Proc. Natl. Acad. Sci. USA Vol. 94, pp. 10379–10384, September 1997 Microbiology

The product of ORF O located within the domain of 1 genome transcribed during latent infection binds to and inhibits in vitro binding of infected cell protein 4 to its cognate DNA site (repressed genes͞gene function͞productive infection͞regulatory proteins)

GLENN RANDALL,MICHAEL LAGUNOFF*, AND BERNARD ROIZMAN†

The Marjorie B. Kovler Viral Oncology Laboratories, University of Chicago, 910 East 58th Street, Chicago IL 60637

Contributed by Bernard Roizman, July 16, 1997

ABSTRACT The partially overlapping ORF P and ORF O lishment of latency is uncertain. Deletion of the promoter or are located within the domains of the 1 coding domains that give rise to the stable intron RNAs decrease genome transcribed during latency. Earlier studies have the number of neurons harboring latent virus and decrease the shown that ORF P is repressed by infected cell protein 4 capacity of latently infected neurons to reactivate virus but do not (ICP4), the major viral regulatory protein, binding to its affect the establishment of latency (11–13). In earlier studies from cognate site at the transcription initiation site of ORF P. The this laboratory we reported that the 8.5 kb of DNA transcribed ORF P protein binds to p32, a component of the ASF͞SF2 during latency contains 16 ORFs encoding at least 50 codons, alternate splicing factors; in cells infected with a recombinant which we have designated ORF A through ORF P. We reported virus in which ORF P was derepressed there was a significant that one of the five ORFs tested, ORF P, situated almost totally decrease in the expression of products of key regulatory genes antisense to the ␥134.5 gene, was repressed in productive infection containing introns. We report that (i) the expression of ORF and expressed only under conditions in which the major regula- O is repressed during productive infection by the same tory protein ICP4 was nonfunctional or if the binding site was mechanism as that determining the expression of ORF P; (ii) mutagenized such that ICP4 could not bind to its cognate site at in cells infected at the nonpermissive temperature for ICP4, the transcription initiation of ORF P (14–16). We should note ORF O protein is made in significantly lower amounts than that the promoter of ORF P was discovered by Bohenzky et al. the ORF P protein; (iii) the results of insertion of a sequence (17), and the existence of the 2.3-kbp mRNA species coterminal encoding 20 amino acids between the putative initiator me- with the 8.5-kb latency-associated transcript, encoding what was thionine codons of ORF O and ORF P suggest that ORF O subsequently shown to be ORF P was described by Yeh and initiates at the methionine codon of ORF P and that the Schafer (18). synthesis of ORF O results from frameshift or editing of its At the time ORF P was discovered, we could not reproduciby RNA; and (iv) glutathione S-transferase–ORF O fusion pro- show the expression of ORF O because its expression was many tein bound specifically ICP4 and precluded its binding to its times lower than that of ORF P. In subsequent studies we cognate site on DNA in vitro. These and earlier results indicate discovered that ORF O was expressed, but that the coding that ORF P and ORF O together have the capacity to reduce domain was smaller and totally overlapped the domain of ORF the synthesis or block the expression of regulatory proteins P (Fig. 1, line 2). Specifically, the nucleotide sequence of ORF O essential for viral replication in productive infection. predicts that the initiator methionine of ORF O is located in the TATA box of ORF P. We show that in fact this methionine is not The herpes simplex 1 and 2 (HSV-1 and HSV-2) cause two used and that the only methionine in a reasonable location to kinds of infections. At the portal of entry into the body, these initiate ORF O translation is that which initiates translation of viruses express Ͼ80 genes, assemble infectious progeny, and ORF P, suggesting that ORF O is expressed by a frameshift or ultimately destroy the infected cells (1). Latent infections usually editing process within the first 35 codons of ORF P mRNA. We take place in neurons of dorsal root ganglia. Thus, the virus also report that fusion proteins comprising ORF O sequences present at the portal of entry infects nerve endings and is interact specifically with ICP4 and interfere in vitro with the transported retrograde to the neuronal nucleus where it multi- binding of ICP4 to its cognate site. plies and destroys the neuron or remains latent (reviewed in ref. 2). In latent state, viral DNA forms an episome and only a small MATERIALS AND METHODS portion of the genome is transcribed. The transcribed domain Cells and Viruses. Rabbit skin and 143tkϪ cells were orig- contained within the inverted repeats flanking the unique long inally obtained from J. McClaren (University of Arizona) and (UL) sequence is Ϸ8.5 kbp. In productively infected cells, the Carlo Croce (Thomas Jefferson Medical College), respec- antisense strand of this domain expresses two ORFs encoding tively. Vero and HeLa cells were from American Type Culture infected cell protein 0 (ICP0) and ␥134.5. In latently infected Collection. HSV-1(F) is the prototype HSV-1 strain used in neurons transcripts of the entire 8.5-kbp DNA stretch (major this laboratory; as is the case with fresh HSV-1 isolates with latency-associated transcript) are present in low abundance (3–7). limited history of replication outside the human host, the ␣4 Transcripts of a 1.5- and 2.0-kbp stretch are present in large gene of HSV-1(F) is temperature sensitive and does not abundance in nuclei and probably represent stable introns (4, 5, repress itself or ORF P at 39.5°C (19). The recombinants 8–10). The role of the 1.5- and 2.0-kbp transcripts in the estab- Abbreviations: HSV-1, herpes simplex virus 1; GST, glutathione The publication costs of this article were defrayed in part by page charge S-transferase; ICP, infected cell protein; tk, thymidine kinase; CMV, . payment. This article must therefore be hereby marked ‘‘advertisement’’ in *Present address: Department of Microbiology and Immunology, accordance with 18 U.S.C. §1734 solely to indicate this fact. University of California, San Francisco, CA 94143. © 1997 by The National Academy of Sciences 0027-8424͞97͞9410379-6$2.00͞0 †To whom reprint requests should be addressed. e-mail: bernard@ PNAS is available online at http:͞͞www.pnas.org. kovler.uchicago.edu.

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derived from HSV-1(F) used in this study fall into two categories, those in which the ICP4 binding site at the tran- scription initiation site of ORF P was destroyed by mutagen- esis, as described elsewhere (14, 16) (e.g., R7530, R7540, R7548), and those which retain the binding site (15) (R7519 and R7520). In addition, several recombinants were tagged with a sequence encoding 20 codons from the glycoprotein B of human CMV and for which a monoclonal antibody (CH28-2) (20) was available from the Goodwin Cancer Re- search Institute, Plantation, FL. A list of recombinants used in this study is given in Table 1. Plasmids. Plasmid pRB4794 (15) containing the 1,800-bp NcoI fragment of BamHI S, spanning the region between the start codons of the ␣0 and ␥134.5 genes, was used as a probe for analysis of ORF P and ORF O recombinant viral DNA; pRB4791 (15) containing a CMV tag in the DraIII site of BamHI S, in the ORF O reading frame, was used to construct virus R7520; pRB4855 (14) containing a 4-base change in the ICP4 binding site which has been shown to derepress ORF P expression. pRB4923 was made by replacing the BspEI–BstEII fragment from pRB4855 with the corresponding fragment from pRB4791. It was used to construct the virus R7540. pBR5181 contains a CMV tag inserted into the SacI sites of pRB4923, in the ORF O reading frame. The CMV tag sequence was GAAAGGACAAAAGCCCAACCTTCTA- GACCGACTCCGACATAGAAAGAACGGGTACCGAC- ACGCAGCT and its complement. It was used to construct virus R7548. pRB5179 contains the 287-bp XmaI fragment from BamHI S inserted in the XmaI site of pGEX-3X (Phar- macia) and was used to purify the glutathione S-transferase (GST)–ORF PC fusion protein (ORF P amino acids 130–225). pRB5180 contains the 420-bp XmaI fragment from BamHI S inserted in the XmaI site of pGEX-4T3 (Pharmacia) and was used to purify GST–ORF PN fusion protein (ORF P amino acids Ϫ10 to 130). pRB4936 contains the same 420-bp XmaI fragment inserted into the XmaI site of pGEX-2T and was used to purify GST–ORF O fusion protein [amino acids 45 to the C terminus of the HSV-1(F) sequence predicted for ORF O]. Construction of R7540 and R7548. Recombinant R7540 con- tains a mutated ICP4 binding site and a CMV tag in the DraIII site of BamHI S, in-frame with ORF O. R7548 contains both of these mutations plus an additional CMV tag insertion in the SacI sites located between the predicted ORF O initiator methionine and the downstream ORF P initiator methionine. The procedure for the construction of the recombinant viruses R7540 and R7548 was similar to that previously described (14, 21, 22). Viral DNA was isolated from infected cells and purified on a 5–20% KAc gradient as described (23). The mutations were verified by sequencing and the recombinant viral DNA was probed for the presence of diagnostic restriction endonuclease sites. In the case of R7540, an EcoRI restriction endonuclease site, diagnostic of FIG. 1. Schematic representations of sequence arrangements of recombinant virus genomes. Lines 1 and 3, representation of the the ICP4 binding site mutation, and an XbaI restriction endonu- HSV-1 (F) genome. The lines represent unique long (UL) and short clease site, diagnostic of the CMV epitope insertion (Fig. 1, lines (US) sequences that are flanked by inverted repeats ab and bЈaЈ and aЈcЈ and ca, respectively, shown as rectangles. Line 2, representation DraIII (Dr) site in frame with ORF O, a mutated ICP4 binding site, of recombinant virus R7520 (15), which contains the cytomegalovirus and an additional CMV gB epitope inserted in the SacI site, also (CMV) epitope inserted in the DraIII site, in-frame with ORF O. The in-frame with ORF O. The second insertion was within the predicted rectangular boxes and arrows represent gene domains and direction of ORF O gene, upstream of the sequences subsequently shown to be the transcription of the ORF O, ORF P, and ␥134.5 genes in the inverted ORF P coding sequences. Lines 4, 6, 8, and 10, the expected sizes of repeat sequence bЈaЈ. The identical sequences in the inverted orien- fragments detected by hybridization of the 1,800-bp NcoI fragment tation map in the ab repeat. The closed circle denotes a wild-type ICP4 with electrophoretically separated digests of viral DNAs with NcoI– binding site. Line 5, the corresponding domain of R3659 (16). The EcoRI (the first band set per virus), diagnostic of the ICP4 binding site StuI–BstEII sequences encoding the ORF O, ORF P, and ␥134.5 genes mutation; or NcoI–XbaI (the second band set per virus), diagnostic of were replaced in both repeats by the chimeric ␣27-tk gene (21). Line the CMV epitope tag insertion. The arrows in these lines denote 7, the corresponding domain of the recombinant virus R7540. The restriction cleavage sites present in the respective viruses and therefore ␣27-tk gene of the recombinant R3659 was replaced with sequences the DNA fragment boundaries. HSV-1(F) would be expected to yield containing a mutated ICP4 binding site with a diagnostic EcoRI bands A and B, respectively; R3659 would be expected to yield bands endonuclease site, and a CMV glycoprotein B (gB) epitope containing C and D; R7540 would be expected to yield bands E, F, and G; R7548 a diagnostic EcoRI site in the DraIII (Dr) site in-frame with ORF O. would be expected to yield bands H, J, K, L, and M, respectively. Dr, Line 9, the corresponding domain of the recombinant virus R7548. DraIII; St, StuI; Nc, NcoI; Bs, BstEII; Sc, SacI; Ec1, the introduced Here the ␣27-tk of R3659 was replaced with the CMV epitope in the EcoRI site. Downloaded by guest on September 28, 2021 Microbiology: Randall et al. Proc. Natl. Acad. Sci. USA 94 (1997) 10381

Table 1. Properties of recombinant viruses respectively). All fragment sizes in Fig. 2 correspond to the Epitope tag predicted patterns shown in Fig. 1. Virus no.* Designation ICP4 site and frame Production of Polyclonal Anti-ORF O Antibody. pRB4936, described above, was transformed into Escherichia coli BL21, † R3659 Deleted None and protein was expressed and purified as recommended by R7519 CMV–ORF P Native BstEII͞ORF P the manufacturer (Pharmacia). Two rabbits were inoculated R7520 CMV–ORF O Native DraIII͞ORF O subcutaneously with 1 mg each of purified fusion protein at R7530 Pϩϩ͞Oϩϩ Mutated None 14-day intervals, as per the normal protocol at Josman Lab- R7540 Pϩϩ Oϩϩ CMV I Mutated DraIII ORFO ͞ ͞ oratories (Napa, CA). The sera used in this study were R7548 Pϩϩ Oϩϩ CMV 1ϩ2 Mutated DraIII ORF O ͞ ͞ collected two weeks after the final immunization. SacI͞ORF O *R7519 and R7520 were described in ref. 15; R7530 was described in RESULTS ref. 14. For R7540 and R7548, see Materials and Methods. ORF O Is Expressed Under the Same Conditions as ORF P. † In R3659 the ORF P͞ORF O and ␥134.5 genes were replaced by the Earlier studies have shown that ORF P is expressed in cells chimeric ␣27-tk gene (14). infected and maintained at 39.5°C, the nonpermissive tempera- 7 and 8), were present. Digestion of R7540 viral DNA with ture for ICP4 in HSV-1(F), or maintained at permissive temper- NcoI–EcoRI resulted in a 930͞940-bp doublet (Fig. 2, lane 5, atures after infection with mutants in which the ICP4 binding site at the transcription initiation site of ORF P was destroyed by bands E͞EЈ), whereas digestion with NcoI–XbaI yielded DNA fragments of 1,225 and 625 bp (Fig. 2, lane 6, bands F and G). In mutagenesis (14–16). The results in Fig. 3 show that the expres- contrast, digestion of HSV-1(F) viral DNA with NcoI–EcoRI or sion of ORF O protein with an apparent Mr of 20,000 is regulated in the same fashion as that of ORF P. Specifically, the tagged NcoI–XbaI results in 1,800-bp DNA fragments (Fig. 2, lanes 1 and ORF O protein was detected only in Vero cells infected with the 2, bands A and B). Digestion of the parental R3659 viral DNA R7520 (CMV–ORF O) recombinant (Fig. 1, line 2), which (Fig. 1, lines 5 and 6) produced 700-bp fragments (Fig. 2, lanes 3 contains a CMV epitope in-frame with ORF O, and maintained and 4, bands C and D) corresponding to the sequence upstream at 39.5°C (Fig. 3 A, lane 7; B, lanes 6 and 9). Inasmuch as both of the thymidine kinase (tk) replacement. As for R7548, an EcoRI ORF P and ORF O were tagged with the same amino acid restriction endonuclease site, diagnostic of the mutation in the sequence and detected with the mAb directed against the epitope, ICP4 binding site, and two XbaI restriction endonuclease sites, we conclude that the results shown in Fig. 3A, lanes 7 and 8, also diagnostic of the two CMV tag insertions (Fig. 1, lines 9 and 10) indicate that the ORF O protein is made in quantities far smaller were present. Digestion of R7548 with NcoI–EcoRI produced than those of ORF P. To test whether the same ICP4 cognate site 930- and 1,000-bp DNA fragments (Fig. 2, lane 7, bands H and was responsible for repression of ORF P and ORF O, we J), whereas cleavage with NcoI–XbaI produced DNA fragments of 1,050, 255, and 625 bp (Fig. 2, lane 8; bands K, L, and M,

FIG. 3. Photograph of infected cell proteins electrophoretically separated on a denaturing polyacrylamide gel and reacted with the CH28-2 mouse mAb to the CMV epitope. (A) Replicate Vero cell cultures grown in 25-cm2 flasks were infected with 10 pfu of HSV-1(F), R7519 (CMV–ORF P), or R7520 (CMV–ORF O) per cell, maintained FIG. 2. Autoradiographic image of electrophoretically separated at 37°C for 4 or 18 h (lanes 1–5) or maintained at 39.5°C for 24 h (lanes viral DNA fragments containing sequences in the domain of the ORF 6–8), harvested, solubilized, electrophoretically separated on 15% O͞ORF P͞␥134.5 genes. Viral DNAs were digested with either NcoI polyacrylamide denaturing gels, transferred to a nitrocellulose sheet, and EcoRI (lanes 1, 3, 5, and 7) or NcoI and XbaI (lanes 2, 4, 6, and and reacted first with mouse mAb CH28-2 (20) and second with 8). They were then subjected to electrophoresis on a 28-cm, 0.85% anti-mouse IgG conjugated to horseradish peroxidase, and horserad- agarose gel and transferred to a Zeta probe (Bio-Rad) by capillary ish peroxidase electrochemiluminescent substrate. Blots were exposed action in 0.5 M NaOH. The membrane was rinsed in 2ϫ SSC (0.3 M to Kodak XAR-5 film for 1 min. (B) Replicate Vero cell cultures grown NaCl͞0.015 M Na citrate), prehybridized in 30% formamide, 6ϫ SSC, in 25-cm2 flasks were infected with 10 pfu of HSV-1(F), R7520 1% milk, 1% SDS, and 100 ␮g single-stranded calf thymus DNA per (CMV–ORF O), or R7540 (Pϩϩ͞Oϩϩ, CMV 1) per cell, maintained ml for 30 min at 68°C. A total of 106 cpm of denatured, 32P-labeled at 37°C for 2, 12, or 22 h (lanes 1–7) or maintained at 39.5°C for 22 h pRB4794 was then added overnight and the blot was rinsed as (lanes 8–10), harvested, solubilized, electrophoretically separated on recommended by the manufacturer. Autoradiographic images on 12.5% polyacrylamide denaturing gels, transferred to a nitrocellulose Kodak XAR-5 film were overexposed to detect smaller fragments. The sheet, and reacted with mouse monoclonal antiserum to CMV gB, then expected sizes of the fragments generated by cleavages (bands A goat anti-mouse IgG conjugated to alkaline phosphatase, and alkaline through M) are shown in Fig. 1. phosphatase substrate. Downloaded by guest on September 28, 2021 10382 Microbiology: Randall et al. Proc. Natl. Acad. Sci. USA 94 (1997)

characterized the recombinant virus R7540 (Pϩϩ͞Oϩϩ, CMV 1), which combines the CMV epitope insertion in-frame with ORF O with the ICP4 binding site mutation previously shown to derepress the expression of ORF P (16). The construction and verification of R7540 (Pϩϩ͞Oϩϩ, CMV 1) are described in Materials and Methods. Vero cells infected with the R7540 (Pϩϩ͞Oϩϩ, CMV 1) recombinant and maintained at both permissive (Fig. 3B, lanes 4 and 7) and nonpermissive tempera- tures (Fig. 3B, lane 10) expressed ORF O. Like ORF P protein (15), the ORF O protein continued to accumulate until at least 22 h after infection. The lower abundance, faster migrating ORF O band is not reproducible and probably represents a degradation product. The results shown here indicate that the expression of ORF O is repressed during productive infection by ICP4 and that mutagenesis of the ICP4 binding site at the transcription initiation site of ORF P derepresses both ORF P shown earlier (14–16) and ORF O. The First Methionine of the ORF Is Not the Initiator Methi- onine of the ORF O Protein. In this series of experiments, we constructed the recombinant virus R7548 in which the ICP4 binding site at the transcription initiation site of ORF P was destroyed by mutagenesis, and two sequences encoding the CMV epitope were inserted in-frame with ORF O, one at the DraIII site as in R7540 (CMV 1) and one at the SacI sites located between the predicted methionine codon at the 5Ј end of the ORF and the initiator methionine codon of the ORF P gene (Fig. 1 line 9). The epitope tag contains no methionine codons in any frame that could alter the translation of downstream sequences. The pre- diction of this experiment was that the second epitope would increase the apparent molecular weight of ORF O as a conse- quence of the addition of 19 amino acids to the protein. The lysates of cells infected with wild-type and recombinant viruses FIG. 4. Photograph of infected cell proteins electrophoretically were subjected to electrophoresis in a denaturing polyacrylamide separated on a denaturing polyacrylamide gel and reacted with (A) rabbit polyclonal antiserum specific for GST–ORF O or with (B) mAb gel, transferred to a nitrocellulose sheet, and probed with either 2 the monoclonal antibody to the CMV epitope or the rabbit CH28-2. Replicate Vero cell cultures grown in 25-cm flasks were infected with 10 pfu of HSV-1(F), R7530 (Pϩϩ͞Oϩϩ), R7540 (CMV polyclonal antibody made against the GST–ORF O chimeric 1), or R7548 (CMV 1ϩ2) per cell, maintained at 37°C for 22 h, protein described in Materials and Methods. As shown in Fig. 4, harvested, solubilized, electrophoretically separated on 12.5% poly- the rabbit polyclonal antibody specifically reacted with the wild- acrylamide denaturing gels, transferred to a nitrocellulose sheet, and type, singly and doubly tagged ORF O proteins (Fig. 4A) whereas reacted with (A) rabbit polyclonal antiserum to ORF O or (B) mouse the mAb reacted only with the tagged proteins (Fig. 4B). ORF O monoclonal antiserum to CMV gB followed by secondary antibodies with a single CMV tag (R7540 CMV 1) migrated more slowly conjugated to alkaline phosphatase as described in the legend to Fig. than the wild-type ORF O (R7530 Pϩϩ͞Oϩϩ), consistent with 3. R7540 (CMV 1) and R7548 (CMV 1ϩ2) both have mutated ICP4 binding sites, designated (Pϩϩ Oϩϩ). the insertion of a 20 amino acid epitope into the coding region of ͞ ORF O (Fig. 4A). The surprising finding was that the electro- anti-ICP4 mAb brought down ICP4 and GST–ORF O but not phoretic mobility of the singly (R7540 CMV 1) and doubly GST–ORF P fusion proteins (data not shown). (R7548 CMV 1ϩ2) tagged ORF was identical. The necessary The purpose of the third series of experiments was to deter- conclusion is that the second tag was inserted into the sequence mine whether the ORF O fusion protein which interacts with that is not translated and by extension, that the initiator codon is ICP4 affects the interaction of ICP4 with its cognate DNA downstream from the site of insertion of the second CMV tag. sequence (Fig. 6). ICP4 binds to both high affinity sites consisting The only methionine between the untranslated and translated of a conserved consensus sequence and weak affinity sites for epitope tags is the initiator methionine of ORF P, suggesting that which no clear consensus sequence has been derived (24, 25). We this is also the initiator methionine of ORF O. The protein then selected for these studies the strong binding site present at the shifts into the ORF O frame by amino acid 35, the site of the transcription initiation site of ORF P (designated probe) and the expressed CMV tag insertion, by an unknown mechanism. corresponding DNA fragment containing a mutagenized ICP4 ORF O Protein Interacts with ICP4 and Blocks the Binding of binding site (designated probe⌬ICP4bs). Reaction of the probe ICP4 to its Cognate DNA Site. The purpose of these studies was DNA with nuclear extracts of HSV-1(F)-infected cells yielded a to determine whether ORF P or ORF O proteins interact with specific ICP4–DNA complex that was supershifted by monoclo- nal antibody H943 to ICP4 (26) (Fig. 6, lanes 2 and 3, respec- any viral proteins expressed during productive infection. In the tively). Concentrations of GST–ORF O greater than 250 ng first series of experiments, replicate HeLa cells were labeled with blocked the binding of ICP4 to the probe (lane 8) whereas 3 ␮g [35S]methionine from 4 to8hafterinfection or mock-infection. of GST–ORF PC had no effect (lane 10). ICP4 did not bind to the The cell lysates were precleared with GST and reacted with either mutagenized sequence (lane 12), consistent with earlier results GST–ORF PC, GST–ORF PN, or GST–ORF O as described in (25, 27). Because GST–ORF O did not bind the DNA probe (lane the legend to Fig. 5. The results (Fig. 5A lane 8) show that only 9), we conclude that ORF O precludes the binding of ICP4 to the GST–ORF O chimeric protein brought down a set of labeled DNA rather than competes with ICP4 for the DNA binding site. proteins with the apparent Mr predicted for ICP4. The bands reacted with the mAb to ICP4 (Fig. 5B, lane 8). GST–ORF O DISCUSSION bound the two forms of ICP4 detectable on this immunoblot with The key findings reported here are that (i) a second ORF, relatively equal affinity. In the second series of experiments, designated ORF O and mapping in the domain of the HSV-1 Downloaded by guest on September 28, 2021 Microbiology: Randall et al. Proc. Natl. Acad. Sci. USA 94 (1997) 10383

FIG.5. (A) Autoradiographic image of [35S]methionine-labeled infected cell proteins from total cell lysates or of proteins bound to GST fusion proteins electrophoretically separated on denaturing gels. (B) Photograph of the same gel reacted with monoclonal antiserum to ICP4. Replicate HeLa cells grown in 150-cm2 flasks were mock- infected (lanes 1–4) or infected with HSV-1(F) (lanes 5–8) and maintained at 37°C. At4hafterinfection the medium was replaced FIG. 6. Autoradiographic image of a gel retardation assay showing the with mixture 199 lacking methionine but supplemented with 1% calf interaction of ICP4 with a high affinity DNA site in the presence or serum and 50 ␮Ci (1 Ci ϭ 37 GBq) of [35S]methionine. After absence of GST-ORF O. Nuclear extracts (1.5 ␮g) were reacted with 2 ϫ additional incubation for 4 h, the cells were harvested, solubilized by 104 cpm of a 32P-labeled probe DNA in 25 ␮l of a solution containing 20 vigorous pipetting in lysis buffer (10 mM Hepes, pH 7.6͞250 mM mM Tris (pH 7.6), 50 mM KCl, 0.05% Nonidet P-40, 5% glycerol, 1 mM NaCl͞10 mM MgCl2͞1% Triton X-100͞0.5 mM phenylmethylsulfonyl EDTA, 1 mg BSA per ml, 10 mM 2-mercaptoethanol, and 3 ␮g flouride͞2 mM benzamidine). The lysates were precleared with 20 ␮g poly(dI⅐dC). The DNA probes were as follows: the 112-bp MscI–SacI of GST and divided into three identical samples to which were added fragment from pRB4794 (13), which is nucleotides Ϫ87 to ϩ25 relative 10 ␮g of GST–ORF P (lanes 2 and 6), GST–ORF P (lanes 3 and 7), C N to the ORF P transcription initiation site (lanes 1–11), and probe⌬ICP4bs or GST–ORF O (lanes 4 and 8) fusion proteins, respectively, and (lanes 12 and 13), which is the same fragment as above from pRB4855 reacted at 4°C for 3 h. Glutathione-conjugated agarose beads were (14), which contains a mutated ICP4 binding site. The probes were then added to the mixtures and allowed to react for 30 min at 4°C. The dephosphorylated by shrimp alkaline phosphatase and 5Ј labeled with beads were collected by low speed centrifugation and washed four [␥-32P]dATP using T4 polynucleotide kinase. The ICP4–DNA complex times with 50 volumes of lysis buffer. The proteins adhering to the was supershifted by the addition of the mAb H943 to the reaction mixture beads were then solubilized, electrophoretically separated on a dena- as originally described by Kristie and Roizman (26). The contents of each turing 15% polyacrylamide gel, transferred to a nitrocellulose sheet, reaction mixture is described above the corresponding lane. NE, nuclear and exposed to Kodak XAR-5 film for 2 days. (B) Photograph of the extract; GST-ORF P, GST-ORF P (Fig. 5). The electrophoretically blot shown in A after reaction first with mouse mAb specific for ICP4, C separated samples in a 4% nondenaturing polyacrylamide gel were dried H1114 (30) (Goodwin Cancer Research Institute), followed by goat- and exposed to Kodak XAR-5 film for 4 h. anti-mouse antibody conjugated to alkaline phosphatase. (ii) The coding domain of the ORF O protein became some- genome transcribed during latency, is expressed only in the what of a mystery because the methionine codon at the 5Ј absence of a functional ICP4 in cells infected and maintained terminus of the ORF is upstream of the transcript shown to be at nonpermissive temperature, or after mutagenesis of the derepressed by mutation of the ICP4 cognate site (14, 18). We ICP4 binding site at the transcription initiation site of ORF P; present evidence suggesting that the methionine codon at the 5Ј (ii) ORF O protein accumulates in significantly lower amounts terminus of the ORF is not the initiator methionine of ORF O than ORF P; (iii) ORF O and ORF P may share the same protein. The key evidence supporting the hypothesis that ORF P initiator methionine codon but differ in size and predicted and ORF O may share the same N-terminal methionine is the amino acid sequence beginning at an amino acid residue N observation that a tag inserted into the sequence between the terminal to amino acid 35; and (iv) in vitro ORF O protein amino terminal codons of the two ORFs did not increase the interacts with ICP4 and blocks it from binding its cognate apparent molecular weight of ORF O protein. The first down- DNA sequence. Relevant to this report are the following. stream methionine codon is that of ORF P; no other methionine (i) In our initial studies, ORF O protein was not detected codon exists within the ORF O gene. Insertion of the epitope tag reproducibly in large part because its abundance was much in-frame with ORF O at codon 35 of ORF P yielded a protein of lower than that of ORF P protein. In numerous assays done a size consistent with the apparent Mr of ORF O protein tagged subsequently, it became apparent that ORF O protein could be with a 20-amino acid sequence. Finally, antibody made against the readily and reproducibly detected by adjusting the assay con- fusion protein consisting of GST fused to the C terminus of ORF ditions to take into account the low abundance of the ORF O O protein reacted with a protein of the predicted size and found protein made in the cell lines tested. ORF O therefore in lysates of cells infected with a virus carrying a mutation at the represents the second gene mapping in the inverted repeats ICP4 binding site at the transcription initiation of ORF P, but not flanking the UL sequences and which is expressed only under in lysates of cells infected with wild-type virus and maintained at conditions in which ICP4 is defective or cannot bind to its the permissive temperature. The observation that ICP4 represses cognate site at the transcription initiation site of ORF P. ORF O as strongly as it does ORF P is consistent with the Downloaded by guest on September 28, 2021 10384 Microbiology: Randall et al. Proc. Natl. Acad. Sci. USA 94 (1997)

hypothesis that the ICP4 binding site is at the transcription 1. Roizman, B. (1996) Proc. Natl. Acad. Sci. USA 93, 11307–11312. initiation site of both genes. The significantly lower levels of 2. Roizman, B. & Sears, A. E. (1996) in Fields’ Virology, eds. Fields, accumulation of ORF O protein are consistent with a post B. N., Knipe, D. M., Howley, P., Chanock, R. M., Hirsch, M. S., transcriptional event such as frameshift or editing of the mRNA Melnick, J. L., Monath, T. P. & Roizman, B. (Lipcincott–Raven, New York), 3rd Ed., pp. 2231–2295. encoding ORF O protein. The limited distance between the 3. Mitchell, W. J., Lirette, R. P. & Fraser, N. (1990) J. Gen. Virol. mapped site of the shift of frames (a maximum of 34 codons, 102 71, 125–132. nt) and the absence of consensus splice acceptor͞donor sites does 4. Spivack, J. G. & Fraser, N. W. (1987) J. Virol. 61, 3841–3847. not support the hypothesis that the synthesis of ORF O is directed 5. Stevens, J. G., Wagner, E. K., Devi-Rao, G. B., Cook, M. L. & by a spliced mRNA. Parenthetically, we have detected only one Feldman, L. T. (1987) Science 235, 1056–1059. derepressed mRNA corresponding to the ORF P transcript (data 6. Wechsler, S. L., Nesburn, A. B., Zwaagstra, J. C. & Ghiasi, H. not shown), this datum is less persuasive than those listed above (1989) Virology 168, 168–172. because ORF O mRNA would be difficult to detect if its 7. Zwaagstra, J. C., Ghiasi, H., Slanina, S. M., Nesburn, A. B., Wheatley, S. C., Lillycrop, K., Wood, J., Latchman, D. S., Patel, abundance reflected that of the protein. K. & Wechsler, S. L. (1990) J. Virol. 64, 5019–5028. (iii) It could be predicted that if productive infection were 8. Devi-Rao, G. B., Goodart, S. A., Hecht, L. M., Rochford, R., to be inhibited at a very early stage, it would be necessary to Rice, M. K. & Wagner, E. K. (1991) J. Virol. 65, 2179–2190. preclude at least the synthesis or function of the ␣ proteins, 9. Farrell, M. J., Dobson, A. T. & Feldman, L. T. (1991) Proc. Natl. particularly ICPs 0, 4, 22, and 27, because these proteins Acad. Sci. USA 88, 790–794. control all subsequent events in the viral replicative cycle. ICP4 10. Wagner, E. K., Devi-Rao, G. B., Feldman, L. T., Dobson, A. T., is the major regulatory protein of the virus and its expression Zhang, Y., Flanagan, W. M. & Stevens, J. G. (1988) J. Virol. 62, is required for the expression of both ␤ and ␥ genes expressed 1194–1202. 11. Block, T. M., Deshmane, S., Masonis, J., Maggiocalda, J., Valyi- later in infection. ICP4 regulates expression of viral genes both Nagi, T. & Fraser, N. W. (1993) Virology 192, 618–630. positively and negatively. ICP0 is a promiscuous transactivator 12. Hill, J. M., Sedarati, F., Javier, R. T., Wagner, E. K. & Stevens, required for efficient expression of viral genes, ICP27 regulates J. G. (1990) Virology 174, 117–125. posttranscriptional processing of mRNAs, and ICP22 regulates 13. Leib, D. A., Bogard, C. L., Kosz-Vnenchak, M., Hicks, K. A., the expression of both early and late gene expression (reviewed Coen, D. M., Knipe, D. M. & Shaeffer, P. A. (1989) J. Virol. 63, in ref. 2). It could also be expected that if HSV-1 encodes 2893–2900. proteins responsible for establishment of latency, the expres- 14. Lagunoff, M., Randall, G. & B. Roizman, B. (1996) J. Virol. 70, sion of these proteins would be repressed during productive 1810–1817. 15. Lagunoff, M. & Roizman, B. (1994) J. Virol. 68, 6021–6028. infection. ORF P and ORF O meet these predictions in that (a) 16. Lagunoff, M. & Roizman, B. (1995) J. Virol. 69, 3615–3623. both genes are repressed during productive infection, and (b) 17. Bohenzky, R. A., Papavassiliou, A. G., Gelman, I. H. & Silver- whereas ORF P protein appears to have a role in blocking the stein, S. (1993) J. Virol. 67, 632–642. synthesis of ICP0 and ICP22 (28), the ORF O protein, at least 18. Yeh, L. & Schaffer, P. A. (1993) J. Virol. 67, 7373–7382. in vitro under conditions tested, appears to affect the ability of 19. Ejercito, P. M., Kieff, E. D. & Roizman, B. (1967) J. Gen. Virol. ICP4 to bind its cognate site on HSV-1 DNA. 2, 357–364. (iv) The central question whether ORF O and ORF P are 20. Liu, F. & Roizman, B. (1991) J. Virol. 65, 206–212. 24, either necessary or sufficient to establish latent infections remains 21. Post., L. E., Mackem, S. & Roizman, B. (1981) Cell 555–565. 22. Post, L. E. & Roizman, B. (1981) Cell 25, 227–232. unresolved. The experiments designed to determine the function 23. Igarishi, K., Fawl, R., Roller, R. J. & Roizman, B. (1993) J. Virol. of ORF P and ORF O have been done mostly in cultured cells and 67, 2123–2132. using in vitro assays. One problem encountered in in vivo studies 24. Faber, S. W. & Wilcox, K. W. (1986) Nucleic Acids Res. 14, is that expression of ORF P and ORF O and of the antisense gene, 6067–6083. ␥134.5, are mutually exclusive (14, 29). Elucidation of the role of 25. Michael, N. & Roizman, B. (1993) Proc. Natl. Acad. Sci. USA 90, these genes in latent infection may require the construction of 2286–2290. novel viruses to dissociate the sense from the antisense genes. In 26. Kristie, T. & Roizman, B. (1986) Proc. Natl. Acad. Sci. USA 83, 3218–3222. addition, several more ORFs within the domain transcribed 27. Leopardi, R., Michael, N. & Roizman, B. (1995) J. Virol. 69, during latency remain to be explored for their contributions to the 3042–3048. establishment or maintenance of the latent state. 28. Bruni, R. & Roizman, B. (1996) Proc. Natl. Acad. Sci. USA 93, 10423–10427. We thank Rosario Leopardi for supportive studies and Alice P. W. 29. Randall, G. & Roizman, B. (1997) J. Virol. 71, in press. Poon for a careful reading of the manuscript. These studies were aided 30. Ackermann, M., Braun, D. K., Pereira, L. & B. Roizman, B. by a grant from the National Cancer Institute (CA47451). (1984) J. Virol. 52, 108–118. 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