Identification and characterization of the herpes simplex virus type 1 ...

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The UL37 open reading frame of the herpes simplex virus type 1 (HSV-1) DNA genome is located between map units 0.527 ... ous lesions to fatal encephalitis.
JOURNAL

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VIROLOGY, Dec. 1990, p. 6101-6109

Vol. 64, No. 12

0022-538X/90/126101-09$02.00/0 Copyright © 1990, American Society for Microbiology

Identification and Characterization of the Herpes Simplex Virus Type 1 Protein Encoded by the UL37 Open Reading Frame LISA S. G. SHELTON, MICHAEL N. PENSIERO, AND FRANK J. JENKINS* Department of Microbiology, Uniformed Services University of the Health Sciences, Bethesda, Maryland 208144799 Received 23 July 1990/Accepted 23 September 1990

The UL37 open reading frame of the herpes simplex virus type 1 (HSV-1) DNA genome is located between units 0.527 and 0.552. We have identified and characterized the UL37 protein product in HSV-1-infected cells. The presence of the UL37 protein was detected by using a polyclonal rabbit antiserum directed against an in vitro-translated product derived from an in vitro-transcribed UL37 mRNA. The UL37 open reading frame encodes for a protein with an apparent molecular mass of 120 kDa in HSV-i-infected cells; the protein's mass was assigned on the basis of its migration in sodium dodecyl sulfate-polyacrylamide gels. The UL37 protein is not present at detectable levels in purified HSV-1 virions, suggesting that it is not a structural protein. Analysis of time course experiments and experiments using DNA synthesis inhibitors demonstrated that the UL37 protein is expressed prior to the onset of viral DNA synthesis, reaching maximum levels late in infection, classifying it as a 'yl gene. Elution of HSV-1-infected cell proteins from single-stranded DNA agarose columns by using a linear KCI gradient demonstrated that the UL37 protein elutes from this matrix at a salt concentration similar to that observed for ICP8, the major HSV-1 DNA-binding protein. In addition, computer-assisted analysis revealed a potential ATP-binding domain in the predicted UL37 amino acid sequence. On the basis of the kinetics of appearance and DNA-binding properties, we hypothesize that UL37 represents a newly recognized HSV-1 DNA-binding protein that may be involved in late events in viral replication. map

in cells infected by HSV-1 in the presence of DNA synthesis inhibitors and therefore was initially identified as an early (,) transcript on the basis of the nomenclature at the time for the different temporal classes of HSV genes. In this study, we report on the identification and characterization of the UL37 protein from HSV-1-infected cells. Our results demonstrate that the UL37 ORF encodes a protein with an apparent molecular mass of 120 kDa, which is nonstructural, belongs to the -yl class of HSV genes, and binds to single-stranded (SS) DNA-agarose columns. A search of the predicted UL37 amino acid sequence reveals a potential ATP-binding site, and computer-assisted analysis comparing the UL37 sequence with the entire varicella-zoster virus (VZV) genome demonstrates that the VZV homolog, gene 21, shares 47% similarity at the amino acid level with UL37. While we currently have not identified a specific function for the UL37 protein, we hypothesize, on the basis of the results presented in this report, that the UL37 protein is a newly recognized HSV-1 DNA-binding protein which may play a role either in viral gene regulation or in the processing of newly synthesized viral DNA.

Herpes simplex virus (HSV) causes a variety of clinical infections ranging from inapparent and self-limiting cutaneous lesions to fatal encephalitis. In addition, HSV establishes and maintains a latent state in the peripheral nerve cells of infected animals from which recrudescent infections arise. A major goal in herpesvirus research is to prevent not only primary HSV infections but also recurrent infections resulting from the reactivation of latent virus. To achieve this goal, it is necessary to understand the molecular biology and pathogenesis of HSV. Elucidation of the function(s) of the proteins encoded by the HSV genome is an important step towards this understanding. The HSV type 1 (HSV-1) genome is a double-stranded, linear DNA molecule of approximately 160 kb (18). The DNA sequence of the entire genome has been determined (23), and subsequent computer analysis suggests the presence of at least 75 separate open reading frames (ORFs). The precise function of many of these genes in either viral replication or viral pathogenicity is not known. Knowledge of the location of the potential ORFs, along with previous information on the genomic location of specific viral mRNAs, permits a focused study on individual HSV gene products which have not been studied previously. One such gene product is defined by the UL37 ORF of the unique long region of the HSV-1 DNA genome. UL37 is located at approximately 0.527 to 0.552 map units on the HSV-1 genome (Fig. 1). Previous analyses of HSV-1 mRNAs encoded in this region by Anderson and co-workers revealed the presence of a 3.8-kb mRNA whose location corresponds to the UL37 ORF (1). In vitro translation of hybrid-selected mRNAs yielded two proteins with molecular masses of 120 and 85 kDa. The 3.8-kb mRNA was detected *

MATERIALS AND METHODS

Cells and viruses. Vero and CV-1 cells (American Type Culture Collection) and human thymidine kinase-negative (TK-) 143 cells were grown in Eagle's minimal essential medium supplemented with 10% (vol/vol) Serum-Plus (Hazleton Corp.) and 50 ,ug of gentamicin (U.S. Biochemicals, Inc., Cleveland, Ohio) per ml. Every fifth passage, the 143 cells were grown in medium containing 50 ,ug of 5-bromodeoxyuridine per ml. The properties of HSV-1(F) have been previously described (12). HSV-1 viral stocks were prepared and the titers were determined on Vero cells. The parent vaccinia virus strain WR was obtained from Bernard Moss

Corresponding author. 6101

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IRL

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FIG. 1. (A) Sequence arrangement of the HSV-1 DNA genome showing the location of the unique sequences of the L and S components (UL and Us) and of the terminal (TRL and TRs) and inverted (IRL and IRS) repeats. (B) Schematic of the HindIII K fragment showing the locations and directions of transcription of the UL37, UL38, and UL39 genes. (C) Schematic representing the UL37 gene. The hatched box indicates the UL37-coding region. H, Hindlll; C, ClaI. The location of the potential ATP-binding domain is indicated by a solid bar. (D) Sequence arrangement of the plasmid PJF34. Solid boxes indicate flanking vaccinia virus TK sequences (TKL and TKR). The hatched box represents the location of the P-galactosidase gene. The open box represents the location of the UL37 ORF.

(National Institutes of Health). All vaccinia viruses were propagated on CV-1 cells. Enzymes and chemicals. Restriction endonucleases and DNA-modifying enzymes were obtained from Bethesda Research Laboratories, Inc. (Gaithersburg, Md.), New England BioLabs (Beverly, Mass.), Boehringer Mannheim Biochemicals (Indianapolis, Ind.), or Promega Corp. (Madison, Wis.). Radiochemicals were purchased from New England Nuclear Corp. (Boston, Mass.) and Amersham (Arlington Heights, Ill.). 5-Bromodeoxyuridine and phosphonoacetic acid (PAA) were obtained from Sigma Chemicals (St. Louis, Mo.). Antisera. Mouse monoclonal antiserum directed against glycoprotein C (gC) and rabbit polyclonal antiserum directed against gD of HSV-1 were obtained from Barry Rouse (University of Tennessee, Knoxville) and Paul Kinchington (Uniformed Services University of the Health Sciences), respectively. Rabbit polyclonal antisera directed against the HSV-1 UL42 protein and the ICP8 protein were obtained from William Ruyechan (USUHS). Construction of recombinant plasmids. Plasmid pRB210 containing the HindIll K fragment of HSV-1(F) (coordinates 0.527 to 0.592 map units) was obtained from Bernard Roizman (University of Chicago). The recombinant plasmid pHC37 was constructed by ligation of a 3.47-kb HindIII-ClaI fragment (coordinates 0.527 to 0.550) from pRB210 into the plasmid vector pBluescript M13 SK+ (Stratagene, La Jolla,

Calif.). The recombinant plasmid pJF34 was constructed by ligation of a Klenow-treated HindIII-ClaI fragment from pHC37 into the SmaI site of the vaccinia virus shuttle vector plasmid pSC11 (5). All plasmids were propagated and DNA were isolated from cesium chloride gradients as previously described (21). In vitro transcription and translation. pHC37 DNA was purified on cesium chloride density gradients (21) and linearized by digestion with EcoRI. Transcription of the linear DNA from the T7 promoter was performed with the Riboprobe System (Promega) according to manufacturer's instructions. Production of full-length transcripts was assessed by parallel reactions containing [32P]UTP (3,000 Ci/mmol). The labeled RNA was analyzed by electrophoresis through a Formalin-agarose gel and detected by autoradiography on Kodak XAR-5 film. In vitro-transcribed UL37 RNA was translated in vitro by using a rabbit reticulocyte translation kit (NEN Research Products, Wilmington, Del.) according to the manufacturer's instructions, including L-[355]methionine incorporation. In vitro-translated products were analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis through 10% polyacrylamide gels cross-linked with bisacrylamide (acrylamide-bisacrylamide, 30:0.8) using a miniprotean apparatus (Bio-Rad Laboratories, Richmond, Calif.). Gels were fixed with methanol-water-acetic acid (5:4:1, vol/vol/ vol) and dried, and 35S-labeled proteins were detected by autoradiography on Kodak XAR-5 film. The efficiency of translation was quantitated by analysis of trichloroacetic acid-precipitable counts. Construction of recombinant vaccinia viruses. A recombinant vaccinia virus expressing UL37 (V37) was constructed essentially as previously described (5, 19, 20). Briefly, CV-1 cells were infected with parental TK+ vaccinia virus (strain WR) followed by transfection with a calcium phosphate DNA precipitate of pJF34 which contains the UL37 gene under the control of the p7.5 vaccinia virus promoter. TKvirus was selected by growth on 143 TK- cells in the presence of 50 ,ug of 5-bromodeoxyuridine per ml, and the resulting progeny were screened for blue-plaque production in the presence of 300 ,ug of the chromogenic substrate 5-bromo-4-chloro-3-indolyl-,-D-galactopyranoside. The recombinant virus VSC11 was constructed as described above for the recombinant virus V37, except that the original vaccinia virus shuttle plasmid, pSC11, was substituted for pHC37. Preparation of rabbit antisera. UL37-specific, polyclonal rabbit antiserum was prepared against UL37 in vitro-translated proteins. The UL37 in vitro translation reaction was

upscaled 32-fold with replacement of labeled methionine with unlabeled L-methionine (Sigma Chemical Co.). The translation products (1 ml total volume) were emulsified with an equal volume of complete Freund adjuvant, and 0.5 ml was injected subcutaneously into the rabbits at multiple subscapular sites at biweekly intervals. Rabbits were bled 2 weeks after each injection. Rabbit inoculations and serum collections were performed by Duncroft Inc. (Lovettsville, Va.). Preparation of cell extracts. For immunoblot analyses, CV-1 cells were infected with either HSV-1(F) or V37 at a multiplicity of infection of 10. At various times postinfection, the cells were washed two or three times with phosphate-buffered saline and solubilized in sample buffer containing 2% SDS, 5% ,-mercaptoethanol, 50 mM Tris hydrochloride (pH 6.8), 5% glycerol, and 0.15 mM bromophenol blue. Mock-infected CV-1 cells were used as a

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negative control. Protein extracts were sonicated and heated 100°C for 2 min prior to electrophoresis. For studies involving the DNA synthesis inhibitor PAA, cells were infected and maintained in the presence of 300 ,ug of PAA per ml. For SS DNA-binding studies, protein extracts were prepared essentially as previously described (33). Briefly, CV-1 cells were infected at a multiplicity of infection of 10, and at 24 hours postinfection (HPI), the infected cells were scraped, pelleted, and quick frozen at -70°C. Prior to use, the cell pellets were resuspended in high-salt buffer (50 mM Tris hydrochloride [pH 7.6], 5 mM EDTA, 0.5 mM dithiothreitol, 0.12 mM phenylmethylsulfonyl fluoride, 1.7 M KCI), sonicated three times for 30 s each time, and centrifuged at 43,000 x g for 30 min to remove cellular components. The supernatants were collected, and DNA and nucleic acid components were precipitated by the addition of 24% polyethylene glycol to a final concentration of 8%. The supernatants were diluted with an equal volume of TEDGP buffer (50 mM Tris hydrochloride [pH 7.6], 5 mM EDTA, 0.5 mM dithiothreitol, 0.12 mM phenylmethylsulfonyl fluoride, 20% glycerol, 150 mM KCI) containing 0.4% Nonidet P-40 and dialyzed overnight against TEDGP buffer containing 0.2% Nonidet P-40. The dialysate was then loaded onto SS DNA-agarose columns. Preparation of HSV-1 virions. Extracellular HSV-1 virions were isolated essentially as previously described (29). Vero or CV-1 cells were infected with HSV-1(F) at a multiplicity of infection of 10. At 24 HPI, infected cells were scraped into the medium and pelleted by low-speed centrifugation. The resulting supernatant was clarified by a second centrifugation at 3,000 x g for 10 min. The supematant containing released virus was then centrifuged at 12,000 x g for 3 h at 4°C. The pellet was resuspended overnight in a Tris EDTA solution. After brief sonication and centrifugation at 2,000 rpm for 5 min, the supernatant was layered onto a 36-ml 5 to 40% (vol/vol) sucrose gradient and centrifuged at 13,000 rpm for 45 min with a Beckman SW28 rotor at 4°C. The virus band was collected and the sucrose was diluted out 1:5 with Tris EDTA. Virions were pelleted by centrifugation at 25,000 rpm for 1 h with an SW28 rotor at 4°C. The virus pellet was resuspended in a small volume of Tris EDTA. SS DNA-agarose column chromatography and DNA polymerase assays. Cell extracts were loaded onto a 30-ml SS DNA-agarose column and washed extensively against TEDGP buffer. Bound proteins were eluted with a linear 0.15 to 1.5 M KCI-TEDGP gradient as previously described (33). A total of 30 5-ml fractions were collected. Individual fractions were assayed for HSV DNA polymerase activity under high-salt (100 mM KCI) conditions specific for the viral enzyme as previously described (34). Immunoblot analysis. Proteins were analyzed by electrophoresis on 1.5-mm-thick 9% polyacrylamide (acrylamidebisacrylamide, 30:0.8) gels containing 0.1% SDS by using a Protean II vertical gel apparatus (Bio-Rad Laboratories). The separated proteins were electrophoretically transferred to nitrocellulose membranes in a buffer containing 0.025 M Tris, 0.213 M glycine, and 20% methanol with a TE50 Transphor apparatus (Hoefer Scientific, San Francisco, Calif.). Transfer efficiency was assessed by observation of the complete transfer of a set of Coomassie blue-prestained marker proteins obtained from Bethesda Research Laboratories. Following transfer, the nitrocellulose membrane was treated with 10% nonfat dry milk in binding buffer (4 mM EDTA, 10 mM Tris hydrochloride [pH 7.6], 0.15 M NaCl, 0.05% Tween 20). The blots were incubated with rabbit sera diluted 1:50 in 1% milk-binding buffer for 1 h at 37°C.

HSV-1 UL37 PROTEIN

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FIG. 2. Autoradiographic images of in vitro translation reactions. Input RNA was as follows. Lane 1, None; lane 2, control yeast RNA; lanes 3 and 4, in vitro transcription products of pHC37. Proteins were separated on an SDS-10% polyacrylamide gel, fixed, and dried as described in Materials and Methods. The molecular sizes of protein standards are indicated on the left.

Unbound antibodies were removed by four 10 to 15 min washes in binding buffer, and bound antibodies were detected by using 1251I-labeled Staphylococcus aureus protein A (4.85 XCi/,Lg; NEN Research Products) in 1% milkbinding buffer for 1 h at 37°C. Unbound protein A was removed by extensive washing in binding buffer, and bound antibodies were detected by autoradiography on Kodak XAR-5 film. Densitometric scans were performed on multiple exposures of the autoradiograms with a Shimatzu laser densitometer. Computer-assisted analysis of DNA and protein sequences. Protein sequence homologies were determined by using the Compare and Dotplot analysis programs of the Genetics Computer Group Sequence Analysis Package (Madison, Wis.) (11). All homology searches were performed by using a window of 19 and stringency of 13. Protein hydrophilicity profiles were determined by using the Protylyze program from Scientific and Educational Software (Stateline, Pa.). The complete HSV-1 DNA sequence was obtained from Duncan McGeoch (Glasgow University, Glasgow, Scotland) and the complete VZV DNA sequence was obtained from Andrew Davidson (Glasgow University). Nucleotide sequence accession number. The complete cytomegalovirus DNA sequence was obtained by direct-line access to GenBank (accession no. X17403).

RESULTS In vitro transcription and translation of the UL37 ORF. The plasmid pHC37 was constructed by cloning a 3.47-kb HindIII-ClaI DNA fragment from pRB210 into the HindIII-ClaI sites of the plasmid vector Bluescript SK+, as described in Materials and Methods. The ClaI site is located 93 bp upstream of the UL37 ATG, whereas the HindIlI site is located 3 bp downstream of the UL37 stop codon. No ATGs are located between the ClaI site and the UL37 ATG.

Insertion of this fragment into the Bluescript plasmid places the UL37 gene under the control of the T7 promoter contained in the plasmid vector. In vitro transcription of pHC37 produced a single transcript of approximately 3.5 kb (data not shown). The products of the in vitro transcription were used as a template for in vitro translation, as described in Materials and Methods, producing a prominent 120-kDa protein band (Fig. 2, lanes 3 and 4) and a series of smaller

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FIG. 3. Autoradiographic images of immunoblot of vaccinia virus-infected cell proteins harvested at 24 HPI. The blot was probed with anti-UL37 antiserum, and antigen-antibody binding was detected with '25I-labeled protein A. Proteins were separated on an SDS-9% polyacrylamide gel. Lane 1, Mock-infected cells; lane 2, VSC11-infected cells; lane 3, V37-1-infected cells; lane 4, V37-2infected cells. The molecular sizes of protein standards are indicated on the left.

bands likely representing premature terminations. These results agree with earlier experiments from another laboratory using hybrid-selected mRNA from HSV-1-infected cells which detected a major 120-kDa band and a minor 85-kDa band (1). The observed size of the in vitro-translated UL37 protein (120 kDa) corresponds well with the predicted size of 120.5 kDa on the basis of the predicted amino acid sequence of the UL37 protein. Production of UL37-specific polyclonal antiserum. The in vitro translation reactions utilizing pHC37 as a substrate were scaled up 32-fold and used to produce rabbit polyclonal antiserum (anti-UL37). Analysis of HSV-1-infected-cell proteins by immunoblot using the anti-UL37 antiserum identified a single band with a molecular mass of 120 kDa that is not present in mock-infected cells and which increases with time postinfection (see Fig. 4A). To develop a system from which large amounts of the UL37 protein could be generated and to confirm the specificity of the anti-UL37 antiserum, a vaccinia virus recombinant expressing UL37 (V37) was constructed. The HindIllClaI fragment from pHC37 was cloned into the unique SmaI site of pSC11. The plasmid pJF34, containing the UL37 ORF under the control of the vaccinia virus p7.5 promoter, was identified by restriction enzyme analysis. pJF34 also contains a P-galactosidase gene under the control of the vaccinia virus pll promoter, and the 3-galactosidase and UL37 genes are flanked by the vaccinia virus TK gene (Fig. 1D). pJF34 was transfected into vaccinia virus-infected cells and TKrecombinants expressing 3-galactosidase were isolated and purified by several rounds of plaque purification. Infectedcell proteins from two of the isolates were analyzed by immunoblot with the anti-UL37 antiserum. UL37 was present in protein extracts from cells infected by both isolates (V37-1 and V37-2) and absent from both mock- and VSC11-infected cells (Fig. 3). VSC11 is a recombinant

vaccinia virus that contains a P-galactosidase gene, is TK-, and was constructed by using the plasmid pSC11 as described in Materials and Methods. The size of the UL37 protein in the vaccinia virus recombinants was identical to that detected in HSV-1-infected cells on the basis of SDSpolyacrylamide gel electrophoresis. Expression of UL37 in HSV-1-infected cells. The expression of UL37 during a lytic HSV-1 replication cycle in infected cells was analyzed by determining the kinetics of appearance of the UL37 protein. The temporal class of HSV genes to which UL37 belongs was specified with the following kinetic data and experiments with viral DNA synthesis inhibitors. For kinetics of appearance, CV-1 cells were infected with HSV-1(F) at a multiplicity of infection of 10 and extracts of cellular proteins were prepared from individual flasks at various times postinfection. The infected-cell proteins were separated on SDS-polyacrylamide gels, transferred to nitrocellulose membranes, and analyzed by immunoblotting with antisera directed against three different HSV-1 proteins. As internal controls for the individual time points, the protein extracts were probed with antisera directed against UL42, a 65-kDa DNA-binding protein (14, 22), and ICP8, the major HSV-1 DNA-binding protein (3, 27, 30). The kinetics of appearance in time course experiments for both of these proteins have been reported previously (15, 26). Both the UL42 and the ICP8 proteins appeared early, being detected by 6 HPI (Fig. 4 and 5). The ICP8 protein was detected as early as 3 HPI by using a longer exposure of the immunoblot (data not shown). Both the UL42 and ICP8 proteins reached maximum levels by 9 to 12 HPI and maintained these levels throughout the entire replication cycle. In contrast, the UL37 protein appeared later in infection, being detected at 9 HPI, and gradually increased in amount, reaching its highest levels very late in infection (24 HPI). The appearance of the UL37 protein during HSV-1 replication is distinct from the appearance of the UL42 and ICP8 proteins (Fig. 5). Since the UL37 protein appears late in infection, it was reasoned that the gene may belong to either the -yl or -y2 class of HSV genes, on the basis of the nomenclature of Roizman (17, 31). The -yl class of HSV genes is defined as those genes whose transcripts are produced in small amounts in the absence of viral DNA synthesis, yet reach their maximum levels of synthesis after the onset of DNA replication. The -y2 class of HSV genes is defined as those genes whose transcripts are made only after the onset of viral DNA replication. To define precisely the temporal class to which UL37 belongs, infected-cell proteins were harvested from CV-1 cells that had been infected and maintained in the presence of 300 ,ug of the DNA synthesis inhibitor, PAA, per ml. The protein extracts were separated on SDS-polyacrylamide gels, transferred to nitrocellulose membranes, and probed by immunoblots with the anti-UL37 antiserum or antiserum directed against either ICP8 or gC of HSV-1. ICP8 has been previously shown to belong to the ,1 class of HSV genes (17), while gC has been shown to belong to the y2 class (13). As a 31 gene, ICP8 was easily detected in both the presence and absence of PAA, with higher levels seen in the presence of PAA, whereas gC, a -y2 gene, was detected only in the absence of PAA (Fig. 6). In contrast, the UL37 protein was detected in both the presence and absence of PAA, with higher levels detected in the absence of PAA. Two additional bands of 42 and 45 kDa were detected when the PAA lanes were probed with the UL37 antiserum (Fig. 6B). While the identity of these bands is not known, their presence in both HSV- and mock-infected lanes indicates that they represent cellular proteins which cross-react with

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HSV-1 UL37 PROTEIN M 3 6 9 12 15 18 24

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FIG. 4. Autoradiographic images of immunoblots of HSV-1infected cell proteins harvested at various times postinfection and probed with anti-UL37 polyclonal rabbit antiserum (A), anti-ICP8 polyclonal rabbit antiserum (B), and anti-UL42 polyclonal rabbit antiserum (C). Proteins were separated on SDS-99o polyacrylamide gels. Antigen-antibody binding was detected with 1251-labeled protein A. The numbers above the lanes refer to times (HPI) at which the cells were harvested. The molecular sizes of protein standards are indicated on the right of each panel. M, Mock infected.

65 K --

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the rabbit polyclonal antiserum. Their presence in only the PAA lanes suggests that they may represent cellular stress proteins induced by PAA. On the basis of the higher levels of the UL37 protein in the absence of PAA and the increased production of the protein late in infection, we have assigned UL37 to the yl class of HSV genes. Analysis of isolated, intact HSV virions. The assignment of UL37 to the yl class raised the possibility that this protein is a component of the HSV virion. To test this hypothesis, intact, enveloped HSV-1 virions were isolated as described in Materials and Methods. Protein extracts from the purified HSV-1 virions were separated on SDS-polyacrylamide gels, and the protein bands were probed in immunoblots with antisera directed against the UL37, ICP8, and gD proteins of HSV-1. The antisera against gD and ICP8 were used as controls for structural and nonstructural HSV proteins, respectively. A Coomassie stain of an SDS-polyacrylamide gel containing the separated virion protein extracts demonstrated a typical protein profile for purified HSV virions, as described previously by Spear and Roizman (35), with very little detectable contamination with cellular proteins (Fig. 7A). Antiserum raised against ICP8, a known HSV nonstructural protein, failed to detect ICP8 in the virion preparation, while easily detecting the presence of ICP8 in infected-cell

protein extracts harvested at 25 HPI (Fig. 7B). Antiserum raised against gD, a component of intact virions, easily detected the mature form of the protein in the virion preparation and detected both the mature and precursor forms in infected-cell extracts (Fig. 7C). Analysis with the anti-UL37 antiserum failed to detect the presence of the UL37 protein in HSV-1 virions (Fig. 7D), strongly suggesting that the UL37 protein is not a structural component of the HSV-1 virion. 100 PJ

u

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FIG. 5. Kinetics of appearance of the UL37, ICP8, and UL42 proteins in HSV-1-infected cells. Multiple exposures of the autoradiograms shown in Fig. 4 were scanned with a laser densitometer. The values shown represent the relative abundance of the detected protein in each lane. Symbols: +, UL37; A, ICP8; 0, UL42.

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29 KSS DNA-agarose chromatography. Since the UL37 gene product does not appear to be a HSV-1 structural protein, other reasonable possibilities for its function would be involvement in either the later phases of viral DNA replication or viral gene expression. Both of these possibilities would likely require the UL37 protein to interact with nucleic acids. To determine if the UL37 protein was capable of binding SS DNA, proteins from HSV-1-infected CV-1 cells were harvested at 24 HPI and loaded onto a 30-ml SS DNA-agarose column. Bound proteins were eluted with a linear 0.15 to 1.5 M KCl gradient. Fractions were collected, separated on SDS-9.3% polyacrylamide gels, and analyzed by immunoblot using anti-UL37 antisera. As controls for these experiments, each fraction was analyzed for the presence of HSV DNA polymerase activity and additional immunoblots using anti-ICP8 antisera were performed. Exposures of the autoradiograms using the UL37 and ICP8 antisera were densitometrically scanned, and the results (along with the DNA polymerase activities of each fraction)

FIG. 6. Immunoblots of mock (M)- and HSV-1-infected cell proteins from cells grown in either the presence (+) or absence (-) of PAA. The blots were probed with anti-ICP8 polyclonal antiserum (A), anti-UL37 polyclonal antiserum (B), and mouse monoclonal antiserum directed against gC (C). Antigen-antibody binding was detected with either 251I-labeled protein A (A and B) or peroxidaseconjugated goat anti-mouse antiserum followed by exposure to the peroxidase substrate 4-chloro naphthol (C). Proteins were separated on SDS-9% polyacrylamide gels. The molecular sizes of protein standards are indicated on the side of each panel.

are shown in Fig. 8. The HSV DNA polymerase activity eluted between 0.3 and 0.4 M KCI, while both the UL37 and ICP8 proteins exhibited an apparently stronger affinity, eluting between 0.6 and 0.7 M KCl. The elution profiles of the HSV DNA polymerase activity and ICP8 protein agree with previously published reports from several laboratories (16, 27, 28, 32, 34). The UL37 protein, from crude extracts, is capable of binding to SS DNA columns with an affinity similar to that of the major HSV-1 DNA protein, ICP8 (Fig.

8). Computer-assisted analysis of the UL37 protein. The publication of the entire DNA sequence for Epstein-Barr virus (EBV) (2), VZV (9), HSV-1 (23), and most recently human CMV (HCMV) (6) has allowed computer-assisted analyses of these genomes and their predicted amino acid sequences. It has become apparent that the VZV genome is closely related to HSV at the genetic level, while EBV and HCMV genomes appear distantly related (6, 8-10, 24). Most of the HSV genes have been found to have counterparts in VZV (8-10, 23) and oftentimes in both EBV (10) and HCMV (6). The predicted homologs to the HSV-1 UL37 gene are gene 21 of VZV (9), BOLF1 of EBV (2), and HCMVUL47 of CMV (6), on the basis of the number of predicted amino acid residues (HSV, 1,123; VZV, 1,038; EBV, 1,239; and CMV, 982), the predicted molecular masses (in kilodaltons) of the

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FIG. 7. Immunoblot analysis of purified HSV-1 virions. Protein extracts from mock-infected (M or MK), HSV-1 25 HPI (25),

proteins (HSV, 120.5; VZV, 115.8; EBV, 132.7; CMV, 110), and their genomic locations and directions of transcription in relation to surrounding genes. Using the programs Compare and Dotplot from the Genetics Computer Group Sequence Analysis Package (11), the HSV-1 UL37 gene was found to have significant homology (47% similarity) at the amino acid level with gene 21 of VZV (data not shown). The genetic similarity between the UL37 and VZV gene 21 proteins is shared throughout the entire length of both proteins. In contrast, the potential EBV homolog, BOLF1, and the

04

potential CMV homolog, HCMVUL47, do not share any noticeable homology at the amino acid level with either UL37 or VZV gene 21 (data not shown). Analysis of the hydropathic profiles of the HSV-1 UL37 and VZV 21 proteins showed the profiles to be very similar, exhibiting a rather uniform mixture of hydrophilicity and hydrophobicity with no obvious highly hydrophobic or hydrophilic domains (data not shown). A search of the UL37 predicted amino acid sequences for several common protein sequence motifs failed to detect sequences corresponding to potential zinc fingers, leucine zippers, or nuclear localization signals. However, a potential ATP-binding site was observed between residues 452 and 490 (Fig. 1), consisting of the sequence NH2-(4 strand)GSNVFG-(a helix)-(, strand)-COOH, which corresponds well with a predicted consensus adenine mononucleotide binding domain of NH2-(0 strand)-GXXXX-(G K a helix)-(0 to 11 amino acids)-(P strand)-COOH, as reported previously by Bradley and co-workers (4). A similar analysis of the predicted amino acid sequences of the VZV 21, EBV BOLF1, and HCMVUL47 genes failed to detect the ATPbinding motif.

or

purified HSV-1 virions (VP) were separated on SDS-9%o polyacrylamide gels. The proteins were transferred to nitrocellulose and probed with anti-ICP8 antiserum (B), anti-gD antiserum (C), or anti-UL37 antiserum (D). Antigen-antibody binding was detected with 1251I-labeled protein A and autoradiography. (A) An identical gel stained with Coomassie blue.

04

DISCUSSION In this paper, we have shown that the predicted protein product of the previously uncharacterized HSV-1 UL37 gene is expressed in HSV-1-infected cells. The UL37 protein has an apparent molecular mass of 120 kDa on SDS-polyacrylamide gels, which is in good agreement with the value of 120.5 kDa predicted from the amino acid sequence. UL37 is expressed in the absence of viral DNA replication and reaches maximum levels late in infection, classifying it as a lyl gene. These results agree with those of Anderson and co-workers (1), who reported that the mRNA from this region was expressed in the presence of DNA synthesis inhibitors but reached higher levels after the onset of viral DNA replication. The UL37 protein was eluted from SS DNA-agarose columns at between 600 and 700 mM KCl, conditions that are similar to those for the major HSV-1 DNA-binding protein, ICP8. This is the first report of the 1200

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FRACTION NUMBER FIG. 8. Chromatography of HSV DNA polymerase, ICP8, and UL37 proteins on SS DNA-agarose. HSV-1-infected cell proteins were isolated by the procedure of Ruyechan (33). Polymerase assays and immunoblots were performed as described in Materials and Methods. For ICP8 and UL37, multiple exposures of autoradiograms were scanned densitometrically. Results are presented as the relative percentage of detectable protein in each fraction. Symbols: +, ICP8; A, HSV polymerase; *, KCI; *, UL37.

6108

SHELTON ET AL.

identification of the UL37 protein as a potential DNAbinding protein. Classification of the UL37 protein as a yl gene which achieves maximum levels very late in infection would suggest that the protein is a structural gene, since the majority of the known HSV-1 structural proteins belong to either the yl or y2 classes. However, analysis of intact, isolated HSV-1 virus particles indicated that within the limits of detection, the UL37 protein is not a component of the virion. While we cannot rule out the presence of minor amounts of the UL37 protein in our preparations, the protein is clearly not a major component of the HSV-1 virion, although it is present in readily detectable amounts in infected cells. The most likely possibility, therefore, is that the UL37 gene product is a nonstructural protein involved in late events of HSV replication. In a lytic HSV-1 replication cycle, during the times at which the UL37 protein is maximally produced (18 to 24 HPI), viral DNA synthesis has already begun and new viral progeny are being formed. It is unlikely therefore that the UL37 protein is involved in the early stages of the replication of viral DNA. If this were the case, we would expect that the UL37 protein would have kinetics of appearance similar to those of ICP8 and UL42 (Fig. 4 and 5), both of which have been previously demonstrated to be essential for viral replication and required for in vitro replication of a plasmidborne HSV origin of replication (ori) (26, 36). The kinetics of appearance for the UL37 protein were clearly distinct from those of the UL42 and ICP8 proteins (Fig. 4 and 5). However, not all HSV genes that appear to be involved in viral DNA replication (e.g., alkaline DNase) are required for the replication of the HSV ori-containing plasmid (25). The observation that the UL37 protein binds to SS DNA columns strengthens the possibility of a role for UL37 in HSV DNA replication. UL37 may play a role in HSV DNA replication by functioning in the processing, cleavage, or packaging of newly synthesized viral DNA. Proteins involved in these functions could be expected to appear late in infection, to bind nucleic acids, and not necessarily to be structural components of the virion. One potential possibility is a protein recently described by Chou and Roizman (7), which interacts with the HSV a sequence. In their study, they reported that two distinct complexes were found which contained HSV-specific proteins and bound to the a sequence. One of the complexes, termed V4, contained two HSV proteins in equimolar concentrations. One protein was identified as ICP1 (UL36) on the basis of its size and reactivity with specific antisera, while the second protein was described as having a molecular mass of 140 kDa, identity unknown. The V4 complex bound to the pac2 and cis cleavage site of the a repeat. Whether UL37 is the 140 kDa protein detected by Chou and Roizman remains to be determined and is currently under investigation. We are currently attempting to determine the relative affinity of the UL37 protein for SS DNA, double-stranded DNA, and RNAs. Our ability to express the UL37 gene in a vaccinia virus recombinant (V37) should greatly facilitate these studies. The recombinant protein will allow a direct determination of UL37-nucleic acid-binding properties without the possibility of modulation due to other HSV-1encoded proteins. A second possibility, on the basis of the kinetics of appearance and nucleic acid-binding activity, is that the UL37 protein has a late regulatory role in viral replication. Study of this possibility would initially best be pursued by the generation of mutation within the UL37 gene. Such mutation are currently being generated.

J. VIROL.

The identification of a potential ATP-binding site in the UL37 protein sequence is also of some interest. The ATPbinding motif contained in the UL37 protein has been reported for several known ATP-binding proteins such as the Escherichia coli tyrosyl tRNA synthetase and porcine lactate dehydrogenase (4). This motif depends heavily on a (predicted) secondary structure of (p strand)-GXXXXG-(a helix)-(,3 strand) which the UL37 amino acid sequence contains. The existence of a putative ATP-binding site also strengthens the hypothesis that UL37 may be involved in an energy-driven step in the cleavage or packaging of viral DNA. The failure to detect this motif in the VZV, EBV, or HCMV protein homologs, however, causes the significance of the presence of the motif in UL37 to remain unclear. Further characterization of the UL37 gene product and attempts to elucidate its function during the lytic-replication cycle of the virus are currently in progress. ACKNOWLEDGMENTS We express our gratitude and thanks to William Ruyechan for generous gifts of antiserum, help with the DNA-binding studies, and long hours of helpful discussions; Barry Rouse for the gC antiserum; and Paul Kinchington for supplying antiserum and preparation of isolated virions. This investigation was supported by Uniformed Services University of the Health Services grants R07396 and C07311. F.J.J. is a Leukemia Society of America Special Fellow. LITERATURE CITED 1. Anderson, K. P., R. J. Frink, G. B. Devi, B. H. Gaylord, R. H. Costa, and E. K. Wagner. 1981. Detailed characterization of the mRNA mapping in the HindIII fragment K region of the herpes simplex virus type 1 genome. J. Virol. 37:1011-1027. 2. Baer, R., A. T. Bankier, M. D. Biggin, P. L. Deininger, P. J. Farrell, T. J. Gibson, G. Hatfull, G. S. Hudson, S. C. Satchwell, C. Seguin, P. S. Tuffnell, and B. G. Barreil. 1984. DNA sequence and expression of the B95-8 Epstein-Barr virus genome. Nature (London) 310:207-211. 3. Bayliss, G. J., H. S. Marsden, and J. Hay. 1975. Herpes simplex virus proteins: DNA-binding proteins in infected cells and in the virus structure. Virology 68:124-134. 4. Bradley, M. K., T. F. Smith, R. H. Lathrop, D. M. Livingston, and T. A. Webster. 1987. Consensus topography in the ATP binding site of the simian virus 40 and polyomavirus large tumor antigens. Proc. Natl. Acad. Sci. USA 84:4026-4030. 5. Chakrabarti, S., K. Brechling, and B. Moss. 1985. Vaccinia virus expression vector: coexpression of ,-galactosidase provides visual screening of recombinant virus plaques. Mol. Cell. Biol. 5:3403-3409. 6. Chee, M. S., A. T. Bankier, S. Beck, R. Bohni, C. M. Brown, R. Cerny, T. Horsnell, C. A. Hutchison III, T. Kouzarides, J. A. Martignetti, E. Preddie, S. C. Satchwell, P. Tomlinson, K. M. Weston, and B. G. Barrell. 1990. Analysis of the protein coding content of the sequence of human cytomegalovirus strain AD169. Curr. Top. Microbiol. Immunol. 154:125-170. 7. Chou, J., and B. Roizman. 1989. Characterization of DNA sequence-common and sequence-specific proteins binding to cis-acting sites for cleavage of the terminal a sequence of the herpes simplex virus 1 genome. J. Virol. 63:1059-1068. 8. Davison, A. J., and D. J. McGeoch. 1986. Evolutionary comparisons of the S segments in the genomes of herpes simplex virus type 1 and varicella-zoster virus. J. Gen. Virol. 67:597-611. 9. Davison, A. J., and J. E. Scott. 1986. The complete DNA sequence of varicella-zoster virus. J. Gen. Virol. 67:1759-1816. 10. Davison, A. J., and P. Taylor. 1987. Genetic relations between varicella-zoster virus and Epstein-Barr virus. J. Gen. Virol. 68:1067-1079. 11. Devereux, J., P. Haeberli, and 0. Smithies. 1984. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 12:387-395.

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24. McGeoch, D. J., and A. J. Davison. 1986. Alphaviruses possess a gene homologous to the protein kinase gene family of eukaryotes and retroviruses. Nucleic Acids Res. 14:1765-1777. 25. Moss, H. 1986. The herpes simplex virus type 2 alkaline DNase activity is essential for replication and growth. J. Gen. Virol. 67:1173-1178. 26. Olivo, P. D., and M. D. Challberg. 1988. Herpes simplex virus DNA replication: identification of the essential genes and their products. In T. Kelly and B. Stillman (ed.), Cancer cells, vol. 6. Eukaryotic DNA replication. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 27. Powell, K. L., and D. J. M. Purifoy. 1976. DNA-binding proteins of cells infected by herpes simplex virus type 1 and type 2. Intervirology 7:225-239. 28. Powell, K. L., and D. J. M. Purifoy. 1977. Nonstructural proteins of herpes simplex virus. I. Purification of the induced DNA polymerase. J. Virol. 24:618-626. 29. Powell, K. L., and D. H. Watson. 1975. Some structural antigens of herpes simplex virus type 1. J. Gen. Virol. 29:167-178. 30. Purifoy, D. J. M., and K. L. Powell. 1976. DNA-binding proteins induced by herpes simplex virus type 2 in HEp-2 cells. J. Virol. 19:717-731. 31. Roizman, B., and A. E. Sears. 1990. Herpes simplex viruses and their replication, p. 1795-1841. In B. N. Fields, D. M. Knipe, and B. Roizman (ed.), Virology, 2nd ed. Raven Press, New York. 32. Ruyechan, W. T. 1983. The major herpes simplex virus DNAbinding protein holds single-stranded DNA in an extended configuration. J. Virol. 46:661-666. 33. Ruyechan, W. T. 1988. N-Ethylmaleimide inhibition of the DNA-binding activity of the herpes simplex virus type 1 major DNA-binding protein. J. Virol. 62:810-817. 34. Ruyechan, W. T., and A. C. Weir. 1984. Interaction with nucleic acids and stimulation of the viral DNA polymerase by the herpes simplex virus type 1 major DNA-binding protein. J. Virol. 52:727-733. 35. Spear, P. G., and B. Roizman. 1972. Proteins specified by herpes simplex virus. V. Purification and structural proteins of the herpesvirion. J. Virol. 9:431-439. 36. Wu, C. A., N. J. Nelson, D. J. McGeoch, and M. D. Challberg. 1988. Identification of herpes simplex virus type 1 genes required for origin-dependent DNA synthesis. J. Virol. 62:435443.