Herpes Simplex Virus Type 1 Oril Is Not Required for Virus Infection In

Home , Pol (HIV), Viral protein

JOURNAL OF VIROLOGY, Nov. 1987, p. 3528-3535 Vol. 61, No. 11 0022-538X/87/113528-08$02.00/0 Copyright C 1987, American Society for Microbiology

Herpes Simplex Virus Type 1 oriL Is Not Required for Virus Replication or for the Establishment and Reactivation of Latent Infection in Mice MARYELLEN POLVINO-BODNAR, PAULO K. ORBERG, AND PRISCILLA A. SCHAFFER* Laboratory of Tumor Virus Genetics, Dana-Farber Cancer Institute, and Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115 Received 11 May 1987/Accepted 31 July 1987

During the course of experiments designed to isolate deletion mutants of herpes simplex virus type 1 in the gene encoding the major DNA-binding protein, ICP8, a mutant, d61, that grew efficiently in ICP8-expressing Vero cells but not in normal Vero cells was isolated (P. K. Orberg and P. A. Schaffer, J. Virol. 61:1136-1146, 1987). d61 was derived by cotransfection of ICP8-expressing Vero cells with infectious wild-type viral DNA and a plasmid, pDX, that contains an engineered 780-base-pair (bp) deletion in the ICP8 gene, as well as a spontaneous -55-bp deletion in OriL. Gel electrophoresis and Southern blot analysis indicated that d61 DNA carried both deletions present in pDX. The ability of d61 to replicate despite the deletion in OriL suggested that a functional OriL is not essential for virus replication in vitro. Because d61 harbored two mutations, a second mutant, ts+7, with a deletion in oriL-associated sequences and an intact ICP8 gene was constructed. Both d61 and ts+7 replicated efficiently in their respective permissive host cells, although their yields were slightly lower than those of control viruses with intact oriL sequences. An in vitro test of origin function of isolated OriL sequences from wild-type virus and ts+7 showed that wild-type OHiL, but not ts+7 OHIL, was functional upon infection with helper virus. In an effort to determine the requirement for OriL in latency, ts+7 was compared with wild-type virus for its ability to establish, maintain, and be reactivated from latent infection in a murine eye model. The mutant was reactivated as efficiently as was wild-type virus from trigeminal ganglia after cocultivation with permissive cells, and each of the seven reactivated isolates was shown to carry the -150-bp deletion characteristic of ts+7. These observations demonstrate that OHL is not required for virus replication in vitro or for the establishment and reactivation of latent infection in vivo.

The genome of herpes simplex virus type 1 (HSV-1) is a While oris sequences are stable upon cloning in bacteria, the linear, double-stranded DNA molecule of approximately larger oriL palindrome suffers deletions with high frequency 160,000 base pairs (bp). It consists of a long unique region when propagated in bacterial vectors (12, 45). Spontaneous (UL) flanked by inverted repeat sequences ab and b'a' and an or engineered deletions within oris and oriL palindromes S component consisting of a short unique region (Us) flanked result in loss of the capacity for autonomous replication (38, by the inverted repeats ac and c'a' (Fig. 1, line 1). During the 45). process of viral DNA synthesis, molecules are generated in The existence of two classes of defective interfering which the long and short components of the genome are particles (class I, whose genomes contain oris sequences, inverted relative to one another such that approximately and class II, whose genomes contain oriL sequences) dem- equimolar amounts of the four possible isomers of viral DNA onstrates that both origins are indeed capable of functioning are produced (1, 36). The viral genome is thought to replicate during productive infection in vitro (7, 8, 19). Little is by a rolling-circle mechanism which yields large head-to-tail known, however, about the requirement for each class of concatemeric intermediates that are subsequently cleaved to origin in the process of viral DNA synthesis (i.e., are oriL generate unit-length molecules (1, 3). Early electron micro- and both copies of oris essential for viral DNA synthesis?) or scopic studies provided evidence for the existence of two whether one class of origin is preferentially used during origins of DNA synthesis in the HSV-1 genome, one near the latent infection. The former question was partially answered center of UL and another near one end of the molecule (9, in a recent report that a mutant of HSV-1 lacking one copy 14). Studies of the genomes of defective interfering particles of oris is viable (23). In this paper, we demonstrate that the generated during serial passage of virus at high multiplicity absence of a functional oriL has little effect on viral replica- of infection and tests of origin function with cloned viral tion in vitro. Furthermore, we show that oriL is not required DNA fragments have shown that HSV-1 DNA contains two for the establishment or reactivation of latent infection in a copies of one origin, termed oris, located in the c and c' murine eye model. inverted repeats, and one copy of a second, more complex origin, termed oriL, located near the center of UL (2, 7, 8, 16-18, 25, 35, 37, 43). MATERIALS AND METHODS For origin function, the diploid oris requires no more than 90 bp, which includes an almost perfect palindromic se- Cells and viruses. African green monkey kidney (CV-1, quence of45 bp (40). oriL shares considerable homology with Vero, and U-47) and human embryonic lung (HEL) cells oris and contains a perfect 144-bp palindrome (12, 28, 45). were propagated and maintained as previously described (44). ICP8-expressing U-47 cells used as permissive hosts for the derivation and propagation of ICP8 deletion mutants * Corresponding author. were derived by cotransfection of Vero cells with the ICP8- 3528 VOL. 61, 1987 oriL DELETION MUTANTS OF HSV-1 3529

a b UL b'a'o' US co ------2 . , . I 1 I 1 0.34 0.35 0.36 0.37 0.38 0.39 0.40 0.41 0.42 0.43 0.44 3.3kb gB 4.2 kb ICP8 4.3kb D 0 ICP8 4.2kb lOkb -.. DNAMI. 5.6 kb ? tsA24- 9kb ? 4 I C*j 0 to -i it to C-i 0 Wm M BS B T p 0 E In (Alf qt CM C*j it) 102 in 5 'W 1 'I II I i I I I 11 1 11 I I I 111- XP K P TP S X X K T SP x'6 XTK TB T SKE B K p Ox 6 pKEF-P4 i--- Bom HI p E E= Eco RI 7 pSGIS-SaIIA ---4 Kr- Kpn I s P- Pst I 8 pDX i A --q s x x s SZSGI I Tz Bst E 11 Xz Xho I FIG. 1. Genomic location of oriL and plasmids used in this study. The prototype arrangement of the HSV-1 genome is shown in line 1. The map units of the expanded, central portion of the long unique region (UL) are shown in line 2. The transcripts known to map to this region of the genome (all of the early or 0 kinetic class [15]) and the location of the oriL (12, 45) are shown in line 3. The length of the transcript is indicated in kilobases (kb). The location of the mutation in the ICP8 ts mutant used in this study, tsA24, appears in line 4 (44). Relevant restriction sites and the corresponding nucleotide numbers (28) are depicted in the next three lines; the symbol A represents deletion in oriL sequences of -55 bp which is present in the parental plasmid, pKEF-P4, from which pSG18-Sa/I A and pDX were derived. containing plasmid pKEF-P4 (Fig. 1, line 6) and pSV2neo viral or cellular DNA were detected by the method of (27, 34). Southern (33). Probes were labeled with [12P]dCTP and The KOS strain of HSV-1 was used as the wild-type virus [32P]dGTP (Amersham Corp., Arlington Heights, 111.) by from which temperature-sensitive QsA24 [44]) and deletion nick translation (24). mutants were derived. Viruses were propagated and assayed Marker transfer and marker rescue. Vero and U-47 cells as previously described (31). were cotransfected with infectious viral (KOS or tsA24) Plasmids and cloning. The map locations oforiL-associated DNA and plasmid (pDX or pKEF-P4) DNAs as previously viral DNA inserts in plasmids used in this study are shown in described (29). Fig. 1. pKEF-P4 (4) and ptkCAToris (not shown) were Cloning and sequencing0fiL from ts'7. Viral DNA from kindly provided by N. DeLuca. The latter plasmid is a ts'7 was digested to completion with KpnI and cloned into derivative of ptkCAT (6) which contains a 220-bp SmaI Kpnl-cleaved pUC18. Clones containing the Kpnl V frag- fragment that contains oris. pSG18-Sall A (21; Fig. 1, line 7) ment were identified by filter hybridization (13) with the contains the intact gene for ICP8 and a spontaneous -55-bp KpnI V fragment of pKEF-P4 as the probe. The Rsal- deletion in oriL- pDX (Fig. 1, line 8) was derived from BamHl fragment of Kpnl V, which contains the large pSG18-SalI A by digestion with Xhol and religation. pUC18 palindrome of oriL, was subcloned into BamHI- and Hincll- (46) was obtained from Pharmacia, Inc. (Piscataway, N.J.). cleaved M13mp18. The Rsal-BamHl fragment cloned into Restriction endonucleases and T4 DNA ligase were ob- M13mp18 was the same size on acrylamide gels (i.e., ±5 bp) tained from New England BioLabs, Inc. (Beverly, Mass.) as the uncloned RsaI-BamHI fragment derived directly from and used as suggested by the manufacturer. viral DNA, indicating that further deletion had not occurred Isolation of viral DNA. Infected-cell DNA was prepared as as a consequence of cloning (data not shown). described previously (5). After proteinase K digestion, viral Sequence analysis was carried out using the chain termi- DNA was separated from cellular DNA by centrifugation in nation method of Sanger et al. (30). The Klenow fragment CsCl gradients (11). KOS DNA was isolated from infected reactions were conducted at 50'C to avoid artifacts gener- HEL cells; tsA24, tsC,, ts+3, and ts'7 DNAs were isolated ated by secondary structures. The products were run on 8% from infected Vero cells; and d6l DNA was isolated from polyacrylamide gels containing 8 M urea and 40% formam- infected U-47 cells. ide. Isolation of viral DNA fragments. HSV DNA fragments for In vitro tests of origin function. The procedure used to test use as probes in Southern blot hybridizations were isolated whether plasmid DNA sequences covalently linked to viral after restriction endonuclease digestion of plasmid DNA and origins of replication were amplified after transfection and electrophoresis through a 0.4% agarose gel. After staining infection with helper virus was similar to that described by with ethidium bromide, bands of interest were excised and other investigators (22, 39, 40, 45), with one significant submitted to three cycles of freezing and thawing (32). modification. Since oriL suffers deletions when propagated Agarose was separated from the DNA solution by centrifu- in bacteria (12, 45), gel-purified viral DNA fragments were gation. Viral DNA fragments used in tests of origin function ligated to the test plasmid, and the ligation mixture was used were isolated after digestion, electrophoresis through a 4% directly in the transfection of CV-1 cells. polyacrylamide gel, and electroelution into a dialysis bag The gel-purified KpnI V fragment (map units 0.406 to (24). In all cases, DNA fragments were ethanol precipitated 0.419) of KOS DNA and of ts'7 DNA (80 ng) was ligated to after purification by passage through an Elutip-d column 420 ng of Kpnl-cleaved pUC18, and 1/20 of the ligation (Schleicher & Schuell, Inc., Keene, N.H.). mixture was electrophoresed in a 0.6% gel to verify that Blot hybridization. Specific DNA sequences in digests of ligation had indeed occurred. One-half of the remaining 3530 POLVINO-BODNAR ET AL. J. VIROL. ligation mixture or 110 ng of plasmid DNA (pUC18, ptkCAToris, or pDX) was coprecipitated with salmon sperm ~~~~IIIL. DNA, and the coprecipitate was added to each of two 0 X oZ; o w replicate 100-mm petri dishes containing 2.5 x 106 CV-1 cells per dish. Four hours after transfection, cells were shocked with 15% glycerol for 1 min. At 10 h posttransfection, cells in one of each pair of replicate dishes were infected with HSV-1 (KOS), at a multiplicity of 10 PFU per cell. At 16 h postinfection, all cells were harvested and washed, and total cellular DNA was extracted by using sodium dodecyl sulfate and proteinase K digestion, phenol and chloroform extrac- tion, and ethanol precipitation. A 10-,ug portion of each sample was then digested with KpnI to excise unit-length pUC18 from replication concatemers and with DpnI (which digests input, methylated plasmid DNA but not newly replicated, unmethylated DNA [20]) to distinguish newly replicated DNA. Control samples containing 135 to 270 ng of V plasmid DNA were also digested with KpnI alone or with I-e04 KpnI plus DpnI. All samples were then electrophoresed in a 0.8% agarose gel and transferred to a nitrocellulose filter, which was probed with 32P-labeled pUC18, and an autoradi- ogram was obtained. Latency testing. Groups of 12 7-week-old CD-1 mice (Charles River Breeding Laboratories, Inc., Kingston, N.Y.) were inoculated with 2 x 106 PFU in 20 p.l of either wild-type virus or ts+7 after corneal scarification. On day 3 postinoc- 832 bp 780 > ulation, eye swabs were taken from four mice and trigeminal bp 4 i ganglia were removed from two mice for each virus (mice used for eye swabs may or may not have been the same as Kpn I KpnI / Bam Hi Xho I those sacrificed for ganglion assays). Eye swabs were assayed directly for infectious virus in Vero cells, and ganglia were frozen, thawed, sonicated, clarified by low-speed centrifugation, and assayed in Vero cells. On day 30 Kpn I or Sal I postinoculation, surviving mice were sacrificed, and ganglia _L 0.406 A 0.418 were removed immediately (within 1 to 2 min of death), cut Hi into eight equal-sized pieces, and cocultivated with Vero arm cells. On day 5 of cocultivation, Vero cells and ganglion FIG. 2. Southern blot analysis of oriL-associated deletions in pieces were scraped into medium, frozen, thawed, and viral and plasmid DNAs. Viral or plasmid DNAs were digested with sonicated. The suspension was clarified by low-speed cen- KpnI only (leftmost two lanes), KpnI and BamHI (next four lanes), trifugation, and the supernatant fluid was assayed for infec- or XhoI (rightmost lane), electrophoresed in an agarose gel, and tious virus in Vero transferred to a nitrocellulose filter. The filter was then probed with cell monolayers. the KpnI-SaIl fragment shown at the bottom of the figure and in Fig. 1, line 5. On the left margin of the figure are indicated the positions RESULTS of the oriL-containing KpnI V fragment and those of the KpnI- BamHI (0.406 to 0.411) fragments of KOS DNA (832 bp [28]) and of Derivation of mutants of HSV-1 carrying deletions in OriL- d61, pKEF-P4, and pDX (780 bp). The size of the latter fragment associated sequences. (i) d61, a double mutant in ICP8 and was estimated by comigration with the sequenced 780-bp (28) XhoI oriL. We have constructed two deletion mutants of HSV-1 in fragment of pKEF-P4, which was visible in the rightmost lane of the the gene encoding the major DNA-binding protein, ICP8, ethidium bromide-stained gel but is not detected using this probe. and verified that one of these, d61, had incorporated the oriL deletion, as well as the ICP8 deletion present in the plasmid used in its construction (27). Briefly, a 780-bp deletion was d61, pKEF-P4, and pDX DNAs were cleaved with KpnI and introduced into ICP8-coding sequences of plasmid pSG18- BamHI and probed with the KpnI-SaII fragment shown at Sall A to yield pDX (Fig. 1, line 8). In addition to the the bottom of Fig. 2, the KpnI-BamHI fragments of d61, engineered deletion, pSG18-SaiI A and consequently pDX pKEF-P4, and pDX exhibited greater mobility (780 bp) than also contain a spontaneous deletion in oriL-associated se- the corresponding KOS fragment did (832 bp). These obser- quences. It was assumed that if oriL were essential for virus vations indicate that d61 had also incorporated the sponta- replication, only the ICP8 deletion in pDX would be incor- neous oriL-associated deletion present in pDX and the porated into the viral genome when ICP8-expressing cells parental plasmid pSG18-SalI A. The efficient replication of were cotransfected with pDX and infectious, wild-type d61 in ICP8-expressing cells suggested the possibility that DNA. Mutant d61 was isolated from the resulting progeny oriL might not be required for viral replication in vitro. The on the basis of its capacity to grow in ICP8-expressing Vero existence of multiple mutations in d61, however, necessi- cells (U-47 [27]) but not in normal Vero cells. Examination of tated the isolation of a second mutant with a mutation in oriL the DNA of d61 revealed that it had incorporated the 780-bp only. engineered deletion in the ICP8 gene (27). Unexpectedly, the (ii) ts+7, a deletion mutant in OHL. A second mutant was KpnI V fragment of d61 (Fig. 2, lane 2) exhibited greater therefore constructed by marker rescue of the ts mutation in mobility than its KOS counterpart did (lane 1). When KOS, the ICP8 mutant, tsA24, with plasmid pKEF-P4 (Fig. 1, line VOL. L61.6iL1987 DELETION MUTANTS OF HSV-1 3531

A permissive U-47 cells. Consistent with the data shown in B Table 1, the plaque sizes of KOS and d21 were slightly larger h than those of ts47 and d61 in Vero and U-47 cells, respec- a. a a.%i* tively. Tests of origin function of isolated viral DNA fragments. To determine whether the deletion in oriL-associated sequences of ts+7 had affected origin function, we carried out in vitro origin function tests similar to those described by others (22, 39, 40. 45). The distinguishing feature of the procedure employed in this study was that uncloned, gel-purified viral DNA fragments were used directly after ligation to test plasmid sequences. thus avoiding the possibility of introduc- Kp I Kp I ing additional deletions during cloning in bacteria. As ex-

I a a pected, pUC18 sequences were not amplified in CV-1 cells :.*X& ^ after mock infection (Fig. 4, lane 1) or superinfection with HSV-1 (lane 2). The requirement for HSV-1-associated

Kpn V factors supplied in trans for o5is and (riL function is seen in lanes 3 and 4 (ptkCAToris) and lanes 5 and 6 (pUC18 ligated to the wild-type o0iL-containing KpnI V fragment), respectively. By contrast, pUC18 sequences ligated to the KpnI V fragment of ts#7 were not amplified after superinfection with HSV-1 (lane 8), nor was pDX, the plasmid used in the 0.406 construction of d61 (lane 10). These tests also confirmed 1ta11 lSt all J- ) previous reports (45) that the oriL deletion present in pKEF- P4 renders it incapable of replicating in experiments such as FIG. 3. (A) KpnI restriction patterns of plasmid and viral DNAs. this (data not shown). These tests thus demonstrate that The position of the undeleted KpnI V fragment of KOS. tsA24. tsCl, neither ts+7 nor pDX possesses an origin of viral DNA and ts+3 is shown on the left. The arrow on the right side of the synthesis that can figure indicates the position of the KpnI V fragment of ts'7. ts.7 and be driven by HSV-1 factors supplied in trans. ts+3 were derived from 39°C plaques generated in the marker rescue of tsA24 with pKEF-P4. tsCt was isolated from 34°C plaques of the Sequencing of oriL deletions in d61 and ts+7. To further same marker rescue. (B) Southern blot analysis of the gel shown in characterize the deletion in ts+7, we cloned the 309-bp panel A probed with the BstE II fragment (shown in the diagram at RsaI-Ba,nHI fragment of the mutant in M13, sequenced it, the bottom of the figure) purified from pKEF-P4 and labeled with 32P and compared this sequence with that of the wild-type virus. by nick translation. The diagram also illustrates the location of MriL The results of this analysis are shown in Fig. 5. Figure 5 within the KpnI V fragment relative to the probe. presents the sequence of the 425-bp BstEII-BacmHI fragment of strain KOS (45), showing the RsaI site at 114 to 118 bp, 6). pKEF-P4 contains a 55-bp deletion in the oriL palindrome the location of the 144-bp inverted repeat, and the locations and an intact ICP8 gene (4, 45). On the basis of our of pertinent promoter regulatory sequences for the divergent experience with d61, we anticipated that the deletion in oriL transcripts specifying the HSV major DNA-binding protein would be transferred to the viral genome concomitantly with and DNA polymerase (10, 15, 26, 41). The deletion in ts+7 is the rescue of the ts mutation by wild-type ICP8 sequences in 148 bp in size and retains only one copy of the sequence pKEF-P4. Of the 14 ts' recombinants examined, one, ts47, CCAC found at positions 156 to 160 and 304 to 308 (Fig. 4). was found to contain a deletion in the KpnI V fragment (Fig. Thus, like other oriL-associated deletions described previ- 3). Unlike the deletion in the parental plasmid pKEF-P4, ously, the ts 7 deletion appears to have occurred between however, the deletion in ts+7 was -150 bp, rather than 55 short repeats (45). The deletion is asymmetric relative to the bp. Two other plaque isolates were picked from the progeny 144-bp inverted repeat, lacking 17 bp immediately to the of the marker transfer experiment: ts*3, from 39°C plates, right of the repeat and all but 13 bp of the 72-bp repeat and tsC1, from 34°C plates. The DNA restriction patterns making up the left half of the palindrome. The deleted and Southern blots shown in Fig. 3 clearly reflect the sequences include the second distal signal upstream of the existence of three classes of the KptI V fragment: undeleted transcriptional start site of the major DNA-binding protein, (KOS, tsA24, tsC1, and ts`3), containing a -55-bp deletion ICP8 (41), and may include as yet unidentified signals (pKEF-P4 and d61), and containing a --150-bp deletion upstream of the transcriptional start site and probably the (ts+7). Thus, tsC1 was phenotypically and genotypically like tsA24 (ts and oriL'), whereas ts+3 was phenotypically and genotypically wild type (ts+, oriL+) and ts+7 was phenotyp- TABLE 1. Burst sizes of viruses containing intact or deleted ically wild type (ts') and genotypically mutant (lacking (IiL-associated sequences oriL). Virus oril- ICP8 Burst size" Replication efficiency of oriL deletion mutants. The two sequences sequences (PFU/cell) independently derived oriL mutants, ts 7 and d61, were KOS Intact Intact 135 capable of efficient replication in one-cycle growth experi- ts+7 Deleted Intact 96 ments in their respective permissive cells (Table 1). Thus, d21 Intact Deleted 62 burst sizes of ts+7 and d61 were similar to those of control d61 Deleted Deleted 12 viruses KOS and d21 in the two cell types. The differences in 11 and relative to KOS and Cells (106) were infected at a multiplicity of 2.5 PFUJ per cell (effective the burst sizes of d21 d61 ts+7 multiplicity, I PFU per cell). washed, incubated at 37°C for 18 h, harvested, probably reflect the requirement for complementation of the and assayed for infectious virus. ICP8-deficient d21 and d61 were tested in former mutant by the resident wild-type ICP8 gene in ICP8-expressing U-47 cells, and KOS and ts-7 were tested in Vero cells. 3532 POLVINO-BODNAR ET AL. J. VIROL.

"ATA" sequence of the DNA polymerase gene at 350 to 354 1 2 3 4 5 6 7 8 9 10 11 12 13 14 bp (10). On the other hand, it is unlikely that the deletion includes any elements critical for transcription of either essential gene as the mutant replicates nearly as well as does wild-type virus (Table 1). ,T4- Latency tests. Having established that ts+7 is replication competent in cell culture, we next tested the requirement for functional oriL in the establishment of latency. For this 0 .*pDX purpose, groups of 7-week-old CD-1 mice were inoculated x with 2 106 PFU per eye of either wild-type virus or ts+7 4 ~~ ., iptkCAToris after corneal scarification. Eye swabs and trigeminal ganglia were on Vero cells for infectious virus assayed directly . 4 during acute infection (day 3), and 12 ganglia from six surviving mice per virus were tested on day 30 by cocultivation for reactivation of latent virus. The results of these tests -pUC18 demonstrate that ts+7 behaved in vivo in a manner similar to 4. 0 li,~~~~~~~~~~~~~ wild-type virus (Table 2). Thus ts+7 was nearly as lethal for CD-1 mice as was wild-type virus. Moreover, it replicated at the site of inoculation and reached trigeminal ganglia, as demonstrated by the presence of virus in eye swabs and ganglia on day 3, and was reactivated from latent infection as am efficiently as was wild-type virus. The DNAs of 7 of 12 virus isolates reactivated from latent infection were tested by Southern blot analysis for the presence of the deletion 9o I a characteristic of ts+7 (Fig. 6). Compared with two isolates derived from mice inoculated with wild-type virus, all seven isolates derived from ts+7-infected mice exhibited the 148-bp 4.* deletion. We conclude from these studies that functional oriL is not required for the establishment or reactivation of latent infection of mice. MVY M V M V M V MVY pUC18 ptkCAT- KOS ts' 7 pDX DISCUSSION oris The data presented herein demonstrate that oriL is not for of HSV in vitro or for the establishment FIG. 4. Origin function test. Total cellular DNAs from CV-1 required growth cells, transfected with the indicated DNAs (lanes 1 to 10) and either and reactivation of latent infection in the murine eye model. mock infected (M) or infected with KOS (V) or plasmid DNAs (lanes These conclusions are based on observations made with 11 to 14), were digested with KpnI only (lane 11) or with KpnI plus two deletion mutants which lack a functional oriL. The first DpnI (all other lanes), electrophoresed in an agarose gel, and of the two mutants isolated, d61, contains mutations in transferred to a nitrocellulose filter. The filter was then probed with ICP8-coding sequences, in oriL-associated sequences, and in 32P-labeled pUC18. Lane 11 contains a mixture of pDX, ptkCAT- several additional sites (27). Although unsuitable for biolog- oris, and pUC18. Lane 12 contains pUC18 only, lane 13 contains ical and molecular characterization because of its multiple ptkCAToris only, and lane 14 shows pDX only. mutations, the replication competence of d61 in ICP8- expressing cells provided the initial clue that HSV-1 can replicate in the absence of a functional oriL. Construction of in bacterial and mammalian cells is unknown. Possible the second oriL deletion mutant, ts+7, provided the neces- mechanisms leading to deletions between short repeats sary tool to examine the replication and latency competence during cloning in bacteria have been discussed by Weller et of HSV-1 lacking a functional oriL. al. (45). DNA sequence analysis and functional characterization of Like all other deleted oriL clones sequenced to date (45) of ts+7 revealed that the 148-bp deletion (i) eliminates all but 11 which the deletions lie at least partially within the inverted bp on the right end of the 144-bp oriL palindrome, (ii) repeat, that of ts+7 eliminated the entire palindrome. Be- occurred between direct repeats, (iii) renders ts+7 oriL cause none of these clones exhibits origin function in assays unresponsive to HSV-1 factors supplied in trans, (iv) has in vitro, the palindrome must contain elements required for little effect on the growth properties of the mutant in vitro, origin function. To date, detailed analysis of the cis-acting and (v) has no detectable effect on the ability of ts+7 to elements necessary for origin function has not been re- establish, maintain, or be reactivated from latent infection. ported, and hence the specific role of the palindrome in It is notable that during the construction of ts+7, the origin function is not known. Distinct homologies between 148-bp deletion in oriL-associated sequences was introduced the highly conserved (22) HSV-1 and HSV-2 oriL and oris by using a plasmid, pKEF-P4, that contains a 55-bp deletion palindromes and papovavirus and adenovirus origins of (45), demonstrating that additional sequences can be lost in DNA replication have been reported (18). Moreover, Elias et mammalian cells during marker transfer of such mutations to al. (personal communication) have reported that selected the viral genome. Of interest is the observation that the large palindromic sequences of HSV-1 and HSV-2 oriL and oris deletion occurred between short direct repeats, as previ- likely constitute the binding site for a viral protein present in ously reported for deletions generated during the cloning of infected cells. oriL in bacteria (45). Whether the mechanism underlying Whatever their role in the initiation of viral DNA replica- deletion of sequences between short repeats applies equally tion, oriL sequences have also been postulated to play a role VOL. 61, 1987 oriL DELETION MUTANTS OF HSV-1 3533

DBP t 10 20 30 40 50 60 70 80 gp 100 ACCACGGGGT GCCGATGAAC CCCGGCGGCT GGCAACGCGG GGTCCCTGCG AGAGGCACAG ATGCTTACGG TCAGGTGCTC CGGCCGGGT GCGTCTGATA TCCTGCCCCA CGGCTACTTG GGGCCGCCGA CCGTTGCGCC CCAGGGACGC TCTCCGTGTC TACGAATGCC AGTCCACGAG GCCCGGCCCA CGCAGACTAT

1 ;0 Rsa 1120 130 140 150 160 170 180 190 200 TGCGGTTGGT ATATGTACAC TTTACCTGGG GGCGTGCCGG ACCGCCCCAG CCCCTCfCAC ACCCCGCGCG TCATCA0CCG GTGG0CGTGG CCGCTATTAT ACGCCAACCA TATACATGTG AAATGGACCC CCGCACGGCC TGGCGGGGTC CGGGA GGTG TGGGCGCCGC AGTAGTCGGC CACCCGCACC GGCGATAATA

ts 7 210 220 230 240 250 260 270 280 290 300 AAAAAAAGTG AGAACGCGAA GCGTTCACTfT99 CCTAA TAATATATAT ATTATTAGGA CAAAGTGCGA ACGCTTCGCG TTCT CATTT TTTTATAATA TTTTTTTCAC TCTTGCGCTT CGCAA CTCAAA CAGGATT ATTATATATA TAATAATCCT GTTTCACGCT TGCGAAGCGC AAGA rGAAA AAAATATTAT pKEF-P4 TpoI I pKEF-P4 10 320 330 3140 350 360~ 370 1--10039040 GCGCAC CCACCGGCTA CGTCACGCTC CTGTCGGCCG CCGGCGGTCC ATAAGCCCGG CCGGCCGGGC CGACGCGAAT AAACCGGGCC GCCGGCCGGG CGCC G GGTGGCCGAT GCAGTGCGAG GACAGCCGGC GGCCGCCAGG TATTCGGGCC GGCCGGCCCG GCTGCGCTTA TTTGGCCCGG CGGCCGGCCC ts*7 4410 420 GCGCCGCGCA GCAGCTCGCC GCCCGG CGCGGCCCGT CGTCGACCGG CGGGCC FIG. 5. Sequence of the 425-bp BstEII-BamHI fragment containing oriL. The bases in bold print (positions 176 to 319) correspond to the 144-bp inverted repeat (45). The sequences in boxes are the short 4- to 6-bp repeats between which deletions in ts+7 and pKEF-P4 occurred, respectively. The location of the RsaI site (position 114 to 118) used to clone the RsaI-BamHI fragment of ts+7 into M13mpl8 for sequencing purposes is also shown. The solid lines beneath bases 106 to 121, 137 to 152, and 176 to 190 represent the proximal, first, and second distal signals, respectively, of the ICP8 promoter (41). The location of the ATA box in the ICP8 promoter is indicated by the dotted line (108 to 114). The transcriptional start site of the ICP8 gene is shown at position 90. The probable ATA box and transcriptional start sites of the polymerase gene are shown at positions 350 to 354 and 370 and 379, respectively (10).

N KOS ts 7 In 0X + in the transcriptional regulation of the two essential genes 1 2 12 3 4 5 6 7 4e- flanking oriL: those encoding the major DNA-binding pro- .p - - tein, ICP8, and DNA polymerase (15). The second distal signal of ICP8 (41) (and possibly regulatory elements of the polymerase gene [10]) is absent in ts+7, yet the virus replicates nearly as efficiently as wild-type virus. As the deletion in ts+7 is among the larger of the HSV-1 oriL deletions to be sequenced and the first to be introduced into the viral genome with little effect on replication competence, the role of palindromic sequences per se in regulation of ICP8 and pol transcription during productive infection re- mains unclear. The measurement of actual levels of transcription of these two genes in ts+7-infected cells is in progress. Although site-directed mutagenesis will be re- Om __m am_ _ _ _ R

TABLE 2. Results of latency tests in CD-1 mice

v Virus titer in specimens w Latent VirusViruLethalityaLethalit Eye swabs" Gangliac infectiond Wild type 9/26 (35) 1.2 x 102 1.5 x 104 12/12 3.7 x 102 2.2 x 104 4.2 x 102 4.7 x 104 1.8 x 103 5.4 x 104 tsI7 3/10 (30) 1.0 x 101 2.3 x 104 10/12 3.1 X 102 2.9 x 104 7.1 x 102 5.8 x 104 1.1 X 1013 6.2 x 104 a Lethality is presented as the ratio of the number of dead animals to the number of animals inoculated with 2 x 106 PFU per eye. b Eye swabs were taken from four mice on day 3 postinoculation. Swabs FIG. 6. Southern blot of DNAs of virus isolates obtained from from both eyes of each mouse were pooled and assayed on Vero cells. Titers trigeminal ganglia of mice inoculated with wild-type virus or ts+7. are total PFU in eye swab suspensions. Two isolates from KOS-infected mice were compared with seven c Two mice were sacrificed on day 3 postinoculation, and individual ganglia isolates from ts+7-infected mice. Total DNAs from cells infected were and directly on Vero cells. Titers are PFU per homogenized plated with each isolate were cleaved with on an ganglion. BamHI, electrophoresed d On day 30 postinoculation, ganglia were removed from six mice (12 agarose gel, and transferred to a nitrocellulose filter, and the filter ganglia) and assayed for reactivatable virus by cocultivation with Vero cells. was probed with the KpnIV fragment which hybridizes to both the No infectious virus was detected in six trigeminal ganglia of mice inoculated BamHI R and V fragments. Control KOS and ts+7 DNAs are shown with wild-type virus, removed on day 30, homogenized, and plated directly. in the right-hand two lanes. 3534 POLVINO-BODNAR ET AL. J. VIROL. quiired to elucidate the specific roles of oriL sequences in ICP4 permissive for early gene expression. J. Virol. 52:767-776. origin function and transcriptional regulation, the availability 6. DeLuca, N. A., and P. A. Schaffer. 1985. Activation of immedi- of an isogenic series of sequenced deletion mutations in oriL ate-early, early, and late promoters by temperature-sensitive and wild-type forms of herpes simplex virus type 1 protein should prove useful, with the unfortunate caveat that their ICP4. Mol. Cell. Biol. 5:1997-2008. introduction into the viral genome by marker transfer may 7. Frenkel, N. 1981. Defective interfering herpesviruses, p. 91-120. result in further sequence loss. In A. J. Nahmias, W. R. Dowdle, and R. F. Schinazi (ed.), The The data presented herein demonstrate that oriL is not human herpesviruses. Elsevier, New York. required for virus replication in vitro, implying that two 8. Frenkel, N., H. Locker, and D. A. Vlazny. 1980. Studies of copies of oris are sufficient for this purpose. In a recent defective herpes simplex viruses. Ann. N.Y. Acad. Sci. report, Longnecker and Roizman reported the viability of a 354:347-370. mutant of HSV-1 strain F, R7023, that contains oriL and only 9. Friedmann, A., J. Shlomai, and Y. Becker. 1977. Electron copy (23). this mutant demon- microscopy of herpes simplex virus DNA molecules isolated one of oris The viability of from infected cells by centrifugation in CsCl density gradients. strates that the two remaining origins (one copy of oris and J. Gen. Virol. 34:507-522. oriL) are sufficient for virus replication. The observations 10. Gibbs, J. S., H. C. Chiou, J. D. Hall, D. W. Mount, M. J. that viable mut4nts of HSV-1 may lack either oriL or one of Retondo, S. K. Weller, and D. M. Coen. 1985. Sequence and two copies of oris raise interesting questions regarding the mapping analyses of the herpes simplex virus DNA polymerase role(s) of the two types of origin in the biology of HSV-1. (i) gene predict a C-terminal substrate binding domain. Proc. Natl. Is either type of origin preferentially used during the lytic or Acad. Sci. USA 82:7969-7973. latent modes of infection, and (ii) why have both been 11. Goldin, A. L., R. M. Sandri-Goldin, M. Levine, and J. C. conserved in the HSV-1 and HSV-2 genomes? Glorioso. 1981. Cloning of herpes simplex virus type 1 se- regard to the former question, we have demonstrated quences representing the whole genome. J. Virol. 38:50-58. With 12. Gray, C. P., and H. C. Kaerner. 1984. Sequence of the putative the replication and latency competence of ts+7 in vivo, origin of replication in the UL region of herpes simplex virus implying that an intact oriL is not required for the establish- type 1 ANG DNA. J. Gen. Virol. 65:2109-2119. ment, maintenance, or reactivation of latency. The only 13. Grunstein, M., and D. Hogness. 1975. Colony hybridization: a other virus for which a latency-specific origin has been method for the isolation of cloned DNAs that contain a specific suggested is Epstein-Barr virus, in which oriP has been gene. Proc. Natl. Acad. Sci. USA 72:3961-3965. shown to function during latent infection (42, 47). In this 14. Hirsch, I., G. Cabral, M. Patterson, and N. Biswal. 1977. Studies system, however, no origins other than oriP have yet been on the intracellular replicating DNA of herpes simplex virus identified (although they may exist), and it has not been type 1. Virology 81:48-61. shown that oriP does not also function during lytic infection. 15. Holland, L. E., R. M. Sandri-Goldin, A. L. Goldin, J. C. into the second question be gained Glorioso, and M. Levine. 1984. Transcriptional and genetic Some insight might by analyses of the herpes simplex virus type 1 genome: coordinates comparison of HSV with other herpesviruses. In this regard, 0.29 to 0.45. J. Virol. 49:947-959. it is notable that the genome of varicella-zoster virus, a virus 16. Kaerner, H. C., I. B. Maichle, A. Ott, and C. H. Schroder. 1979. which shares extensive biological and DNA structural simi- Origin of two different classes of defective HSV-1 Angelotti larities with HSV, contains a diploid oris but lacks an oriL DNA. Nucleic Acids Res. 6:1467-1478. equivalent (39). 17. Kaerner, H. C., A. Ott-Hartmann, R. Schatten, C. H. Schroder, and C. P. Gray. 1981. Amplification of a short nucleotide sequence in the repeat units of defective herpes simplex virus ACKNOWLEDGMENTS type 1 Angelotti DNA. J. Virol. 39:75-81. We thank N. DeLuca for valuable discussions, M. Bush, D. Coen, 18. Knopf, C. W., B. Spies, and H. C. Kaerner. 1986. The DNA J. Jacobson, D. Knipe, K. Tyler, E. Villareal, and D. Yager for replication origins of herpes simplex virus type 1 strain assistance in latency tests of ts+7, and M. Cook for manuscript Angelotti. Nucleic Acids Res. 14:8655-8667. preparation. 19. Knopf, C. W., G. Strauss, A. Ott-Harman, R. Schatten, and This investigation was supported by Public Health Service Pro- H. C. Kaerner. 1983. Herpes simplex virus defective genomes: gram Project grant no. CA21082 from the National Cancer Institute structure of HSV-1 ANG defective DNA of class II and encoded and grant no. A124010 from the National Institute of Allergy and polypeptides. J. Gen. Virol. 64:2455-2470. Infectious Diseases. M.P.-B. is supported by a Special Fellowship 20. Lacks, S., and B. Greenberg. 1977. Complementary specificity from the Leukemia Society of America. P.K.O. was supported by of restriction endonucleases of Diplococcus pneumoniae with Postdoctoral Fellowship no. DRG-840 from the Damon Runyon- respect to DNA methylation. J. Mol. Biol. 114:153-168. Walter Winchell Cancer Fund. 21. Lee, C. K., and D. M. Knipe. 1983. Thermolabile in vivo DNA-binding activity associated with a protein encoded by LITERATURE CITED mutants of herpes simplex virus type 1. J. Virol. 46:909-919. 1. Ben-Porat, T. 1982. Organization and replication of herpesvirus 22. Lockshon, D., and D. A. Galloway. 1986. Cloning and charac- DNA, p. 147-172. In A. S. Kaplan (ed.), Organization and terization of oriL2, a large palindromic DNA replication origin of replication of viral DNA. 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