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.