A Virus with a Mutation in the ICP4-Binding Site in the L/ST Promoter of Herpes Simplex Virus Type 1, but Not a Virus with a Mutation in Open Reading Frame P, Exhibits Cell-Type-Specific Expression of γ134.5 Transcripts and Latency-Associated Transcripts

Lily Yeh Lee; Priscilla A Schaffer

doi:10.1128/jvi.72.5.4250-4264.1998

. 1998 May;72(5):4250–4264. doi: 10.1128/jvi.72.5.4250-4264.1998

A Virus with a Mutation in the ICP4-Binding Site in the L/ST Promoter of Herpes Simplex Virus Type 1, but Not a Virus with a Mutation in Open Reading Frame P, Exhibits Cell-Type-Specific Expression of γ₁34.5 Transcripts and Latency-Associated Transcripts

Lily Yeh Lee ¹, Priscilla A Schaffer ^1,^*

PMCID: PMC109655 PMID: 9557715

Abstract

The herpes simplex virus type 1 L/S junction-spanning transcripts (L/STs) are a family of multisized transcripts expressed at high levels in cells infected with mutant viruses that (i) do not express ICP4, (ii) specify forms of ICP4 unable to bind to the consensus ICP4 binding site, or (iii) contain mutations in the ICP4 binding site located at the transcriptional start site of the L/STs. By extension, the failure to detect the L/STs in wild-type virus-infected cells is due to the repressive effect of ICP4 bound to its cognate binding site upstream of the L/ST transcription initiation site. ORF-P, the first and largest open reading frame (ORF) encoded by the L/STs, overlaps >90% of the ORF encoding ORF-34.5, a putative neurovirulence factor, which is transcribed from the opposite DNA strand. Viruses with mutations in the overlapping region of ORF-P and ICP34.5 exhibit premature shutoff of infected-cell protein synthesis and are highly attenuated following intracranial inoculation of juvenile mice. To determine whether the premature protein shutoff and neuroattenuated phenotypes of ORF-P ORF-34.5 double mutants are a consequence of alterations in ORF-P, ORF-34.5, or both, viruses containing mutations only in ORF-P or only in the ICP4 binding site in the L/ST promoter were isolated and characterized. Mutant virus L/ST-n38 contains a single-base-pair transition mutation in ORF-P codon 38, resulting in translational termination of the ORF-P protein (OPP). This mutation does not alter the amino acid sequence of ICP34.5. Expression of a truncated form of OPP by mutant virus L/ST-n38 did not result in premature shutoff of infected-cell protein synthesis and produced no other observable phenotype relative to wild-type virus in in vitro tests. Moreover, the 50% lethal dose (LD₅₀) of L/ST-n38 was comparable to that of wild-type virus following intracranial inoculation of 3-week-old mice, as were the latency and reactivation phenotypes of the virus. These properties of L/ST-n38 indicate that the attenuated phenotype of ORF-P ORF-34.5 double mutants is a consequence of mutations that affect the function of ICP34.5 and not the function of OPP. Mutant virus L/ST-4BS contains four single-base-pair substitutions in the ICP4 binding site in the L/ST promoter that abrogate the binding of ICP4 to this site, leading to high-level expression of the L/STs and OPP. L/ST-4BS induced premature shutoff of viral and cellular protein synthesis and was slightly growth restricted in cells of neural lineage (SK-N-SH human neuroblastoma cells) but was wild type for these two parameters in cells of nonneural lineage (immortalized primate Vero cells). Of particular interest was the observation that L/ST-4BS exhibited cell-type-specific expression of both the γ₁34.5 transcripts and the latency-associated transcripts (LATs). Thus, expression of these transcripts was barely detectable in cells of neural lineage (NB41A3 mouse neuroblastoma cells) but was wild type in Vero cells. In vivo, L/ST-4BS was reactivated from mouse trigeminal ganglia with reduced efficiency and delayed kinetics relative to wild-type virus. L/ST-4BS was completely attenuated for neurovirulence (LD₅₀ > 10⁶ PFU) relative to wild-type virus (LD₅₀ < 900 PFU), although the four single-base-pair substitutions lie outside the coding region for the neurovirulence factor, ICP34.5. Collectively, the complex in vitro and in vivo phenotypes of L/ST-4BS can be attributed to (i) disruptions of the ICP4 binding site in the L/ST promoter and subsequent overexpression of the L/STs and OPP; (ii) alterations in ORF-O, which is also mutated in L/ST-4BS; or (iii) alterations in other cryptic genes or cis-acting elements.

Herpes simplex virus type 1 (HSV-1) is a neurotropic virus that causes a variety of clinically significant diseases ranging from superficial cutaneous lesions to life-threatening encephalitis (49). The designation of HSV-1 as a neurotropic virus is based on its ability to produce both lytic and latent infections of neurons of the peripheral and central nervous systems. The extent to which a given strain of HSV-1 can gain access to and travel within neurons (neuroinvasiveness) and the relative efficiency with which a strain produces neuronal damage and death (neurovirulence) are major determinants of the disease-producing capabilities of that strain. Identification of the viral genes that control neuroinvasiveness and neurovirulence and elucidation of the functions of the protein products of these genes are necessary to understand the mechanism by which HSV-1 causes disease.

By definition, any viral gene which, when mutated, results in the failure of the virus to enter or spread in neurons or to replicate in epithelial or neuronal cells can be said to affect neuroinvasiveness or neurovirulence, respectively. Some viral genes, however, have unique properties which appear to function exclusively or predominantly in neurons. Notably, a number of viral genes which affect neurovirulence and which, collectively, determine whether infection will be productive or latent are located within the abb′a′c′ca repeat regions flanking the unique long and short regions of the HSV-1 genome (Fig. 1) (reviewed in reference 50).

The genes specifying the L/S junction-spanning transcripts (L/STs) and the γ₁34.5 gene, are located within these repeat regions and constitute the subject of this paper. The L/STs are a series of 5′-coterminal polyadenylated RNAs transcribed from the same DNA strand as the latency-associated transcripts (LATs) (Fig. 1) (50). The L/STs range in size from 2.3 to >9.0 kb and are expressed at detectable levels only by mutant viruses that express no functional ICP4 or that lack an intact ICP4 binding site at the transcription initiation site of the L/ST gene (50). These properties suggest that ICP4 bound to its cognate binding site in the L/ST promoter blocks the expression of the L/STs. The L/STs overlap the 3′ terminal 2.3 kb of the 8-kb minor LAT on the same strand, and, critical to the subject of this paper, the L/STs overlap and are antisense to the γ₁34.5 gene (Fig. 1).

The first and largest open reading frame (ORF) in the L/STs is ORF-P, which specifies the ORF-P protein (OPP) (Fig. 1C and Fig. 10). ORF-P is conserved in all HSV-1 strains whose genomes have been sequenced in this region. OPP is expressed at detectable levels only under conditions which allow expression of the L/STs (26, 50). Overlapping the 5′ half of ORF-P, and encoded by the same DNA strand but in another reading frame, is ORF-O, which encodes the ORF-O protein (OOP) (Fig. 1C). In strain 17syn+, ORF-O is approximately 300 codons in length. In strains KOS, F, MGH10, and CVG2, a single-base-pair substitution terminates ORF-O after approximately 160 codons. Randall et al. have recently provided evidence suggesting that, like ORF-P, ORF-O is downregulated by ICP4 (34). The translational start codon of ORF-O lies immediately downstream of the TATA box in the L/ST promoter; thus, ORF-O is probably encoded by the LATs rather than the L/STs. Recently, however, Randall et al. have suggested that in strain F, ORF-O and ORF-P initiate from the same methionine codon and that the initiation of ORF-O at this codon results from frameshift or editing of its RNA (34).

The L/STs overlap and are antisense to the γ₁34.5 gene. The γ₁34.5 gene is located between the genes specifying two major immediate-early (IE) transcriptional regulatory proteins, ICP0 and ICP4 (Fig. 1). The ∼8-kb LAT, specified by the opposite strand, overlaps and is antisense to the entire ICP0 and γ₁34.5 genes. The γ₁34.5 gene encodes ICP34.5, a protein with an apparent molecular mass of 44 kDa (2, 11, 15). The γ₁34.5 ORF (ORF-34.5) is conserved in all HSV-1 strains that have been sequenced to date (14, 18). Viruses with deletions in ORF-34.5 are replication competent in vitro; however, they are highly attenuated for neurovirulence in vivo following intracranial inoculation of mice (6, 14, 35, 46–48). Other properties of γ₁34.5 mutant viruses include (i) more efficient replication at some primary sites of infection (e.g., the footpad and vagina) than at others (e.g., the eye) (49), (ii) defective replication in the sensory ganglia of mice, guinea pigs, and rabbits and in the central nervous system of mice (6, 14, 30–32, 35, 45, 48); (iii) wild-type growth in most cell types but restricted growth in SK-N-SH (human neuroblastoma) (13), CV-1 (African green monkey kidney) (32), murine 10T1/2 (stationary-phase primary mouse embryo), and 3T6 cells (6, 7); (iv) premature shutoff of all protein synthesis (cellular and viral) following infection of SK-N-SH cells (12, 13); and (v) reduced establishment of latency compared to wild-type virus (31, 45).

During the course of the studies of ICP34.5 just described, a number of mutant viruses were constructed and characterized. By virtue of the complete overlap of the γ₁34.5 gene with the L/ST and the 8-kb LATs, mutations introduced into the γ₁34.5 gene were also introduced into sequences specifying the L/STs and the LATs. Consequently, “γ₁34.5 mutant viruses” are either ORF-34.5 ORF-P double mutants or ORF-34.5 ORF-P ORF-O triple mutants (Fig. 1).

Because ORF-O is probably contained in the LATs rather than the L/STs, as noted above, we focused the present investigation on the contribution of ORF-P to the phenotype of ORF-34.5 ORF-P double-mutant viruses. We approached this problem by using two genetic strategies. In the first, we mutated ORF-P without altering ORF-34.5, and in the second, we altered the transcriptional regulation of the L/STs by greatly increasing the levels of L/ST and ORF-P expression. We report here the construction and characterization of two mutant viruses: one, L/ST-n38, containing a nonsense mutation that terminates ORF-P translation at amino acid 38 but does not alter the sequence of ICP34.5 encoded by the complementary DNA strand (Fig. 1G; also see Fig. 10), and another, L/ST-4BS, containing a mutation in the L/ST promoter that derepresses L/ST expression by abrogating ICP4 binding to its cognate binding site near the transcription initiation site of the L/STs (Fig. 1G; also see Fig. 10). The properties of L/ST-n38 demonstrate that expression of a truncated OPP produces no observable phenotype relative to wild-type virus in vitro or in vivo. By contrast, L/ST-4BS, which is mutated in the ICP4 binding site in the L/ST promoter and overexpresses the L/STs and OPP, is highly attenuated following intracranial inoculation of juvenile mice, yet replicates efficiently in the mouse eye and trigeminal ganglia (TGG) without eliciting an adverse host response. Of special interest was the observation that L/ST-4BS induced cell-type-specific expression of both γ₁34.5 transcripts and LATs since both types of transcript were abundant in Vero cells but barely detectable in mouse neuroblastoma cells (NB41A3). The significance of these findings to the assignment of specific functions to OPP and ICP34.5 can be summarized as follows: (i) ICP34.5 and not ORF-P is the neurovirulence factor responsible for attenuation of ORF-P ORF-34.5 double mutants and (ii) a factor or element affected by the 4BS mutation mediates cell-type-specific expression of the γ₁34.5 gene and the LATs.

MATERIALS AND METHODS

Cells and viruses.

African green monkey kidney cells (Vero; ATCC CCL 81) and human embryonic lung cells (HEL cells) were grown and maintained in Dulbecco’s modified Eagle’s medium as previously described (38). Mouse neuroblastoma cells (NB41A3; ATCC CCL147) were propagated in Ham’s F10 medium supplemented with 2.5% fetal calf serum and 15% horse serum. Human neuroblastoma cells (SK-N-SH) were kindly provided by Patrick Vaughn (University of California, Irvine, Calif.) and were propagated as described above for Vero and HEL cells. Rat pheochromocytoma cells (PC12) were the gift of John Wagner (Cornell University Medical College, New York, N.Y.) and were propagated and maintained as described previously (21).

The KOS strain of HSV-1 (41) and mutants derived from KOS were used in these studies. The ICP4 nonsense mutant, n12, and the ICP0 null mutant, 7134, were grown and assayed as previously described (9, 17). Mutant virus L/ST-n38 contains a nonsense mutation in ORF-P, and mutant virus L/ST-4BS contains four single-base-pair mutations in the ICP4 binding site in the L/ST promoter. L/ST-n38R is a rescuant of L/ST-n38, and L/ST-4BSR is a rescuant of L/ST-4BS. The 17syn+ wild-type strain of HSV-1 and the ICP34.5 nonsense mutant, 1771, derived from 17syn+, were generous gifts of Alasdair MacLean (MRC Virology Unit, Glasgow, United Kingdom) and have been described previously (30).

Plasmids.

For subcloning purposes, pBamK (Fig. 1F), containing the KOS BamHI K fragment from pSG28 (40), and pNco (Fig. 1G), containing the 1.75-kbp NcoI subclone of pBamK, were constructed as described previously (50). Plasmid pL/ST-n38 (Fig. 1G) is identical to pNco, except that it contains a single-base-pair transition in ORF-P. Mutagenesis was accomplished by in vitro phagemid mutagenesis with pNco as the parental plasmid and the mutant oligonucleotide 5′-ACCAGGTGACGCACCCGG-3′. The transition mutation from G to A (underlined) eliminates the cleavage site for the restriction enzyme PflMI. Plasmid pL/ST-4BS^m (Fig. 1G) is identical to pNco except for four single-base-pair changes in the ICP4 binding site near the transcriptional start site of the L/STs. The mutations were introduced by in vitro phagemid mutagenesis with pNco as the parental plasmid and the mutant oligonucleotide 5′-GCGGCTCCTGCCAGCGCTCCTCCGGAGAGC-3′. The mutation of ATCGTCTCT to AGCGCTCCT generates a new restriction site for the endonuclease Eco47III.

For in vitro transcription and translation of L/ST ORF-P, a 2.6-kb SacI-NcoI fragment derived from pBamK was cloned into pGEM5Zf(+) (Promega Corp., Madison, Wis.) to generate plasmid pL/ST-Nxi (Fig. 1H). To construct pL/ST-NxiLM3 (Fig. 1H), the 0.7-kb SacI-BstEII fragment of pL/ST-Nxi was replaced by the corresponding fragment from pL/ST-n38. Plasmid pL/ST-NxiLM3 was used to test the efficacy of the stop codon.

The plasmids used in transient-expression assays were as follows. Plasmid pWR-CAT, an expression vector carrying a promoterless chloramphenicol acetyltransferase (CAT) gene, was described previously (33). A modified vector, pWR-CAT⁺ (Fig. 1I), was used as the parent plasmid for all transient-expression plasmids constructed in this study. Plasmid pWR-CAT⁺ was constructed by cloning the EcoRI (blunt ended with Klenow)-HindIII linker from pGEM3ZF(+) into the Ecl136II-HindIII sites of pWR-CAT. Plasmid pL/ST-U-CAT (Fig. 1I), containing the L/ST promoter region, was constructed by cloning a fragment spanning the NcoI site at −935 through the entire 5′ untranslated region of the L/STs to position +122 in pWR-CAT⁺. In pL/ST-4BS^m-CAT (Fig. 1I), the L/ST promoter (−935 to +1) in pL/ST-U-CAT was replaced by the corresponding fragment (NcoI-BspEI) from pL/ST-4BS^m, producing a plasmid in which the reporter CAT gene is driven by the L/ST promoter carrying the mutated ICP4 binding site. Effector plasmids, pSH, containing the wild-type ICP0 gene, and pn11, containing the wild-type ICP4 gene, have been described previously (9, 16).

To generate L/ST mutant viruses, the following plasmids were constructed. p0V (Fig. J) contains the 7.1-kb PstI-DraI fragment from pSG28 cloned into the PstI-Ecl136II sites in pGEM3Zf(+) (Promega). p0V-n38 contains the 0.7-kb Tsp509I-DraIII fragment from pLST-n38 cloned into the EcoRI-PflMI sites of p0V. p0V-4BS contains the 0.3-kb MscI-PflMI fragment from pLST-4BS^m cloned into the same sites in p0V. p0V-4/38 contains the 0.7-kb Tsp509I-DraIII fragment from pLST-n38 cloned into the EcoRI-PflMI sites of p0V-4BS.

Riboprobes EBN9-LAT, EBNc3-LAT(S), 4Sma, and BbSLAT have been described previously (Fig. 1D) (50). EBN9-LAT and EBNc3-LAT(S) were used to detect the L/STs; 4Sma was used to detect the ICP4 transcript; BbSLAT was used to detect the LATs. Riboprobe EBSN34.5, used to detect ICP34.5 transcripts, was derived from plasmid pEBSN34.5 (Fig. 1E) containing the SacI-NotI fragment of pNco (50).

Plasmids pEBNc3-LAT(S), pEBSN34.5, and pEBD1Ms (Fig. 1E) were also nick translated and used as probes in Southern blot analysis. pNco, pEBNc3-LAT(S), and pEBSN34.5 were described above. pEBD1Ms contains the 1.3-kb DraI-MscI fragment spanning the a sequences cloned into the HincII site of pGEM3Zf(+).

To express and purify OPP for the production of anti-OPP antibodies, a number of plasmids specifying glutathione S-transferase (GST) fusion proteins were constructed. In plasmid pG-L/ST (Fig. 1K), the ORF-P sequence, from its translational start site through the DraI restriction site in the a sequences, was cloned in frame to the 3′ end of the GST ORF in pGEX-2TK (Pharmacia Biotech Inc., Piscataway, N.J.) with BamHI linkers. To generate pG-L/ST-C (Fig. 1K), the plasmid expressing only the carboxyl-terminal 52 amino acids of ORF-P, a 278-bp NotI-EcoRI fragment from pG-L/ST, was blunt ended with Klenow enzyme and cloned in frame with GST in pGEX-2TK.

Plasmid mutagenesis.

Competent Escherichia coli CJ236 (dut ung) was transformed with plasmid pNco, and single-stranded phagemid DNA was prepared as instructed by the manufacturer (Promega). Oligonucleotide-directed mutagenesis was then performed by the Kunkel method (23). The mutations were confirmed by the absence (pL/ST-n38) or appearance (pL/ST-4BS^m) of specific restriction enzyme sites and by DNA sequencing.

In vitro transcription and translation.

Linearized template DNA (2 μg) was transcribed in vitro with SP6 polymerase to generate capped RNA as specified by the manufacturer (Promega). Half of the RNA was used for in vitro translation; the other half was used for Northern blot analysis. In vitro translation was performed, as specified by the manufacturer (Promega), with rabbit reticulocyte lysate and either [³⁵S]cysteine or [³⁵S]methionine (New England Nuclear, Boston, Mass.). Aliquots (2.5 μl) of in vitro-translated proteins were analyzed by denaturing polyacrylamide gel electrophoresis.

Gel mobility shift assays.

To generate probes for use in-gel mobility shift assays and competition experiments, single-stranded 30-mer oligonucleotides corresponding to the plus and minus strands of the wild-type and mutated ICP4 binding sites were synthesized by the Molecular Biology Core Facility at the Dana-Farber Cancer Institute. The nucleotide sequence of the wild-type binding site is 5′-GCGGCTCCTGCCATCGTCTCTCCGGAGAGC-3′, and that of the mutated binding site is 5′-GCGGCTCCTGCCAGCGCTCCTCCGGAGAGC-3′. (The core ICP4 binding site is underlined.) Complementary single-stranded probes were annealed by heating to 98°C for 5 s followed by slow cooling to 68°C, incubation at 68°C for 1 h, and slow cooling to room temperature. Double-stranded probes were separated from unannealed single-stranded oligonucleotides by polyacrylamide gel electrophoresis (PAGE), cut out of the gel, and electroeluted overnight at 35 V. Gel-purified double-stranded probes were radiolabeled with T4 polynucleotide kinase (New England Biolabs, Beverley, Mass.) and [γ-³²P]ATP (New England Nuclear).

As the source of proteins, Vero cells were mock infected or infected with KOS or n12, an ICP4 null mutant (17), at a multiplicity of infection of 10 PFU/cell. Extracts were prepared at 8 h postinfection (p.i.) by the method of Andrews and Faller (3). Individual binding-reaction mixtures contained 6 to 7 μg of protein extract, 1 ng of ³²P-labeled probe, 20 mM HEPES, 50 mM NaCl, 1 mM EDTA, 10% glycerol, and 4 μg of poly(dI-dC) (Boehringer Mannheim, Indianapolis, Ind.). The binding-reaction mixtures were incubated for 30 min at 25°C. For antibody supershift assays, 1 μl of 58S (a monoclonal antibody to ICP4 [43]) was added after the 30-min binding reaction and the mixture was incubated for an additional 10 min at 25°C. Protein-DNA complexes were separated by electrophoresis in 4% polyacrylamide gels (80:1 acrylamide/bisacrylamide) prepared and run in 0.5 × Tris-borate-EDTA buffer (TBE) (39). The gels were prerun at 170 V for 1 h. In competition experiments, radiolabeled probe and unlabeled competitor probe were premixed before the addition of buffer and extract. The gels were dried on filter paper and exposed to XAR-5 film (Kodak, Rochester, N.Y.).

Transient-expression assays.

Vero cells (10⁶ cells) were plated in 60-mm-diameter petri dishes 24 h prior to transfection. The medium was replaced 3 h prior to transfection. Reporter and effector plasmids were transfected by the calcium phosphate–bis-ethane sulfonic acid method described by Ausubel et al. (4). Cell lysates for determination of CAT activity were prepared approximately 40 h posttransfection and assayed by the phase extraction method (42) as modified by Frazier et al. (20). For transfection/superinfection assays, monolayers were infected with virus at an MOI of 10 PFU/cell 24 h after transfection and lysates were prepared at 3, 6, 12, and 24 h p.i.

Isolation of mutant and rescuant viruses.

To isolate mutant viruses, L/ST-n38 and L/ST-4BS, Vero cells (5 × 10⁵ cells) were seeded in 60-mm-diameter dishes and cotransfected 24 h later with 1 μg of infectious 7134 DNA and 10 μg of a DNA fragment containing the mutation (i.e., the 7,002-bp PstI-EcoRV fragment from p0V-n38 or the 7,129-bp PstI-EcoRI fragment from p0V-4BS [Fig. 1J]). Six days later, when cytopathic effects were generalized, the cultures were harvested, frozen and thawed, sonicated for 1 min, clarified by low-speed centrifugation, plated on Vero cell monolayers, and overlaid with methylcellulose. After incubation at 37°C for 4 days, 2 ml of 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-Gal) (300 μg/ml) in neutral red was added to each 35-mm petri dish. Parental 7134 virus would yield blue plaques because the ICP0 sequence has been replaced by lacZ. Recombinant plaques would be white if the wild-type ICP0 sequences had replaced lacZ sequences. In this way, the n38 and 4BS mutations were “piggybacked” simultaneously into the genome in a subset of recombinants. White plaques were picked and assayed by Southern blot analysis for the presence of the desired mutation in both copies of the b repeat sequences. Mutants were subjected to two additional rounds of plaque purification. One viral isolate obtained from cotransfection of infectious 7134 and linearized p0V-n38 DNAs was designated L/ST-n38, whereas an isolate from the cotransfection of 7134 and p0V-4BS DNAs was designated L/ST-4BS.

To isolate rescuant viruses, L/ST-n38R and L/ST-4BSR, cotransfections were performed as described above with 1 μg of infectious L/ST-n38 or L/ST-4BS DNA and 0.5 μg of the wild-type 1.75-kB NcoI fragment from p0V. Rescuants generated from cotransfections were identified by Southern blot analysis, plaque purified two additional times, and designated L/ST-n38R and L/ST-4BSR, respectively.

Southern blot analysis was used to confirm the genotype of L/ST-n38, 4ST-4BS, and rescuants thereof. For this purpose, 5 × 10⁵ Vero cells were infected with 0.01 PFU of the designated viruses per cell. When cytopathic effects were generalized, the cells were scraped into medium, frozen at −70°C, thawed, sonicated for 1 min, pelleted, and resuspended in 400 μl of lysis buffer (10 mM Tris-HCl [pH 8], 10 mM EDTA [pH 8], 2% sodium dodecyl sulfate [SDS], 400 μg of proteinase K per ml.). Lysis was carried out overnight at 37°C. DNA was extracted once with phenol and twice with phenol-chloroform-isoamyl alcohol (25:24:1), ethanol precipitated, and resuspended in deionized, distilled H₂O. One-third of the DNA yield was digested by restriction enzymes, separated in an 0.8% agarose gel, and transferred onto a nitrocellulose membrane by the method of Southern (39, 44). The membranes were probed with nick-translated pEBSN34.5, pEBNc3-LAT(S), or pEBD1Ms. Briefly, the nick translation reaction mixture contained 0.5 μg of plasmid DNA, 40 nM each dATP and dTTP (Pharmacia), about 625 nM each [α-³²P]dCTP and [α-³²P]dGTP (New England Nuclear), 2 μU of DNase I (Promega), and 10 U of E. coli DNA polymerase I (New England Biolabs) in 1 × polymerase buffer (10 mM Tris-HCl [pH 7.5], 5 mM MgCl₂, 7.5 mM dithiothreitol). The reaction mixture was incubated at 16°C for 1 h. Free nucleotides were removed with Sephadex G-25 Quick-Spin (TE) columns (Boehringer-Mannheim). To confirm the presence of the mutation in each of the L/ST mutant viruses, L/ST-n38 and L/ST-4BS, a 1.75-kb NcoI fragment containing the mutation was cloned as p38Nco and p4BSNco, respectively, and sequenced (Fig. 1L). The 1.75-kb fragment corresponds to the fragment present in pNco.

Western blot analysis.

Cells were infected at a multiplicity of 10 PFU/cell and harvested at the indicated times p.i. Lysates were prepared as follows. Infected monolayers were washed twice with cold phosphate-buffered saline (PBS) (39), and scraped into PBS in the presence of protease inhibitors (10 μg of TPCK [N-tosyl-l-phenylalanine chloromethyl ketone] per ml and 2 mM phenylmethylsulfonyl fluoride [PMSF]). The cells were pelleted and resuspended in a small volume of PBS-TPCK-PMSF, aliquoted, and quick-frozen in liquid nitrogen. Aliquots were thawed only once for use. The protein concentration was determined with the Bio-Rad protein assay reagent, as specified by the manufacturer. Cell lysis was completed by the addition of SDS-PAGE loading buffer (50 mM Tris, 2% SDS, 0.7 M β-mercaptoethanol, 2.75% sucrose). Proteins were separated by denaturing polyacrylamide gel electrophoresis (10) and transferred to Immobilon-P membranes (Millipore Corp., Bedford, Mass.).

Western blot analysis was performed as follows. Blots were blocked for 2 h with 3% bovine serum albumin (BSA) in PBST (PBS containing 0.05% Tween 20). The primary antibody was diluted in 3% BSA–PBST and incubated with the blot for 1 h. The blot was then washed with two quick rinses, two 5-min washes, and three 10-min washes with PBST; it was then blocked again with 3% BSA–PBST for 20 min. The same procedure was repeated with the secondary antibody, rabbit anti-mouse immunoglobulin G (Cappel Laboratories, Durham, N.C.), when applicable, and with horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G (Bio-Rad). Immunodetection was performed with the ECL chemiluminescence reagent from Amersham Life Science (Arlington Heights, Ill.) and visualized with BioMax-MR film (Kodak).

Mouse monoclonal antibodies H1112 and H1113 are specific for ICP0 and ICP27, respectively (1) (Goodwin Institute for Cancer Research, Plantation, Fla.). Monoclonal antibody 58S, specific for ICP4, was generated from a hybridoma cell line obtained from the American Type Culture Collection (43). Rabbit polyclonal antibody R45, which is directed against gD, was kindly provided by R. Courtney (Pennsylvania State University Health Science Center, Hershey, Pa.). Rabbit polyclonal antibody D111, directed against OPP, was generated in this laboratory as described below.

G-L/ST-C fusion protein purification.

To prepare the G-L/ST-C fusion protein, an overnight culture of bacteria transformed with pG-L/ST-C was diluted 1:50 and allowed to grow until its optical density at 595 nm reached 1.0. Fusion protein expression was induced by the addition of isopropyl-β-d-thiogalactopyranoside (IPTG) to a final concentration of 1 mM. At 2 h after induction, bacteria were pelleted and resuspended in Tris-buffered saline (TBS) (39) containing 10 mM EDTA, 75 μg of TPCK per ml, 2 mM PMSF, and 2 mM leupeptin. The bacteria were lysed by sonication, incubation with Triton X-100 (final concentration, 1%), and rocking for 30 min at 4°C. The lysate was cleared by centrifugation for 15 min at 26,890 × g in an SS34 rotor. Glycerol was added to the supernatant fluid to a final concentration of 10%. The G-L/ST-C fusion protein was purified on glutathione-Sepharose 4B beads (Pharmacia) as specified by the manufacturer instructions and eluted from beads with 20 mM glutathione in 100 mM Tris [pH 8]–120 mM NaCl.

Preparation of antibodies to OPP.

Rabbit polyclonal antibodies were raised against the purified G-L/ST-C fusion protein by HRP, Inc (Denver, Pa.). Two New Zealand White rabbits were inoculated intradermally with 250 μg of G-L/ST-C each and given booster doses three times at 2-week intervals by subcutaneous dorsal injections of 125 μg of G-L/ST-C prior to obtaining antisera. Antisera from both rabbits reacted with G-L/ST-C in Western blot analysis. Anti-G-L/ST-C antibodies were purified from one of the antisera, DF111, by the blot purification method of Harlow and Lane (22).

Analysis of infected-cell polypeptides.

Vero cells (5 × 10⁵ cells) or SK-N-SH cells (1 × 10⁷ cells) were seeded in 35-mm-diameter petri dishes 24 h prior to infection. The cells were infected with the indicated viruses at a multiplicity of 10 PFU/cell. At the designated times p.i., the culture medium was replaced with 1 ml of methionine-minus medium (ICN Biomedicals, Inc., Costa Mesa, Calif.) supplemented with 50 μCi of [³⁵S]methionine (New England Nuclear). Monolayers were labeled for 30 min at 37°C and then processed as described above to obtain protein lysates. Individual proteins in lysates (5 μg of Vero and 10 μg of SK-N-SH cell lysate) were separated by SDS-PAGE and analyzed by autoradiography as described previously (24).

PAGE.

SDS-PAGE of infected cell lysates was performed by the method of Laemmli (24). Protein samples were boiled for 5 min in sample buffer (50 mM Tris, 2% SDS, 0.7 M β-mercaptoethanol, 2.75% sucrose, Coomassie blue). In vitro-translated proteins were also analyzed on Tricine gels. The running gel contained 20% acrylamide (acrylamide/bisacrylamide ratio, 30:1), 1 M Tris (pH 8.45), 0.1% SDS, and 16.7% glycerol. The stacking gel contained 4% acrylamide, 0.75 M Tris (pH 8.45), and 0.075% SDS. Samples were boiled in Tricine gel sample buffer (0.125 M Tris [pH 6.8], 4% SDS, 0.7 M β-mercaptoethanol, 10% glycerol, Coomassie blue). The anode buffer contained 200 mM Tris (pH 8.9). The cathode buffer contained 100 mM Tris, 100 mM Tricine, and 0.1% SDS. The pH of the cathode buffer was approximately 8.25 unadjusted. All the polyacrylamide gels were cross-linked with ammonium persulfate and N,N,N′,N′-tetramethylethylenediamine (TEMED).

Northern blot analysis.

Total-cell RNA was isolated and Northern blot analysis was performed as previously described (50) with two exceptions. First, for Northern blot analysis, 7.5 μg of RNA was used per lane, and second, after overnight hybridization of the ³²P-labeled probe, the final wash in 0.1× SSC–0.1% SDS was performed twice for approximately 50 min per wash. (0.1× SSC is 15 mM NaCl plus 1.5 mM sodium citrate [pH 7].) Riboprobes were transcribed from the SP6 promoter in the vector as instructed by the manufacturer (Promega).

Animal procedures.

For neurovirulence assays, 3-week-old female BALB/C mice (Charles River Breeding Laboratories, Inc., Kingston, N.Y.) were anesthetized with sodium pentobarbital and inoculated intracranially with 10-fold dilutions of virus stock (n = 6 to 10 per dilution) in a volume of 30 μl. The mice were monitored for 21 days, and the 50% lethal dose was calculated by the method of Reed and Muench (36).

For tests of latency and reactivation, 7-week-old randomly bred CD-1 mice (Charles River Breeding Laboratories) were anesthetized with sodium pentobarbital, both corneas were scarified, and 10⁶ PFU of virus in 10 μl was added to each eye as described previously (28). At the indicated times p.i., both eyes of four mice were swabbed with cotton swabs dampened in cell culture medium. The material on the swab was suspended in 1 ml of cell culture medium. Two mice were also sacrificed at each time point, and their TGG were excised and frozen. Ganglion homogenates were prepared by thawing and homogenizing with 10 strokes of a Kontes pellet pestle (Fisher Scientific, Pittsburgh, Pa.). Eye swab material and ganglion homogenates were assessed for infectious virus by standard plaque assays in Vero cell monolayers.

For reactivation assays, mice were sacrificed 30 days after infection. Each TGG was removed, cut into eight pieces, and explanted into one well of a 24-well dish containing 2 × 10⁵ Vero cells in suspension. On each day postexplant, for 15 or 21 days, 150 μl of culture medium was removed and assayed for infectious virus. Fresh medium (150 μl) was added back to the cocultivation to maintain the medium volume. To assay for infectious virus, 10⁵ Vero cells were infected in suspension with the 150 μl of the cocultivation medium; cytopathic effects were scored (+ or −) on day 5 after infection.

RESULTS

Sequence analysis of the 2.3-kb L/ST region.

The DNA sequences used throughout these studies were derived from HSV-1 strain KOS. Since only the sequence of the L/ST promoter region of strain KOS had been published prior to these studies (5), we subcloned and sequenced the region corresponding to the 2.3-kb L/ST. Comparison of the KOS DNA sequence with that of HSV-1 strains 17syn+, F, MGH-10, and CVG-2 revealed a nearly identical sequence, confirming that this region of the genome is highly conserved among various strains of HSV-1 (data not shown). The L/ST promoter region, as well as sequences containing the 5′ end of the L/STs, are also highly homologous to the corresponding region in HSV-2 strain HG52 (26, 29). As shown by the alignment of amino acid sequences, OPP of strain KOS is nearly identical to OPP of other HSV-1 strains (Fig. 2). The only major strain-specific difference lies in the variable number of AGV repeats near the amino terminus of the protein. These repeats correspond to the previously reported ATP repeats in the carboxy terminus of ICP 34.5 (14, 18, 26, 29).

FIG. 2 — Alignment of the ORF-P amino acid sequence in five strains of HSV-1. The amino acid sequences of HSV-1 strain KOS ORF-P are aligned with ORF-P amino acid sequences from strains 17syn+ (18), F, MGH10, and CVG2 (26). Consensus amino acids are denoted by asterisks beneath the sequences. Periods represent gaps. The codon number of the last amino acid on each line is shown on the right. The numbers at the lower right represent the length of ORF-P in each viral strain.

The nonsense mutation (n38) terminates the translation of ORF-P at amino acid 38.

To elucidate the functions of the L/STs and OPP, we introduced (i) nonsense mutations into both copies of ORF-P to yield a truncated protein consisting of only the amino terminus of OPP (the n38 mutation does not affect the amino acid sequence of ICP34.5 or the regulation of L/ST expression), and (ii) mutations into both copies of the ICP4 binding sites near the transcriptional start site of the L/STs that significantly enhance L/ST expression (and hence OPP expression) by eliminating ICP4-mediated repression.

To create the n38 nonsense mutation, we introduced a single G-to-A nucleotide transition in ORF-P, creating a termination codon at position 38 of this 225-codon ORF (Fig. 1M). This mutation disrupts a PflMI restriction enzyme cleavage site, thereby facilitating the identification of the mutation in the plasmid and in mutant viral constructs. Unlike previously published mutations in this region of the genome, the n38 mutation is a single-phase nonsense mutation. Therefore, other proteins that may be encoded by this sequence on the same or opposite DNA strands would not be prematurely terminated. The n38 mutation does not alter ORF-34.5 coding specificity because the single-base-pair transition occurs at the wobble position of codon 180. The mutation does alter ORF-O, however, changing amino acid 93 from Arg to Thr.

Prior to introduction of the mutation into the viral genome, the effect of the substitution on OPP synthesis was examined by in vitro transcription-translation. In the uncoupled in vitro transcription-translation system, the lengths of the transcript and the translated protein are dictated by the location of the restriction enzyme cleavage site used to linearize the expression plasmid. pLST-Nxi and pLST-NxiLM3, containing wild-type and mutant ORF-P sequences, respectively, were linearized at the restriction sites shown in Fig. 3A. In vitro-transcribed RNAs generated from the linearized plasmids were divided into aliquots for use in Northern analysis and in vitro translation. As shown by the blot in Fig. 3B, which was probed with riboprobe EBN9-LAT for L/ST sense RNA (Fig. 1D), linearization of the plasmid containing wild-type ORF-P at consecutive restriction sites downstream of the L/ST cap site resulted in the synthesis of progressively longer RNAs (Fig. 3B, lanes 1 to 6). As one would expect of a nonsense mutation that does not affect transcription, in vitro transcription from the mutant plasmid yielded RNAs (lanes 7 and 8) that were the same size as the corresponding wild-type RNAs (lanes 5 and 6). Because the polyadenylation site of the 2.3-kb L/ST lies between the DraI and MscI sites (Fig. 3A), RNAs synthesized from the MscI-linearized plasmid (MscI-RNAs) represent the entire coding capacity of the 2.3-kb L/ST (Fig. 3B, lanes 6 and 8).

An aliquot of each in vitro-transcribed RNA was used for in vitro translation (Fig. 3C). No protein was detected when PflMI-RNA was translated because the 37-amino-acid product does not contain [³⁵S]Cys and therefore was not labeled (lane 1). Progressively larger protein products were detected when progressively longer wild-type RNAs were translated (lanes 2 to 6). Translation of both DraI-RNA and the much longer MscI-RNA yielded the same sized protein of approximately 26 kDa, since both restriction sites lie downstream of the ORF-P translation termination site (lanes 5 and 6). The molecular mass of this protein is similar to the theoretical molecular mass of OPP (23 kDa), suggesting that the observed protein band was derived from ORF-P.

Unlike in vitro translation of wild-type RNA, translation of n38 RNA yielded no 26-kDa band, demonstrating that the n38 mutation terminated translation (Fig. 3C, lanes 7 and 8). In this in vitro system, translation of n38 should have produced a short (37-amino-acid) peptide. To determine if this was the case, protein translated from n38 DraI-RNA and n38 MscI-RNA in the presence of [³⁵S]Met was analyzed on a 20% denaturing Tricine polyacrylamide gel. A band corresponding to the 37-amino-acid peptide product of n38 RNA was detected in this test (data not shown). Because the RNAs containing the n38 mutation encode an otherwise full-length ORF-P, these results confirm that the single G-to-A transition terminated the translation of ORF-P.

The ICP4 binding-site mutation (4BS) abrogates ICP4 binding at the L/ST transcriptional start site and significantly enhances expression from the L/ST promoter.

To disrupt ICP4-mediated repression of L/ST expression, we introduced four single-base-pair substitution mutations into the ICP4 binding site in the L/ST promoter (Fig. 1M). These mutations created a new Eco47III restriction enzyme site, facilitating the identification of this mutation in plasmid and recombinant viral constructs. The effects of these mutations were first evaluated in vitro by gel mobility shift and transient-expression assays before they were introduced into the viral genome.

In gel mobility shift assays, proteins from infected-cell lysates formed a supershiftable complex with a labeled probe containing the wild-type ICP4 binding site but not with a probe containing the four single-base-pair substitution mutations (Fig. 4A). This complex was supershifted by an ICP4-specific antibody. These findings were confirmed in a series of transient-expression and transfection/superinfection assays designed to determine the effect of the 4BS mutation on transcription from the L/ST promoter. In the first series of experiments, Vero cells were cotransfected with reporter constructs containing the L/ST promoter with the wild-type (pL/ST-U-CAT) or mutated (4BS; pL/ST-4BS^m-CAT) ICP4 binding site and effector plasmids, pn11 and pSH, expressing ICP4 and ICP0, respectively. At 40 h posttransfection, the cells were harvested and processed for CAT activity. Whereas expression from the wild-type promoter was repressed in the presence of ICP4, expression from the promoter containing the 4BS mutation was strongly activated, demonstrating that ICP4 is able to activate the L/ST promoter but through a mechanism not involving the ICP4 consensus binding site in the L/ST promoter (Fig. 4B). To ascertain whether the 4BS mutation specifically affected regulation by ICP4 and not all regulators of gene expression, we cotransfected the wild-type and 4BS reporter plasmids with the ICP0-expressing plasmid, pSH. The responses of the wild-type and 4BS constructs to ICP0 were essentially identical (Fig. 4C) but were distinct from the responses of these plasmids to ICP4 (Fig. 4B). Thus, as reported previously, expression from both plasmids was high following cotransfection with low levels of ICP0 whereas higher levels of ICP0 resulted in lower levels of CAT activity (5). These results suggest that the 4BS mutation does not deregulate the L/ST promoter indiscriminately and that the ICP4 binding site is not a target of ICP0 or ICP4 activation. Together with the findings from gel mobility shift assays, the results of transient-expression assays with a 4BS reporter construct indicate that ICP4 cannot bind to the mutated binding site to repress L/ST expression.

FIG. 4 — Effects of the mutant ICP4 binding site on expression from the L/ST promoter. (A) Gel shift analysis. Radiolabeled 30-bp probe DNAs containing either the wild-type (WT) or mutated (4BS Mutant) ICP4 binding site were incubated with mock-, n12-, or KOS-infected Vero cell lysates prepared at 8 h p.i. and enriched for nuclear proteins. Anti-ICP4 monoclonal antibody (58S MAb) was added to the incubation mixture where indicated (lanes 2, 4, 6, 9, 11, and 13). The four major complexes detected are labeled a through d. (B to D) Transient-expression assays. Vero cells were transfected with pL/ST-U-CAT (WT), containing the wild-type ICP4 binding site, or pL/ST-4BS^m-CAT (4BS MUT), containing the mutated ICP4 binding site. Reporter plasmids were cotransfected with increasing amounts of the ICP4-expressing plasmid, pn11 (16) (B) or the ICP0-expressing plasmid, pSH (9) (C). Cells were harvested at 40 h posttransfection or at the times indicated, and CAT enzyme activity was assayed. Data points represent the average activities in two separately transfected plates. Transfection of pWR-CAT⁺, the promoter-less reporter plasmid, in the presence or absence of the effector plasmids, yielded background levels of CAT activity (data not shown). At 24 h after transfection of reporter plasmids alone, Vero cells were mock superinfected or superinfected with n12 or KOS at an MOI of 10 PFU/cell (D). The cells were harvested at 3, 6, 12, and 24 h p.i. A 9 h p.i., an additional data point was determined for 4BS reporter-transfected and KOS-superinfected Vero cells.

The cotransfection experiments examined the effects of specific viral regulatory proteins on the L/ST promoter containing the 4BS mutation. Because L/ST promoter activation following superinfection may better reflect promoter function during natural viral infection, we performed a transient-expression/superinfection assay in which transfected Vero cells were mock infected or infected with KOS or n12 virus 24 h after transfection. At 3, 6, 12, and 24 h p.i., the cells were processed for CAT activity. As shown in Fig. 4D, expression from the wild-type promoter construct was repressed by superinfection with KOS (ICP4⁺) but not n12 (ICP4⁻). Expression from the mutated 4BS construct was also high following superinfection with either KOS or n12, demonstrating that introduction of the 4BS mutation into the L/ST promoter derepressed expression in the context of wild-type viral superinfection. The mutated 4BS construct was more active following superinfection with KOS than with n12, consistent with the data presented in Fig. 4B and demonstrating that ICP4 and ICP0 (KOS) or only ICP0 (n12) can activate the mutated L/ST promoter in the context of viral infection (10).

The introduction of the L/ST mutations n38 and 4BS into the viral genome was facilitated by the availability of 7134, an ICP0-null mutant virus in which both copies of the ICP0-coding region have been replaced by the E. coli lacZ coding sequence (9). Thus, the L/ST mutations were “piggybacked” into both copies of the L/ST gene simultaneously with the rescue of both copies of ICP0, producing the white-plaque phenotype. The mutations in the resulting viruses (L/ST-n38 and L/ST-4BS) were also rescued by homologous recombination.

L/ST mutant viruses replicate efficiently in cells of neural and nonneural origin.

ORF-34.5/ORF-P double mutants replicate to wild-type levels in permissive cells (e.g., Vero cells) but are growth restricted in certain neural cell lines (e.g., SK-N-SH cells) (12, 13). To assess the ability of mutant viruses L/ST-n38 and L/ST-4BS to replicate in Vero and SK-N-SH cells, single-cycle growth experiments were performed with the wild-type virus, the two mutants, and the rescuant viruses. In Vero cells, L/ST-n38, L/ST-4BS, and the rescuants replicated as efficiently as wild-type virus, indicating that no spurious mutations in genes that affect replication efficiency were present in any of the viruses (Fig. 5A). In SK-N-SH cells, however, L/ST-n38 replicated to wild-type levels whereas L/ST-4BS replicated to somewhat lower levels (Fig. 5B). This experiment was repeated twice with identical results. The modest reduction in replication efficiency of L/ST-4BS in SK-N-SH cells was not observed with another ICP4 binding-site mutant virus, R7530 (25).

FIG. 5 — Growth curves of L/ST mutant viruses in cell culture. Monolayers of Vero cells (A) or SK-N-SH cells (B) were infected at an MOI of 2.5 PFU/cell with KOS, L/ST-n38, L/ST-n38R, L/ST-4BS, or L/ST-4BSR as described in Materials and Methods. Total virus was harvested at the indicated times p.i. and quantified by a standard plaque assay on Vero cell monolayers. Titers are the average obtained in two tests.

To determine if the modest reduction in the growth of L/ST-4BS in SK-N-SH cells was specific for cells of neural origin, single-cycle growth experiments were also performed in undifferentiated and nerve growth factor-differentiated PC12 cells, mouse neuroblastoma cells (NB41A3), and dividing and resting HEL cells as a control nonneural cell type. No major differences were observed in the growth patterns of L/ST-4BS, L/ST-n38, or KOS in any of the cell lines tested (data not shown), suggesting that the modest reduction in replication efficiency of L/ST-4BS in SK-N-SH is specific for this cell type.

To further examine the general properties of the L/ST mutant viruses, patterns of protein synthesis in infected Vero cell extracts were analyzed at 3, 7, or 12 h p.i. by SDS-PAGE and by Western blot analysis with antibodies specific for ICP0, ICP4, ICP27, and glycoprotein D. Consistent with the similarities in the growth patterns of the L/ST mutants in Vero cells, the protein profiles of the mutants and their respective rescuants were indistinguishable from that of wild-type virus (data not shown).

Transcription of genes in the abb′a′c′ca repeats in cells of nonneural and neural lineage by L/ST mutant viruses.

L/ST expression by the mutant viruses, KOS, and the rescuant viruses was examined by Northern blot hybridization of total RNA harvested from infected Vero and NB41A3 cells (Fig. 6A). The ICP4 nonsense mutant n12 was included as an L/ST-expressing control (50).

(i) L/STs.

The L/STs were not detected in RNA from L/ST-n38-infected Vero or NB41A3 cells (Fig. 6A, lanes 4 and 11); this was expected since the L/ST promoter in L/ST-n38 contains a wild-type ICP4 binding site and is therefore subject to repression by ICP4. Similarly, the L/STs were not detected in KOS-infected (lanes 2 and 9) or L/ST-n38R-infected (lanes 5 and 12) cells for the same reason. As expected from the results of transient-expression assays, the L/STs were expressed in L/ST-4BS-infected cells (lanes 6 and 13). In Vero cells, L/ST-4BS expressed lower levels of the 2.3-kb L/ST than did n12, whereas the opposite was true in NB41A3 cells. L/ST-4BS also expressed higher levels of L/STs than did n12 in differentiated and undifferentiated PC12 cells and in SK-N-SH cells (data not shown). In addition to the abundant 2.3-kb transcript, two smaller transcripts (<1.35 kb) were detected in L/ST-4BS-infected Vero and NB41A3 cells (lanes 6 and 13, respectively). The presence of these smaller transcripts in both infected Vero and NB41A3 cells argues that they represent independent transcripts and not degradation products of the larger L/STs. Finally, L/ST expression was not detected in L/ST-4BSR infected cells, confirming that the expression of the L/STs in L/ST-4BS was due to the 4-bp substitution in the ICP4 binding site (lanes 7 and 14).

(ii) γ₁34.5 transcripts.

Using a DNA probe specific for γ₁34.5 transcripts, Chou and Roizman (15) detected two infected-cell-specific RNAs of 1.3 and 5.2 kb and one nonspecific 3.5-kb RNA in wild-type virus (strain F)-infected Vero cells. As shown in Fig. 6B, the abundant 1.3-kb γ₁34.5 RNA and two less prominent 2.5- and 4-kb transcripts were detected in infected Vero cells with the EBSN34.5 riboprobe (lanes 2 and 4 through 7). Further mapping experiments are needed to determine whether the 2.5- and 4-kb transcripts are γ₁34.5 specific or nonspecific. Levels of the 1.3-kb γ₁34.5 RNA detected in cells infected with wild-type virus and L/ST-n38 were similar (lanes 2 and 4) but somewhat higher in L/ST-4BS-infected cells, indicating that the n38 mutation had no major effect and that the 4BS mutation had, if anything, an enhancing effect on the levels of γ₁34.5 RNA in Vero cells. As anticipated, transcripts were not detected in n12-infected cells with the γ₁34.5 probe (lane 3), since ICP4-null viruses do not express late genes.

Three transcripts (1.3, 4, and 5.2 kb) were readily detected in KOS-infected (lane 9), L/ST-n38-infected (lane 11), and L/ST rescuant virus-infected (lanes 12 and 14) NB41A3 cells with the EBSN34.5 riboprobe. In contrast, only barely detectable levels of γ₁34.5 transcripts were observed in L/ST-4BS-infected NB41A3 cells (lane 13). Similarly, only very low levels of γ₁34.5 transcripts were observed in L/ST-4BS-infected PC12 cells compared with those in KOS-infected PC12 cells (data not shown). In contrast, nearly equal levels of γ₁34.5 transcripts were detected in Vero cells infected with KOS (lane 2) or L/ST-4BS (lane 6). These findings demonstrate that γ₁34.5 transcript accumulation in L/ST-4BS-infected cells is cell type specific. Figure 6B also shows that the levels of γ₁34.5-specific transcripts detected were uniformly greater in NB41A3 cells than in Vero cells and that the sizes of the transcripts detected in the two cell types differed in that the 5.2-kb transcript was evident in NB41A3 cells but not in Vero cells.

(iii) ICP0 and ICP4 transcripts.

Northern blot analysis of total RNA harvested from infected Vero cells at 6 h p.i. demonstrated that ICP0 and ICP4 RNAs were expressed by both L/ST-n38 and L/ST-4BS at levels comparable to those of wild-type virus (Fig. 6C, lanes 2, 4, and 6). Because these transcripts were present at very low levels in NB41A3 cells at 6 h p.i., a considerably longer autoradiographic exposure was necessary to visualize the bands in lanes 8 to 14. As in Vero cells, levels of ICP0 and ICP4 transcripts were similar to those seen in KOS-infected cells. Although abundant ICP4 transcripts were present in n12-infected Vero cells, no functional ICP4 protein was made because of the nonsense mutation in the ICP4 gene; hence, ICP0 and ICP4 transcription is not repressed and these transcripts are made at higher-than-wild-type levels. Notably, overexpression of ICP0 and ICP4 transcripts by n12 relative to KOS was evident in Vero cells but not in NB41A3 cells (or PC12 cells [data not shown]). Thus, overexpression of these two transcripts is cell type specific.

(iv) LATs.

Because the L/STs may function during HSV latency (50), it was of interest to determine if the n38 or 4BS mutations have an effect on LAT expression in vitro. We therefore performed Northern blot analysis with a LAT-specific riboprobe, BbSLAT, with total RNA from Vero and NB41A3 cells harvested at 18 h p.i. with the L/ST mutant and rescuant viruses (Fig. 6D). RNAs from KOS-infected cells (lanes 2 and 9) and n12-infected cells (lanes 3 and 10) were included as LAT-positive and LAT-negative controls, respectively.

In Vero cells, L/ST-n38 and L/ST-4BS and their respective rescuant viruses (lanes 4 to 7) induced the synthesis of wild-type or near-wild-type levels of the LATs, although we have consistently noted a slightly lower level of LAT expression during infection of these cells with L/ST-4BS (lane 6).

In NB41A3 cells, the LATs were reproducibly detected during infection with KOS, L/ST-n38, and the L/ST rescuant viruses (lanes 9, 11, 12, and 14); however, only a faint LAT signal was detected in repeat experiments in RNA from L/ST-4BS-infected cells (lane 13). (The low LAT signal seen for L/ST-n38R in lane 12 was not reproducible; near-wild-type levels of LAT expression in NB41A3 cells are more characteristic of this virus.) Since the overall intensities of the LAT signals in NB41A3 cells were considerably lower than those in Vero cells, the low signal intensity in L/ST-4BS-infected NB41A3 cells (lane 13) may reflect the slight reduction observed in Vero cells noted above (lane 6). Whatever the basis for this reduction, the 4BS mutation had a major effect on LAT expression in NB41A3 cells relative to wild-type virus and L/ST-n38. Similar results were observed in PC12 cells. We conclude from these tests that LAT expression, like the expression of γ₁34.5 transcripts, is cell type specific, being significantly higher in nonneural cells (Vero) than in cells of neural lineage (NB41A3 and PC12).

OPP expression by L/ST mutant viruses.

Having demonstrated high-level expression of the L/STs by L/ST-4BS but not by L/ST-n38 in both Vero and NB41A3 cells, we attempted to assess the kinetics and levels of expression of OPP in Vero cells. To facilitate these studies, rabbit antibody (DF111) was raised against the C-terminal 52 amino acids of ORF-P fused to GST. Using this antibody, we first examined the ability of the L/ST mutant viruses and their respective rescuants to express OPP. Extracts of KOS- and n12-infected Vero cells were included as OPP-negative and -positive controls, respectively. As shown in Fig. 7, OPP was not detected in Vero cells infected with KOS, L/ST-n38, and L/ST-4BSR, viruses that do not express the L/STs as demonstrated by Northern blot analysis (Fig. 6A). In cells infected with L/ST-4BS, three proteins of approximately 24, 29, and 33 kDa were detected (Fig. 7, lanes 10 to 12). The size of the 24-kDa band is consistent with the molecular mass of OPP (23 kDa) and with the size of the OPP band observed by in vitro transcription-translation (26 kDa) (Fig. 3). The size and relative abundance of each protein species at 6, 12, and 18 h p.i. suggest that the 24-kDa protein is modified posttranslationally during the course of lytic infection. As anticipated, the 24-kDa protein was also detected in n12-infected Vero cells, although the appearance of the larger (29-kDa) species was delayed and these proteins were present in lower abundance relative to that in L/ST-4BS-infected cells. OPP expression by n12 and L/ST-4BS was also detected in NB41A3 cells, nerve growth factor-differentiated and undifferentiated PC12 cells, and SK-N-SH cells (data not shown), demonstrating that like expression of the L/STs, OPP expression is not cell type specific.

FIG. 7 — Expression of ORF-P protein by the L/ST mutant viruses. Vero cells were mock infected or infected with KOS, n12, L/ST-n38, L/ST-4BS, L/ST-4BSR, or L/ST-4/38 at a multiplicity of 10 PFU/cell. Infected-cell proteins were prepared at the indicated times p.i., separated by SDS-PAGE, and transferred to a polyvinylidene difluoride membrane. ORF-P protein was detected by Western blot analysis with rabbit polyclonal antibody, DF111, diluted 1:1,000.

L/ST-4BS but not L/ST-n38 induces premature shutoff of protein synthesis in SK-N-SH cells.

Gradual shutoff of host cell protein synthesis is observed after HSV infection of cultured cells (19). In contrast to wild-type virus infection, ORF-34.5/ORF-P double-mutant viruses induce the shutoff of all protein synthesis (viral and host cell) at early times (3 to 6 h p.i.) in SK-N-SH cells relative to other cell types (13). To examine protein shutoff in L/ST-n38- and L/ST-4BS-infected Vero and SK-N-SH cells, we performed metabolic labeling experiments. An ICP34.5-null mutant, 1771, derived from strain 17syn+ was included as a positive control, and wild-type strains KOS and 17syn+ were included as negative controls. In Vero cells, infection with the L/ST mutant viruses, both rescuants and the positive and negative control viruses, produced wild-type (KOS or 17syn+) protein profiles, characterized by a gradual shift from host to viral protein synthesis over the 12-h period (Fig. 8A). In SK-N-SH cells, L/ST-n38 and both rescuant viruses produced KOS wild-type-like protein profiles (Fig. 8B). In contrast, L/ST-4BS and 1771 infection resulted in rapid and extensive shutoff of both viral and cellular protein synthesis relative to their respective wild-type viruses. Rescue of the 4BS mutation to produce the wild-type genotype (L/ST-4BSR) returned the protein synthesis profile to that seen with wild-type virus. The results obtained with L/ST-n38 suggest that the premature protein shutoff phenotype of the ORF-P ORF-34.5 double-mutant viruses in SK-N-SH cells is not likely to be a consequence of mutations in ORF-P but, rather, is a consequence of mutations in ORF-34.5. The molecular basis for the premature protein shutoff phenotype of L/ST-4BS is not clear and will be discussed below.

FIG. 8 — Kinetics of the shutoff of viral and cellular protein synthesis in Vero and SK-N-SH cells infected with L/ST mutant viruses. Cells were mock infected (M) or infected with 10 PFU of the indicated viruses per cell, labeled with [³⁵S]methionine for 30 min at 3, 7, or 12 h p.i., and processed for PAGE as described in Materials and Methods. (A) Vero cells, 5 μg of protein per lane. (B) SK-N-SH cells, 10 μg of protein per lane.

L/ST-4BS but not L/ST-n38 is attenuated for neurovirulence in mice.

Having shown that the protein shutoff phenotype is characteristic of L/ST-4BS but not L/ST-n38, we tested these mutants in vivo for their neurovirulence phenotypes and their ability to establish and reactivate from latency in mice.

To determine if the n38 and 4BS mutations result in the attenuated phenotype characteristic of ORF-P ORF-34.5 double-mutant viruses, we assessed the neurovirulence of the L/ST mutant viruses by intracranial inoculation of BALB/c mice. The results of two independent experiments are shown in Table 1. Consistent with published data, wild-type strain 17syn+ was more neurovirulent than KOS and the 17syn+ mutant, 1771, was highly attenuated in these tests. L/ST-n38, although somewhat less virulent than KOS in experiment 1 and somewhat more virulent than KOS in experiment 2, is clearly a neurovirulent virus, demonstrating that the mutation of ORF-P in ORF-P ORF-34.5 double mutants is not responsible for the attenuated phenotypes of these viruses.

TABLE 1.

Neurovirulence of L/ST mutant viruses in mice

Virus	LD₅₀^a after i.c. inoculation in:
Virus	Expt 1	Expt 2
KOS	72	898
L/ST-n38	537	273
L/ST-n38R	ND^b	626
L/ST-4BS	>2.4 × 10⁶	>2.6 × 10⁶
L/ST-4BSR	ND	997
17syn+	1.1	ND
1771	>7.4 × 10⁵	ND

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Mice were infected as described in the text. The 50% lethal doses (LD₅₀) of the indicated viruses were calculated by the method of Reed and Muench (36). i.c., intracranical.

ND, not determined.

The reduced levels of γ₁34.5 transcripts (Fig. 6B) and the premature shutoff of protein synthesis in SK-N-SH cells observed in in vitro tests of L/ST-4BS (Fig. 8B) predict that L/ST-4BS might behave in vivo like ORF-P ORF-34.5 double-mutant viruses. As predicted, L/ST-4BS was completely attenuated in both experiments (Table 1), causing no detectable morbidity or mortality in mice. Rescue of the mutation in L/ST-4BS restored the wild-type neurovirulence phenotype, confirming that the attenuated phenotype is due to the 4BS mutation.

L/ST-4BS replicates in the mouse eye and in TGG during acute infection following corneal inoculation.

Because the attenuation of L/ST-4BS in mice could, in theory, be due to a general defect in the ability of this virus to replicate in mice, we examined the ability of KOS, L/ST-4BS, and L/ST-4BSR to replicate in the mouse eye and in TGG following corneal inoculation. Tear film from four mice and TGG from two mice were collected daily during acute infection (the first 7 days p.i.) and assayed for infectious virus by a standard plaque assay on Vero cell monolayers. The arithmetic mean titers of multiple samples at each time point are shown in Fig. 9. In tear film, KOS and L/ST-4BSR reached peak titers of approximately 10⁵ PFU/ml on day 1 p.i., and these titers were maintained at high levels through day 6 p.i., dropping slightly on day 7 p.i. (Fig. 9A). Replication of these viruses in TGG was evident by day 2 and peaked between days 3 and 4 p.i. (Fig. 9B). In both tear film and TGG, L/ST-4BS replicated to near-wild-type levels during the first few days p.i. but exhibited an earlier and more rapid decline in titer thereafter. Rescue of the mutation in L/ST-4BS restored wild-type levels of replication in TGG as in eyes. Notably, these results demonstrate that L/ST-4BS is capable of significant replication (albeit less efficiently than the wild type) in the peripheral nervous system.

FIG. 9 — Production of infectious virus in mouse eyes and TGG during acute infection and in explanted ganglia during reactivation. Mice were inoculated with 2 × 10⁶ PFU of KOS, L/ST-n38, L/ST-4BS, or L/ST-4BSR per eye. (A) Eye swabs were taken 1 to 3 h p.i. and then daily from 1 to 7 days p.i. Each point represents the arithmetic mean titer of eight eye swabs (four mice). (B) TGG were removed and assayed directly for the presence of infectious virus on days 1 through 7 p.i. Each point represents the arithmetic mean titer for TGG (two mice). The titers were determined by a standard plaque assay on Vero cells. Data points below the asterisk represent undetectable levels of virus. (C) At 30 days p.i., TGG were removed and cocultivated with Vero cells. Aliquots of the cocultivation medium were then assayed for infectious virus on the indicated days postexplant. Each point represents the cumulative percentage of cocultivated ganglia scoring positive for infectious virus. In experiment 1, the total numbers of TGG tested were as follows: KOS, 18; L/ST-n38, 18; L/ST-4BS, 24. In experiment 2, the total numbers of TGG used were as follows: KOS, 18; L/ST-n38, 19; L/ST-4BS, 18; L/ST-4BSR, 14.

L/ST-4BS exhibits delayed reactivation from latency in the mouse ocular model.

Because the L/STs and/or OPP may be involved in some way in the establishment or reactivation of latency, we examined the ability of the L/ST mutant viruses to reactivate from latently infected mouse TGG. In two independent experiments, mice were inoculated with 2 × 10⁶ PFU/eye. On day 30 p.i., TGG were removed and cocultivated with Vero cells. The cocultivation medium was then assayed daily for the presence of infectious, reactivated virus. The cumulative percentage of cocultivated ganglia which scored positive for infectious virus in both experiments is shown in Fig. 9. Consistent with its wild-type phenotype in vitro, L/ST-n38 reactivated with wild-type kinetics and efficiency in both experiments. L/ST-4BS also reactivated but with delayed kinetics and reduced efficiency relative to wild-type virus in both experiments. In experiment 1, reactivation of L/ST-4BS was delayed somewhat and was less efficient than that of the wild type (71% TGG [17 of 24]) by day 15 postexplant versus 100% of TGG [24 of 24] by day 6). In this experiment, it was not determined whether the TGG remaining on day 15 harbored latent L/ST-4BS virus; therefore, in the second experiment, explant cocultures were monitored for an extended period (21 days postexplant). In this experiment, L/ST-4BS ultimately reactivated from 89% of TGG (16 of 18), a level comparable to KOS (100% [18 of 18 TGG]) and L/ST-n38 (100% [19 of 19 TGG]) in the 21-day experiment. The delayed kinetics of L/ST-4BS reactivation noted in the first experiment were also evident in the second experiment. Rescue of the 4BS mutation restored the wild-type reactivation profile (L/ST-4BSR, experiment 2). Viral DNA isolated from half of the reactivated cultures in experiment 1 and all of the cultures in experiment 2 was analyzed by Southern blot analysis; the L/ST mutations were present in reactivated mutant viruses in all cases (data not shown).

DISCUSSION

The abb′c′a′ca repeats of the HSV-1 genome contain genes and cis-acting elements that play central roles in establishing the programs of gene expression characteristic of productive infection and latency. The large number of these genes and elements and the extent to which they overlap have complicated the genetic and functional analysis of this region of the genome in that mutations introduced into one gene or element invariably alter the nucleotide sequence of other overlapping genes and elements. Such has been the case in studies of the γ₁34.5 gene, which is encoded on the DNA strand opposite that specifying the L/STs and OPP (Fig. 10). For these reasons, we endeavored to design mutations that have minimal effects on ORF-34.5 in an effort to elucidate the function(s) of the L/STs and OPP.

OPP does not play a major role in neurovirulence or latency in murine models.

To examine the contributions of mutations in ORF-P to the in vitro premature protein shutoff and in vivo neurovirulence and latency phenotypes of ORF-34.5 ORF-P double mutants, we constructed mutant virus L/ST-n38, containing a single-base-pair single-frame nonsense mutation at codon 38 in ORF-P. Before the n38 mutation was introduced into the viral genome, the phenotype of a plasmid carrying this mutation was established in vitro. In transient-expression assays, the n38 mutation terminated the translation of ORF-P prematurely but had no effect on the transcription of the L/STs (Fig. 3). In the context of viral infection, the n38 mutation (i) had no detectable effect on virus replication in neural (SK-N-SH) or nonneural (Vero) cells (Fig. 5); (ii) had no effect on the levels of the L/STs, γ₁34.5, ICP0, or ICP4 transcripts or the LATs in neural (NB41A3) or nonneural (Vero) cells (Fig. 6); and (iii) did not produce premature shutoff of infected SK-N-SH cell protein synthesis (Fig. 8). Consistent with the absence of a unique phenotype in in vitro tests, L/ST-n38 was wild type with regard to neurovirulence, latency, and reactivation in mice. Although the n38 mutation in ORF-P is a single-base-pair, single-frame nonsense mutation, the G-to-A nucleotide transition also induced an Arg-to-Thr change at amino acid 93 of ORF-O (Fig. 10). If OOP is involved in any of the functions measured by our in vitro or in vivo assays, the Arg-to-Thr substitution in L/ST-n38 had no detectable effect on these functions.

What, then, is the function of OPP? Using a yeast two-hybrid system, Bruni and Roizman have presented evidence that OPP interacts with the p32 component of the splicing factor, SF2/ASF (8). If this interaction proves to be specific, its functional consequences for HSV replication remain to be determined. Based on studies of ORF-P ORF-34.5 double mutants, these investigators have implicated OPP in blocking the synthesis of the spliced transcripts of the ICP0 and 22 genes early (8 h) but not late (18 h) in infection. As noted above (Fig. 6), we detected no difference in the levels of ICP0 transcripts (or proteins [data not shown]) at early times (6 h) p.i. in L/ST- n38-infected Vero and NB41A3 cells relative to wild-type virus-infected cells. Thus, at this writing, no phenotype has been demonstrated for L/ST-n38 and the function of OPP remains unknown. Although it does not appear to be a major player in neurovirulence or in latency and reactivation in mice, our results do not preclude the possibility that OPP functions during productive or latent infections in humans.

L/ST-4BS exhibits in vitro and in vivo properties characteristic of ORF-P ORF-34.5 double mutants.

The second mutation used to examine the function of ORF-P ORF-34.5 double mutants, 4BS, alters the ICP4 binding site 4 bp upstream of the transcriptional start site of the L/STs. As anticipated, tests conducted before the transfer of this mutation into the viral genome demonstrated that the 4BS mutation abrogates the binding of ICP4 to its cognate repressive binding site (Fig. 4A). Notably, this mutation had no effect on the ability of ICP4 or ICP0 to activate the expression of a reporter gene from the same mutant promoter in cotransfection and transfection/superinfection assays, demonstrating that the sequences responsible for ICP0- ICP4-mediated activation of the L/ST promoter do not include the repressive ICP4 binding site (Fig. 4B to D).

The in vitro and in vivo phenotypic properties of L/ST-4BS are summarized in Table 2. Several points can be made from these findings. First, four of the five in vitro properties of LST-4BS reflect deficiencies in gene expression in neural with respect to nonneural cells, implicating the site of the 4BS mutation as a critical determinant of the interactions of HSV-1 with neural cells. This hypothesis is borne out by the neuroattenuated phenotype of L/ST-4BS. Although L/ST-4BS induces wild-type levels of ICP0 and ICP4 transcripts in both neural (NB41A3) and nonneural (Vero) cells, these immediate-early transcripts were expressed at markedly reduced levels in NB41A3 cells relative to Vero cells infected with all viruses tested, implicating neural factors specifically in the low-level expression of these two immediate-early genes.

TABLE 2.

Phenotypic properties of mutant virus L/ST-4BS

Properties in vitro	Properties in vivo
Expresses higher levels of a unique subset of the L/STs in neural (NB41A3) cells than in nonneural (Vero) cells	Is completely attenuated for neurovirulence Replicates less efficiently in mouse eyes and TGG than does wild-type virus Exhibits delayed reactivation kinetics and reduced reactivation efficiency compared with wild-type virus
Replicates less efficiently in neural (SK-N-SH) cells than in nonneural (Vero) cells
Induces premature protein shutoff in neural (SK-N-SH) cells but not in nonneural (Vero) cells
Induces barely detectable levels of γ₁34.5 transcripts and LATs in neural (NB41A3) cells but wild-type levels in nonneural (Vero) cells
Induces wild-type levels of ICP0 and ICP4 transcripts in neural (NB41A3) and nonneural (Vero) cells

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Second, the 4BS mutation, which resulted in the (expected) high-level expression of the L/STs, was (unexpectedly) accompanied by the nearly complete absence of γ₁34.5 transcripts and the LATs in neural (NB41A3, SK-N-SH, and PC12) but not nonneural (Vero) cells. We conclude, therefore, that expression of these two families of transcripts is regulated differentially in the two cell types (Fig. 6B and D) and that the genomic locus in which the 4BS mutation in L/ST-4BS lies controls the expression of both the γ₁34.5 transcripts and the LATs in neural cells.

What is the molecular basis of the nearly total absence of γ₁34.5 transcripts and the LATs in L/ST-4BS-infected neural cells? Based on the location of the 4BS mutation (Fig. 10), several possibilities present themselves. First, because the 4BS mutation lies in the ICP4 binding site in the L/ST promoter, overexpression of the L/STs and the multiple proteins which they potentially encode may downregulate γ₁34.5 and LAT expression. Clearly, however, the putative L/ST-encoded downregulatory protein(s) is not OPP, since the levels of γ₁34.5 transcripts and the LATs were nearly wild type in L/ST-n38-infected NB41A3 cells, which produce a severely truncated and probably nonfunctional form of OPP. Second, the 4BS mutation in ORF-O sequences may produce this result. As noted above and as shown in Fig. 10, the 4BS mutation lies in sequences encoding OOP. (Although Randall et al. [34] have recently presented evidence suggesting that translation of ORF-O actually begins at the ORF-P translational start site in HSV-1 strain F, no evidence to support this contention has yet been obtained for strain KOS or other HSV-1 strains.) If OOP is encoded by the LATs, as suggested above, and if OOP activates the expression of both γ₁34.5 and the LATs by a positive-feedback mechanism, the 4BS mutation in ORF-O would result in the failure of OPP to activate γ₁34.5 transcription and LAT expression. Third, the 4BS mutation may lie in an unrecognized gene or element that controls the expression of γ₁34.5 and LAT transcription.

Notably, the reduced levels of γ₁34.5 transcripts and the LATs in L/ST-4BS-infected NB41A3 cells are not likely to be due to the instability of these transcripts as a consequence of the 4BS mutation (which alters the nucleotide sequence of both classes of transcript), because these mutated transcripts were as stable as their wild-type counterparts in L/ST-4BS-infected Vero cells. Similarly, the reduced levels of γ₁34.5 transcripts and the LATs in L/ST-4BS-infected neural cells is not likely to be due to antisense repression by the L/STs (as has been suggested recently [35]), because wild-type levels of these transcripts, which lie ∼0.5 kb upstream of the 4BS mutation, were produced in L/ST-4BS-infected Vero cells, which also expressed high levels of the L/STs (Fig. 6).

Whatever the mechanism underlying their repression, the absence of γ₁34.5 transcripts in L/ST-4BS-infected NB41A3 cells is consistent with the premature shutoff of infected-cell protein synthesis in neural cells and the neuroattenuation characteristic of ORF-P ORF-34.5 double mutants. In addition, the absence of the LATs in L/ST-4BS-infected neural cells is consistent with the delay in and reduced efficiency of reactivation, which is characteristic of LAT-minus viruses (27).

The results of our characterization of L/ST-4BS are in general agreement with the published properties of another virus mutated in the ICP4 binding site in the L/ST promoter, R7530 (also called ORFP⁺⁺ [25]). The most significant difference between the two viruses is that R7530 induced no detectable γ₁34.5 transcripts yet premature shutoff of protein synthesis in SK-N-SH cells was not observed, whereas L/ST-4BS exhibited greatly reduced levels of γ₁34.5 transcripts as well as premature shutoff of host protein synthesis. It may be that the two properties (synthesis of γ₁34.5 transcripts and premature protein shutoff, which are thought to represent cause and effect, respectively) are regulated differentially. Whatever the answer to this puzzling question, it is likely that the differences in the phenotypes of the two mutant viruses reflect differences in the specific mutations introduced into the ICP4 binding sites of strains F and KOS (25), as well as differences in the properties of the wild-type strains from which the two mutants were derived.

We conclude from our studies of L/ST-n38 and L/ST-4BS that (i) mutations in ORF-34.5 and not in ORF-P are responsible for the attenuated phenotype of ORF-34.5 ORF-P double mutants, (ii) the function of OPP remains unknown, (iii) expression of the γ₁34.5 gene and the LATs in cells of neural lineage is regulated directly or indirectly by elements in the locus marked by the 4BS mutation, (iv) expression of the γ₁34.5 transcripts and the LATs is regulated similarly in cells of neural lineage, and (v) γ₁34.5 and LAT transcription is regulated differently in cells of neural and nonneural lineage.

ACKNOWLEDGMENTS

We thank Robert Jordan and Feng Yao for helpful discussions; David Levy, Anh Nguyen-Huynh, Jill Samale, and Josh Pepe for assistance with the animal experiments; Elli Mendoza, Luis Schang, Bill Halford, Amy Francis, and Jennifer Isler for critical reading of the manuscript; and Paige Hasling for preparation of the manuscript.

These studies were supported by research grants R37 CA20260 from the National Cancer Institute and P01 NS35138 from National Institute of Neurological Disorders and Stroke. L.Y.L. was supported in part by National Science Foundation predoctoral fellowship 75982 and by a Ryan Predoctoral Fellowship, Division of Medical Sciences, Harvard University.

REFERENCES

1.Ackermann M, Braun D K, Pereira L, Roizman B. Characterization of herpes simplex virus 1 α proteins 0, 4, and 27 with monoclonal antibodies. J Virol. 1984;52:108–119. doi: 10.1128/jvi.52.1.108-118.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
2.Ackermann M, Chou J, Sarmiento M, Lerner R A, Roizman B. Identification by antibody to a synthetic peptide of a protein specified by a diploid gene located in the terminal repeats of the L component of the herpes simplex virus genome. J Virol. 1986;58:843–850. doi: 10.1128/jvi.58.3.843-850.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
3.Andrews N C, Faller D V. A rapid micropreparation technique for extraction of DNA-binding proteins from limiting numbers of mammalian cells. Nucleic Acids Res. 1991;19:2499. doi: 10.1093/nar/19.9.2499. [DOI] [PMC free article] [PubMed] [Google Scholar]
4.Ausubel F M, Brent R, Kingston R E, Moore D D, Seidman J G, Smith J A, Struhl K, editors. Current protocols in molecular biology. New York, N.Y: John Wiley & Sons, Inc.; 1988. [Google Scholar]
5.Bohenzky R A, Papavassiliou A G, Gelman I H, Silverstein S. Identification of a promoter mapping within the reiterated sequences that flank the herpes simplex virus type 1 UL region. J Virol. 1993;67:632–642. doi: 10.1128/jvi.67.2.632-642.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
6.Bolovan C A, Sawtell N A, Thompson R L. ICP34.5 mutants of herpes simplex virus type 1 strain 17syn+ are attenuated for neurovirulence in mice and for replication in confluent primary mouse embryo cell cultures. J Virol. 1994;68:48–55. doi: 10.1128/jvi.68.1.48-55.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
7.Brown S M, Harland J, MacLean A R, Podlech J, Clements J B. Cell type and cell state determine differential in vitro growth of non-neurovirulent ICP34.5-negative herpes simplex virus types 1 and 2. J Gen Virol. 1994;75:2367–2377. doi: 10.1099/0022-1317-75-9-2367. [DOI] [PubMed] [Google Scholar]
8.Bruni R, Roizman B. Open reading frame P—a herpes simplex virus gene repressed during productive infection encodes a protein that binds a splicing factor and reduces synthesis of viral proteins made from spliced mRNA. Proc Natl Acad Sci USA. 1996;93:10423–10427. doi: 10.1073/pnas.93.19.10423. [DOI] [PMC free article] [PubMed] [Google Scholar]
9.Cai W, Schaffer P A. Herpes simplex virus type 1 ICP0 plays a critical role in the de novo synthesis of infectious virus following transfection of viral DNA. J Virol. 1989;63:4579–4589. doi: 10.1128/jvi.63.11.4579-4589.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
10.Cai W, Schaffer P A. Herpes simplex virus type 1 ICP0 regulates expression of immediate-early, early, and late genes in productively infected cells. J Virol. 1992;66:2904–2915. doi: 10.1128/jvi.66.5.2904-2915.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
11.Chou J, Kern E R, Whitley R J, Roizman B. Mapping of herpes simplex virus-1 neurovirulence to γ134.5, a gene nonessential for growth in culture. Science. 1990;250:1262–1266. doi: 10.1126/science.2173860. [DOI] [PubMed] [Google Scholar]
12.Chou J, Poon A P, Johnson J, Roizman B. Differential response of human cells to deletions and stop codons in the γ134.5 gene of herpes simplex virus. J Virol. 1994;68:8304–8311. doi: 10.1128/jvi.68.12.8304-8311.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
13.Chou J, Roizman B. The γ134.5 gene of herpes simplex virus 1 precludes neuroblastoma cells from triggering total shutoff of protein synthesis characteristic of programed cell death in neuronal cells. Proc Natl Acad Sci USA. 1992;89:3266–3270. doi: 10.1073/pnas.89.8.3266. [DOI] [PMC free article] [PubMed] [Google Scholar]
14.Chou J, Roizman B. The herpes simplex virus 1 gene of ICP34.5, which maps in inverted repeats, is conserved in several limited-passage isolates but not in strain 17syn+ J Virol. 1990;64:1014–1020. doi: 10.1128/jvi.64.3.1014-1020.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
15.Chou J, Roizman B. The terminal a sequence of the herpes simplex virus genome contains the promoter of a gene located in the repeat sequences of the L component. J Virol. 1986;57:629–637. doi: 10.1128/jvi.57.2.629-637.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
16.DeLuca N A, Schaffer P A. Activities of herpes simplex virus type 1 (HSV-1) ICP4 genes specifying nonsense peptides. Nucleic Acids Res. 1987;15:4491–4511. doi: 10.1093/nar/15.11.4491. [DOI] [PMC free article] [PubMed] [Google Scholar]
17.DeLuca N A, Schaffer P A. Physical and functional domains of the herpes simplex virus transcriptional regulatory protein ICP4. J Virol. 1988;62:732–743. doi: 10.1128/jvi.62.3.732-743.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
18.Dolan A, McKie E, MacLean A R, McGeoch D J. Status of the ICP34.5 gene in herpes simplex virus type 1 strain 17. J Gen Virol. 1992;73:971–973. doi: 10.1099/0022-1317-73-4-971. [DOI] [PubMed] [Google Scholar]
19.Fenwick M L. The effects of herpesviruses on cellular macromolecular synthesis. In: Fraenkel-Conrat H, Wagner R R, editors. Comprehensive virology. Vol. 19. New York, N.Y: Plenum Press; 1984. pp. 359–390. [Google Scholar]
20.Frazier D P, Cox D, Godshalk E M, Schaffer P A. Identification of cis-acting sequences in the promoter of the herpes simplex virus type 1 latency-associated transcripts required for activation by nerve growth factor and sodium butyrate in PC12 cells. J Virol. 1996;70:7433–7444. doi: 10.1128/jvi.70.11.7433-7444.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
21.Greene L A, Tischler A S. PC12 pheochromocytoma cultures in neurobiological research. Adv Cell Neurobiol. 1982;3:373–414. [Google Scholar]
22.Harlow E, Lane D, editors. Antibodies, a laboratory manual. Cold Spring Harbor, N.Y: Cold Spring Harbor Laboratory; 1988. [Google Scholar]
23.Kunkel T A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci USA. 1985;82:488–492. doi: 10.1073/pnas.82.2.488. [DOI] [PMC free article] [PubMed] [Google Scholar]
24.Laemmli U K. Cleavage of the structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970;227:680–684. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
25.Lagunoff M, Randall G, Roizman B. Phenotypic properties of herpes simplex virus 1 containing a derepressed open reading frame P gene. J Virol. 1996;70:1810–1817. doi: 10.1128/jvi.70.3.1810-1817.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
26.Lagunoff M, Roizman B. Expression of a herpes simplex virus 1 open reading frame antisense to the γ134.5 gene and transcribed by an RNA 3′ coterminal with the unspliced latency-associated transcript. J Virol. 1994;68:6021–6028. doi: 10.1128/jvi.68.9.6021-6028.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
27.Leib D A, Bogard C L, Kosz-Vnenchak M, Hicks K A, Coen D M, Knipe D M, Schaffer P A. A deletion mutant of the latency-associated transcript of herpes simplex virus type 1 reactivates from the latent state with reduced frequency. J Virol. 1989;63:2893–2900. doi: 10.1128/jvi.63.7.2893-2900.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
28.Leib D A, Coen D M, Bogard C L, Hicks K A, Yager D R, Knipe D M, Tyler K L, Schaffer P A. Immediate-early regulatory gene mutants define different stages in the establishment and reactivation of herpes simplex virus latency. J Virol. 1989;63:759–768. doi: 10.1128/jvi.63.2.759-768.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
29.McGeoch D J, Cunningham C, McIntyre G, Dolan A. Comparative sequence analysis of the long repeat regions and adjoining parts of the long unique regions in the genomes of herpes simplex viruses types 1 and 2. J Gen Virol. 1991;72:3057–3075. doi: 10.1099/0022-1317-72-12-3057. [DOI] [PubMed] [Google Scholar]
30.McKie E A, Hope R G, Brown S M, MacLean A R. Characterization of the herpes simplex virus type 1 strain 17+ neurovirulence gene RL1 and its expression in a bacterial system. J Gen Virol. 1994;75:733–741. doi: 10.1099/0022-1317-75-4-733. [DOI] [PubMed] [Google Scholar]
31.Perng G-C, Ghiasi H, Slanina S M, Nesburn A B, Wechsler S L. High-dose ocular infection with a herpes simplex virus type 1 ICP34.5 deletion mutant produces no corneal disease or neurovirulence yet results in wild-type levels of spontaneous reactivation. J Virol. 1996;70:2883–2893. doi: 10.1128/jvi.70.5.2883-2893.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
32.Perng G-C, Thompson R L, Sawtell N M, Taylor W E, Slanina S M, Ghiasi H, Kaiwar R, Nesburn A B, Wechsler S L. An avirulent ICP34.5 deletion mutant of herpes simplex virus type 1 is capable of in vivo spontaneous reactivation. J Virol. 1995;69:3033–3041. doi: 10.1128/jvi.69.5.3033-3041.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
33.Ralph W M, Jr, Cabatingan M S, Schaffer P A. Induction of HSV-1 immediate-early gene expression by a cellular activity expressed in Vero and NB41A3 cells following growth arrest-release. J Virol. 1994;68:6871–6882. doi: 10.1128/jvi.68.11.6871-6882.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
34.Randall G, Lagunoff M, Roizman B. The product of ORF O located within the domain of herpes simplex virus type 1 genome transcribed during latent infection, binds to and inhibits binding of infected cell protein 4 to its cognate DNA site. Proc Natl Acad Sci USA. 1997;94:1079–1084. doi: 10.1073/pnas.94.19.10379. [DOI] [PMC free article] [PubMed] [Google Scholar]
35.Randall G, Roizman B. Transcription of the derepressed open reading frame P of herpes simplex virus type 1 precludes the expression of the antisense γ134.5 gene and may account for the attenuation of the mutant virus. J Virol. 1997;71:7750–7757. doi: 10.1128/jvi.71.10.7750-7757.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
36.Reed L J, Muench H. A simple method of estimating fifty percent endpoints. Am J Hyg. 1938;27:493–497. [Google Scholar]
37.Robertson L M, MacLean A R, Brown S M. Peripheral replication and latency reactivation kinetics of the non-neurovirulent herpes simplex virus type 1 variant 1716. J Gen Virol. 1992;73:967–970. doi: 10.1099/0022-1317-73-4-967. [DOI] [PubMed] [Google Scholar]
38.Sacks W R, Greene C C, Aschman D P, Schaffer P A. Herpes simplex virus type 1 ICP27 is an essential regulatory protein. J Virol. 1985;55:796–805. doi: 10.1128/jvi.55.3.796-805.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
39.Sambrook J, Fritsch E F, Maniatis T. Molecular cloning: a laboratory manual. 2nd ed. Cold Spring Harbor, N.Y: Cold Spring Harbor Laboratory Press; 1989. [Google Scholar]
40.Sandri-Goldin R M, Levine M, Glorioso J C. Method for introduction of mutations in physically defined regions of the herpes simplex virus genome. J Virol. 1981;38:41–49. doi: 10.1128/jvi.38.1.41-49.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
41.Schaffer P A, Carter V C, Timbury M C. Collaborative complementation study of temperature-sensitive mutants of herpes simplex virus types 1 and 2. Virology. 1978;27:490–504. doi: 10.1128/jvi.27.3.490-504.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
42.Seed B, Sheen J Y. A simple phase-extraction assay for chlorampheniol acetyltransferase activity. Gene. 1988;67:271–277. doi: 10.1016/0378-1119(88)90403-9. [DOI] [PubMed] [Google Scholar]
43.Showalter S D, Zweig M, Hampar B. Monoclonal antibodies to herpes simplex virus type 1 proteins, including the immediate-early protein ICP4. Infect Immun. 1981;34:684–692. doi: 10.1128/iai.34.3.684-692.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
44.Southern E M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975;98:503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
45.Spivack J G, Fareed M U, Valyi-Nagy T, Nash T C, O’Keefe J S, Gesser R M, McKie E A, MacLean A R, Fraser N W, Brown S M. Replication, establishment of latent infection, expression of the latency-associated transcripts and explant reactivation of herpes simplex virus type 1 γ34.5 mutants in a mouse eye model. J Gen Virol. 1995;76:321–332. doi: 10.1099/0022-1317-76-2-321. [DOI] [PubMed] [Google Scholar]
46.Taha M Y, Clements G B, Brown S M. The herpes simplex virus type 2 (HG52) variant JH2604 has a 1488 bp deletion which eliminates neurovirulence in mice. J Gen Virol. 1989;70:3072–3078. doi: 10.1099/0022-1317-70-11-3073. [DOI] [PubMed] [Google Scholar]
47.Taha M Y, Clements G B, Brown S M. A variant of herpes simplex virus type 2 strain HG52 with a 1.5 kb deletion in RL between 0 to 0.02 and 0.81 and 0.83 map units is non-neurovirulent for mice. J Gen Virol. 1989;70:705–716. doi: 10.1099/0022-1317-70-3-705. [DOI] [PubMed] [Google Scholar]
48.Valyi-Nagy T, Fareed M U, O’Keefe J S, Gesser R M, MacLean A R, Brown S M, Spivack J G, Fraser N W. The herpes simplex virus type 1 strain 17+ γ34.5 deletion mutant 1716 is avirulent in SCID mice. J Gen Virol. 1994;75:2059–2063. doi: 10.1099/0022-1317-75-8-2059. [DOI] [PubMed] [Google Scholar]
49.Whitley R J, Kern E R, Chatterjee S, Chou J, Roizman B. Replication, establishment of latency, and induced reactivation of herpes simplex virus γ134.5 deletion mutants in rodent models. J Clin Invest. 1993;91:2837–2843. doi: 10.1172/JCI116527. [DOI] [PMC free article] [PubMed] [Google Scholar]
50.Yeh L, Schaffer P A. A novel class of transcripts expressed with late kinetics in the absence of ICP4 spans the junction between the long and short segments of the herpes simplex virus type 1 genome. J Virol. 1993;67:7373–7382. doi: 10.1128/jvi.67.12.7373-7382.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B1] 1.Ackermann M, Braun D K, Pereira L, Roizman B. Characterization of herpes simplex virus 1 α proteins 0, 4, and 27 with monoclonal antibodies. J Virol. 1984;52:108–119. doi: 10.1128/jvi.52.1.108-118.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B2] 2.Ackermann M, Chou J, Sarmiento M, Lerner R A, Roizman B. Identification by antibody to a synthetic peptide of a protein specified by a diploid gene located in the terminal repeats of the L component of the herpes simplex virus genome. J Virol. 1986;58:843–850. doi: 10.1128/jvi.58.3.843-850.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B3] 3.Andrews N C, Faller D V. A rapid micropreparation technique for extraction of DNA-binding proteins from limiting numbers of mammalian cells. Nucleic Acids Res. 1991;19:2499. doi: 10.1093/nar/19.9.2499. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B4] 4.Ausubel F M, Brent R, Kingston R E, Moore D D, Seidman J G, Smith J A, Struhl K, editors. Current protocols in molecular biology. New York, N.Y: John Wiley & Sons, Inc.; 1988. [Google Scholar]

[B5] 5.Bohenzky R A, Papavassiliou A G, Gelman I H, Silverstein S. Identification of a promoter mapping within the reiterated sequences that flank the herpes simplex virus type 1 UL region. J Virol. 1993;67:632–642. doi: 10.1128/jvi.67.2.632-642.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B6] 6.Bolovan C A, Sawtell N A, Thompson R L. ICP34.5 mutants of herpes simplex virus type 1 strain 17syn+ are attenuated for neurovirulence in mice and for replication in confluent primary mouse embryo cell cultures. J Virol. 1994;68:48–55. doi: 10.1128/jvi.68.1.48-55.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B7] 7.Brown S M, Harland J, MacLean A R, Podlech J, Clements J B. Cell type and cell state determine differential in vitro growth of non-neurovirulent ICP34.5-negative herpes simplex virus types 1 and 2. J Gen Virol. 1994;75:2367–2377. doi: 10.1099/0022-1317-75-9-2367. [DOI] [PubMed] [Google Scholar]

[B8] 8.Bruni R, Roizman B. Open reading frame P—a herpes simplex virus gene repressed during productive infection encodes a protein that binds a splicing factor and reduces synthesis of viral proteins made from spliced mRNA. Proc Natl Acad Sci USA. 1996;93:10423–10427. doi: 10.1073/pnas.93.19.10423. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B9] 9.Cai W, Schaffer P A. Herpes simplex virus type 1 ICP0 plays a critical role in the de novo synthesis of infectious virus following transfection of viral DNA. J Virol. 1989;63:4579–4589. doi: 10.1128/jvi.63.11.4579-4589.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B10] 10.Cai W, Schaffer P A. Herpes simplex virus type 1 ICP0 regulates expression of immediate-early, early, and late genes in productively infected cells. J Virol. 1992;66:2904–2915. doi: 10.1128/jvi.66.5.2904-2915.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B11] 11.Chou J, Kern E R, Whitley R J, Roizman B. Mapping of herpes simplex virus-1 neurovirulence to γ134.5, a gene nonessential for growth in culture. Science. 1990;250:1262–1266. doi: 10.1126/science.2173860. [DOI] [PubMed] [Google Scholar]

[B12] 12.Chou J, Poon A P, Johnson J, Roizman B. Differential response of human cells to deletions and stop codons in the γ134.5 gene of herpes simplex virus. J Virol. 1994;68:8304–8311. doi: 10.1128/jvi.68.12.8304-8311.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B13] 13.Chou J, Roizman B. The γ134.5 gene of herpes simplex virus 1 precludes neuroblastoma cells from triggering total shutoff of protein synthesis characteristic of programed cell death in neuronal cells. Proc Natl Acad Sci USA. 1992;89:3266–3270. doi: 10.1073/pnas.89.8.3266. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B14] 14.Chou J, Roizman B. The herpes simplex virus 1 gene of ICP34.5, which maps in inverted repeats, is conserved in several limited-passage isolates but not in strain 17syn+ J Virol. 1990;64:1014–1020. doi: 10.1128/jvi.64.3.1014-1020.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B15] 15.Chou J, Roizman B. The terminal a sequence of the herpes simplex virus genome contains the promoter of a gene located in the repeat sequences of the L component. J Virol. 1986;57:629–637. doi: 10.1128/jvi.57.2.629-637.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B16] 16.DeLuca N A, Schaffer P A. Activities of herpes simplex virus type 1 (HSV-1) ICP4 genes specifying nonsense peptides. Nucleic Acids Res. 1987;15:4491–4511. doi: 10.1093/nar/15.11.4491. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B17] 17.DeLuca N A, Schaffer P A. Physical and functional domains of the herpes simplex virus transcriptional regulatory protein ICP4. J Virol. 1988;62:732–743. doi: 10.1128/jvi.62.3.732-743.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B18] 18.Dolan A, McKie E, MacLean A R, McGeoch D J. Status of the ICP34.5 gene in herpes simplex virus type 1 strain 17. J Gen Virol. 1992;73:971–973. doi: 10.1099/0022-1317-73-4-971. [DOI] [PubMed] [Google Scholar]

[B19] 19.Fenwick M L. The effects of herpesviruses on cellular macromolecular synthesis. In: Fraenkel-Conrat H, Wagner R R, editors. Comprehensive virology. Vol. 19. New York, N.Y: Plenum Press; 1984. pp. 359–390. [Google Scholar]

[B20] 20.Frazier D P, Cox D, Godshalk E M, Schaffer P A. Identification of cis-acting sequences in the promoter of the herpes simplex virus type 1 latency-associated transcripts required for activation by nerve growth factor and sodium butyrate in PC12 cells. J Virol. 1996;70:7433–7444. doi: 10.1128/jvi.70.11.7433-7444.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B21] 21.Greene L A, Tischler A S. PC12 pheochromocytoma cultures in neurobiological research. Adv Cell Neurobiol. 1982;3:373–414. [Google Scholar]

[B22] 22.Harlow E, Lane D, editors. Antibodies, a laboratory manual. Cold Spring Harbor, N.Y: Cold Spring Harbor Laboratory; 1988. [Google Scholar]

[B23] 23.Kunkel T A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci USA. 1985;82:488–492. doi: 10.1073/pnas.82.2.488. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B24] 24.Laemmli U K. Cleavage of the structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970;227:680–684. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]

[B25] 25.Lagunoff M, Randall G, Roizman B. Phenotypic properties of herpes simplex virus 1 containing a derepressed open reading frame P gene. J Virol. 1996;70:1810–1817. doi: 10.1128/jvi.70.3.1810-1817.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B26] 26.Lagunoff M, Roizman B. Expression of a herpes simplex virus 1 open reading frame antisense to the γ134.5 gene and transcribed by an RNA 3′ coterminal with the unspliced latency-associated transcript. J Virol. 1994;68:6021–6028. doi: 10.1128/jvi.68.9.6021-6028.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B27] 27.Leib D A, Bogard C L, Kosz-Vnenchak M, Hicks K A, Coen D M, Knipe D M, Schaffer P A. A deletion mutant of the latency-associated transcript of herpes simplex virus type 1 reactivates from the latent state with reduced frequency. J Virol. 1989;63:2893–2900. doi: 10.1128/jvi.63.7.2893-2900.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B28] 28.Leib D A, Coen D M, Bogard C L, Hicks K A, Yager D R, Knipe D M, Tyler K L, Schaffer P A. Immediate-early regulatory gene mutants define different stages in the establishment and reactivation of herpes simplex virus latency. J Virol. 1989;63:759–768. doi: 10.1128/jvi.63.2.759-768.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B29] 29.McGeoch D J, Cunningham C, McIntyre G, Dolan A. Comparative sequence analysis of the long repeat regions and adjoining parts of the long unique regions in the genomes of herpes simplex viruses types 1 and 2. J Gen Virol. 1991;72:3057–3075. doi: 10.1099/0022-1317-72-12-3057. [DOI] [PubMed] [Google Scholar]

[B30] 30.McKie E A, Hope R G, Brown S M, MacLean A R. Characterization of the herpes simplex virus type 1 strain 17+ neurovirulence gene RL1 and its expression in a bacterial system. J Gen Virol. 1994;75:733–741. doi: 10.1099/0022-1317-75-4-733. [DOI] [PubMed] [Google Scholar]

[B31] 31.Perng G-C, Ghiasi H, Slanina S M, Nesburn A B, Wechsler S L. High-dose ocular infection with a herpes simplex virus type 1 ICP34.5 deletion mutant produces no corneal disease or neurovirulence yet results in wild-type levels of spontaneous reactivation. J Virol. 1996;70:2883–2893. doi: 10.1128/jvi.70.5.2883-2893.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B32] 32.Perng G-C, Thompson R L, Sawtell N M, Taylor W E, Slanina S M, Ghiasi H, Kaiwar R, Nesburn A B, Wechsler S L. An avirulent ICP34.5 deletion mutant of herpes simplex virus type 1 is capable of in vivo spontaneous reactivation. J Virol. 1995;69:3033–3041. doi: 10.1128/jvi.69.5.3033-3041.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B33] 33.Ralph W M, Jr, Cabatingan M S, Schaffer P A. Induction of HSV-1 immediate-early gene expression by a cellular activity expressed in Vero and NB41A3 cells following growth arrest-release. J Virol. 1994;68:6871–6882. doi: 10.1128/jvi.68.11.6871-6882.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B34] 34.Randall G, Lagunoff M, Roizman B. The product of ORF O located within the domain of herpes simplex virus type 1 genome transcribed during latent infection, binds to and inhibits binding of infected cell protein 4 to its cognate DNA site. Proc Natl Acad Sci USA. 1997;94:1079–1084. doi: 10.1073/pnas.94.19.10379. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B35] 35.Randall G, Roizman B. Transcription of the derepressed open reading frame P of herpes simplex virus type 1 precludes the expression of the antisense γ134.5 gene and may account for the attenuation of the mutant virus. J Virol. 1997;71:7750–7757. doi: 10.1128/jvi.71.10.7750-7757.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B36] 36.Reed L J, Muench H. A simple method of estimating fifty percent endpoints. Am J Hyg. 1938;27:493–497. [Google Scholar]

[B37] 37.Robertson L M, MacLean A R, Brown S M. Peripheral replication and latency reactivation kinetics of the non-neurovirulent herpes simplex virus type 1 variant 1716. J Gen Virol. 1992;73:967–970. doi: 10.1099/0022-1317-73-4-967. [DOI] [PubMed] [Google Scholar]

[B38] 38.Sacks W R, Greene C C, Aschman D P, Schaffer P A. Herpes simplex virus type 1 ICP27 is an essential regulatory protein. J Virol. 1985;55:796–805. doi: 10.1128/jvi.55.3.796-805.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B39] 39.Sambrook J, Fritsch E F, Maniatis T. Molecular cloning: a laboratory manual. 2nd ed. Cold Spring Harbor, N.Y: Cold Spring Harbor Laboratory Press; 1989. [Google Scholar]

[B40] 40.Sandri-Goldin R M, Levine M, Glorioso J C. Method for introduction of mutations in physically defined regions of the herpes simplex virus genome. J Virol. 1981;38:41–49. doi: 10.1128/jvi.38.1.41-49.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B41] 41.Schaffer P A, Carter V C, Timbury M C. Collaborative complementation study of temperature-sensitive mutants of herpes simplex virus types 1 and 2. Virology. 1978;27:490–504. doi: 10.1128/jvi.27.3.490-504.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B42] 42.Seed B, Sheen J Y. A simple phase-extraction assay for chlorampheniol acetyltransferase activity. Gene. 1988;67:271–277. doi: 10.1016/0378-1119(88)90403-9. [DOI] [PubMed] [Google Scholar]

[B43] 43.Showalter S D, Zweig M, Hampar B. Monoclonal antibodies to herpes simplex virus type 1 proteins, including the immediate-early protein ICP4. Infect Immun. 1981;34:684–692. doi: 10.1128/iai.34.3.684-692.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B44] 44.Southern E M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975;98:503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]

[B45] 45.Spivack J G, Fareed M U, Valyi-Nagy T, Nash T C, O’Keefe J S, Gesser R M, McKie E A, MacLean A R, Fraser N W, Brown S M. Replication, establishment of latent infection, expression of the latency-associated transcripts and explant reactivation of herpes simplex virus type 1 γ34.5 mutants in a mouse eye model. J Gen Virol. 1995;76:321–332. doi: 10.1099/0022-1317-76-2-321. [DOI] [PubMed] [Google Scholar]

[B46] 46.Taha M Y, Clements G B, Brown S M. The herpes simplex virus type 2 (HG52) variant JH2604 has a 1488 bp deletion which eliminates neurovirulence in mice. J Gen Virol. 1989;70:3072–3078. doi: 10.1099/0022-1317-70-11-3073. [DOI] [PubMed] [Google Scholar]

[B47] 47.Taha M Y, Clements G B, Brown S M. A variant of herpes simplex virus type 2 strain HG52 with a 1.5 kb deletion in RL between 0 to 0.02 and 0.81 and 0.83 map units is non-neurovirulent for mice. J Gen Virol. 1989;70:705–716. doi: 10.1099/0022-1317-70-3-705. [DOI] [PubMed] [Google Scholar]

[B48] 48.Valyi-Nagy T, Fareed M U, O’Keefe J S, Gesser R M, MacLean A R, Brown S M, Spivack J G, Fraser N W. The herpes simplex virus type 1 strain 17+ γ34.5 deletion mutant 1716 is avirulent in SCID mice. J Gen Virol. 1994;75:2059–2063. doi: 10.1099/0022-1317-75-8-2059. [DOI] [PubMed] [Google Scholar]

[B49] 49.Whitley R J, Kern E R, Chatterjee S, Chou J, Roizman B. Replication, establishment of latency, and induced reactivation of herpes simplex virus γ134.5 deletion mutants in rodent models. J Clin Invest. 1993;91:2837–2843. doi: 10.1172/JCI116527. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B50] 50.Yeh L, Schaffer P A. A novel class of transcripts expressed with late kinetics in the absence of ICP4 spans the junction between the long and short segments of the herpes simplex virus type 1 genome. J Virol. 1993;67:7373–7382. doi: 10.1128/jvi.67.12.7373-7382.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]

PERMALINK

A Virus with a Mutation in the ICP4-Binding Site in the L/ST Promoter of Herpes Simplex Virus Type 1, but Not a Virus with a Mutation in Open Reading Frame P, Exhibits Cell-Type-Specific Expression of γ134.5 Transcripts and Latency-Associated Transcripts

Lily Yeh Lee

Priscilla A Schaffer