B. (1985). lsomerization of herpes simplex virus 1 genome: Identification of the c&acting and recombination sites within the domain of the a sequence. Ce//41, 803-811, CHOU, J., and ROIZMAN, B. (1986). The terminal a sequence of the herpes simplex virus genome contains a promoter of a gene located in the repeat sequences in the L component. J. Viral. 57, 629-637. DAVISON, A. 1. (1984). Structure of the genome termini of varicellazoster virus. J. Gen. Viral. 65, 1969-l 977. DAVISON, A. J., and WILKIE, N. M. (1981). Nucleotide sequences of the joint between the L and S segments of herpes simplex virus types 1 and 2. J. Gen. Viral. 55, 3 15-331. DEISS, L. P., CHOU, J., and FRENKEL, N. (1986). Functional domains within the a sequence involved in the cleavage-packaging of herpes simplex virus DNA. J. l&o/. 59, 605-618. DEISS, L. P., and FRENKEL, N. (1986). Herpes simplex virus amplicon: Cleavage of concatemeric DNA is linked to packaging and involves amplification of the terminally reiterated a sequence. J. Viral. 57, 933-941. ELIAS, P., O’DONNELL, M. E., MOCARSKI, E. S., and LEHMAN, I. R. (1986). A DNA binding protein specific for an origin of replication of herpes simplex virus type 1. froc. Nat/. Acad. Sci. USA 83, 63226326. HALL, C. V., JACOB, P. E.. RINGOLD, G. M., and LEE, F. (1983). Expression and regulation of Escherichia co/i /acZ gene fusions in mammalian cells. J. Mol. Appl. Genet. 2, 101-l 09. HAMMERSCHMIDT, W., LUDWIG, H., and BUDK, H. J. (1988). Specificity of cleavage in replicative-form DNA of bovine herpesvirus 1. 1. Viral. 62, 1355-l 363. LOCKER, H., and FRENKEL, N. (1979). Baml, Kpnl. and Sal1 restriction enzyme maps of the DNAs of herpes simplex virus strains Justin and F: Occurrence of heterogeneities in defined regions of the viral DNA. J. Virol. 32,429-441. MARKS, J. R., and SPECTOR, D. H. (1988). Replication of the murine cytomegalovirus genome: Structure and role of the termini in the generation and cleavage of concatenates. Virology 162,98-l 07. MOCARSKI, E. S., DEISS, L. P.. and FRENKEL. N. (1985). Nucleotide sequence and structural features of a novel Us-a junction present in a defective herpes simplex virus genome. J. Viral. 55, 140-l 46. MOCARSKI, E. S., Lu, A. C., and SPAETE, R. R. (1987). Structure and variability of the a sequence in the genome of human cytomegalovirus (Towne strain). 1. Gen. Viral. 68, 2223-2230. MOCARSKI, E. S., POST, L. E., and ROIZMAN, B. (1980). Molecular engineering of the herpes simplex virus genome: Insertion of a second
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