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J Virol. Aug 2001; 75(15): 6808–6816.
PMCID: PMC114407

Origin Binding Protein-Containing Protein-DNA Complex Formation at Herpes Simplex Virus Type 1 oriS: Role in oriS-Dependent DNA Replication

Abstract

Initiation of herpes simplex virus type 1 (HSV-1) DNA replication during productive infection of fibroblasts and epithelial cells requires attachment of the origin binding protein (OBP), one of seven essential virus-encoded DNA replication proteins, to specific sequences within the two viral origins, oriL and oriS. Whether initiation of DNA replication during reactivation of HSV-1 from neuronal latency also requires OBP is not known. A truncated protein, consisting of the C-terminal 487 amino acids of OBP, termed OBPC, is the product of the HSV UL8.5 gene and binds to origin sequences, although OBPC's role in HSV DNA replication is not yet clear. To characterize protein-DNA complex formation at oriS in cells of neural and nonneural lineage, we used nuclear extracts of HSV-infected nerve growth factor-differentiated PC12 and Vero cells, respectively, as the source of protein in gel shift assays. In both cell types, three complexes (complexes A, B, and C) which contain either OBP or OBPC were shown to bind specifically to a probe which contains the highest-affinity OBP binding site in oriS, site 1. Complex A was shown to contain OBPC exclusively, whereas complexes B and C contained OBP and likely other cellular proteins. By fine-mapping the binding sites of these three complexes, we identified single nucleotides which, when mutated, eliminated formation of all three complexes, or complexes B and C, but not A. In transient DNA replication assays, both mutations significantly impaired oriS-dependent DNA replication, demonstrating that formation of OBP-containing complexes B and C is required for efficient initiation of oriS-dependent DNA replication, whereas formation of the OBPC-containing complex A is insufficient for efficient initiation.

Herpes simplex virus type 1 (HSV-1) encodes seven proteins required for replication of its 152-kb double-stranded DNA genome (27). During productive infection, initiation of HSV DNA replication occurs at viral origins which include one copy of oriL, located in the unique long region of the genome, and two copies of oriS, located in the repeat sequences flanking the unique short region of the genome (22, 25). The core element of oriS consists of a 90-bp sequence that includes a 45-bp imperfect palindrome, whereas the core element of oriL consists of a 144-bp perfect palindrome (Fig. (Fig.1).1). Despite these differences, oriL and oriS share extensive nucleotide sequence homology, and both origins contain binding sites for the origin binding protein (OBP), encoded by the UL9 gene (8, 9, 19). OBP binds specifically to sequences within the origins, termed sites I, II, and III, which differ slightly in nucleotide sequence and thus in binding affinity for OBP (site I > site II > site III) (11). The imperfect oriS palindrome contains one copy each of sites I, II, and III, whereas the perfect oriL palindrome contains two copies each of sites I and III (Fig. (Fig.1).1).

FIG. 1
Comparison of sizes and sequences in HSV-1 oriL and oriS. The DNA sequences of the core origin of oriL (a 144-bp perfect palindrome) and oriS (a 45-bp imperfect palindrome) of HSV-1 KOS are shown. Black dots indicate the nucleotide differences between ...

The functional significance of the presence of three origins of DNA replication within the HSV-1 genome is not clear. Mutant viruses lacking either oriL or both copies of oriS have no obvious growth defects during productive infection of cells in culture, suggesting that the two types of origin are able to substitute functionally for each other (13, 21). However, there is evidence that oriL and oriS differ with respect to the efficiency of origin function in neural (PC12) and nonneural (Vero) cells. Specifically, in undifferentiated PC12 cells and in Vero cells, oriL and oriS function with similar efficiency in in vitro assays. In nerve growth factor (NGF)-differentiated PC12 (Nd-PC12) cells, the efficiency of oriS function is significantly reduced whereas oriL function is the same as in undifferentiated PC12 or Vero cells. Furthermore, addition of the synthetic glucocorticoid dexamethasone (DEX) enhances oriL function and further represses oriS function. Although the mechanism responsible for the repression of oriS function in PC12 cells in response to NGF-induced differentiation is unclear, the enhancement of oriL function in Nd-PC12 cells in response to DEX was shown to be mediated through a perfect glucocorticoid response element (GRE) present in oriL (10). oriS contains a degenerate GRE that may be responsible for the DEX-induced repression of oriS function in Nd-PC12 cells. Based on these findings, the differences in nucleotide sequence between oriL and oriS clearly have significant functional consequences. Moreover, cell-type-specific functional differences likely have significant implications for the biology of HSV (e.g., the ability to establish and reactivate from latent infection in neurons). One possible explanation for these functional differences between oriL and oriS is that formation of OBP-containing protein-DNA complexes differs at oriL versus oriS and/or in cells of neural versus nonneural lineage.

In the current model of HSV DNA replication, OBP functions as a DNA replication initiator protein by binding to HSV origins, initiating unwinding of origin DNA, and recruiting additional viral DNA replication proteins to the initiation site (reviewed in reference 4). Thus, the ability of OBP to bind to viral origins was shown to be essential for HSV DNA replication, as mutations within the origins themselves or within OBP that abrogate origin binding inhibit origin-dependent DNA replication (12, 23, 24). DNA footprinting and electron microscopy have demonstrated that binding of OBP to sites I and II in oriS loops and distorts the A+T-rich apex of the origin, which is thought to allow for subsequent DNA unwinding via the ATP-dependent helicase activity of OBP (14, 15). Binding of OBP to viral origins is also thought to facilitate recruitment of other essential replication proteins (i.e., the product of the UL8, UL42, and UL29 genes) to the sites of initiation via direct protein-protein interactions (3, 16, 17).

A truncated form of OBP, termed OBPC, has also been shown to bind to oriL and oriS (2). OBPC, encoded by the UL8.5 gene, is the product of a unique delayed-early transcript that originates within the open reading frame (ORF) of the gene encoding OBP (UL9) (1). Because the genes encoding OBP and OBPC are translated in the same reading frame, the amino acid sequence of OBPC is identical to the C-terminal 487 amino acids of OBP; however, OBPC lacks the N-terminal domain of OBP, which includes five of the six conserved helicase motifs, the ATP-binding and leucine zipper motifs, as well as domains that mediate interactions with a component of the helicase-primase complex (UL8) and the DNA polymerase processivity factor (UL42). Based on the observations that OBPC localizes to the nuclei of HSV-infected cells and binds to origin DNA, it has been postulated that OBPC may play a role in HSV DNA replication (2). Notably, overexpression of OBPC or C-terminal peptides of OBP has been shown to inhibit origin-dependent viral DNA replication and plaque formation by infectious HSV-1 DNA, presumably by occupying OBP binding sites and interfering with the initiation process (2, 20). Ultimately, determination of the precise role of OBPC in HSV DNA replication will require isolation of an OBPC mutant. Construction of this virus has not yet been achieved, however, because the genes encoding OBP and OBPC overlap and their ORFs are translated in the same reading frame. Consequently, mutagenesis of the nucleotide sequence of the OBPC promoter and transcriptional and translational start sites (which would be needed to abrogate expression of OBPC) also alters the amino acid sequence of OBP. Since maintaining a functional form of OBP is critical to differentiating the roles of OBP and OBPC, construction of an OBP+ OBPC virus is a formidable challenge under these circumstances. Numerous efforts to introduce mutations into the wobble position of OBP codons (i.e., into the promoter and start sites of the OBPC gene) that do not alter the amino acid sequence of OBP but eliminate OBPC expression have met with only limited success. Given the difficulty in isolating a suitable OBPC mutant virus, we have been forced to evaluate OBPC function by using alternative approaches, including characterization of mutations in site I that eliminate binding of OBP but not OBPC to HSV origins.

To characterize the formation of protein complexes at oriS and to determine whether these complexes differ when proteins are derived from neural versus nonneural cells, we used nuclear extracts of HSV-infected Nd-PC12 and Vero cells as the source of protein and a DNA sequence containing oriS site I (Fig. (Fig.1)1) as the probe in gel shift assays. Using nuclear extracts of both cell types tested, three HSV-specific protein complexes, A, B, and C, were shown to bind specifically to the site I probe. The three complexes exhibited similar migration patterns when proteins were derived from Nd-PC12 or Vero cells. Mapping of the precise nucleotide binding sites of the three complexes revealed that complex A (containing only OBPC) binds to a site lying entirely within the shared binding site of two OBP-containing complexes, B and C. We generated single-nucleotide substitution mutations which eliminate formation of all three complexes or of complexes B and C only and determined their effects on oriS-dependent DNA replication. Both mutations reduced oriS-dependent DNA replication significantly in in vitro assays.

MATERIALS AND METHODS

Cells and viruses

PC12 cells (a gift from John Wagner, Cornell University Medical College, New York, N.Y.) were grown as described previously (10). PC12 cells were induced to differentiate by incubation in medium containing NGF (2.5S; Collaborative Biomedical Products, Bedford, Mass.) at a concentration of 100 ng/ml for 6 days, with one medium change on day 3 postplating. In experiments in which PC12 cells were treated with DEX, 0.5 μM DEX was added to the medium at the time of infection as previously described (10). Vero cells (ATCC CCL-81) were propagated and maintained as described previously (7). The wild-type strain of HSV-1, KOS, was grown and assayed as previously described (7).

Plasmids and mutagenesis.

A plasmid containing wild-type oriS (pOS822 [26]) was used in this study. Two single-nucleotide substitution mutations described below, termed mutOBP and mutR(C-G), were introduced into pOS822 by the Quick Change mutagenesis method (Stratagene, La Jolla, Calif.), with the addition of 8 μl of 25% glycerol and 3 μl of dimethyl sulfoxide to each 50-μl reaction. Reactions underwent 12 amplification cycles in a thermal cycler block (MHJ Research, Watertown, Mass.), using the following parameters: 95°C for 30 s, 55°C for 1 min, and 68°C for 12 min.

Gel mobility shift assays.

Nuclear extracts were prepared from 3 × 106 HSV-infected (multiplicity of infection of 10 PFU/cell) Nd-PC12 and Vero cells at 12 h postinfection (hpi), and protein concentrations were measured as described previously (10).

The oligonucleotide probes used in gel shift assays were synthesized by the Nucleic Acid Facility at the University of Pennsylvania Cancer Center. All probes were double-stranded 24-mer oligonucleotides consisting of either wild-type or mutant oriS sequences containing the highest-affinity 10-bp OBP-binding site, site I. The sequences of these wild-type and mutant site I probes are shown in Fig. Fig.44 and and5.5. Each oligonucleotide and its complement were gel purified, annealed to each other, and labeled using T4 polynucleotide kinase and [γ-32P]ATP (Dupont NEN, Boston, Mass.) as described elsewhere (9). DNA binding reactions were performed as follows Nuclear extract (5 μg) was incubated with 105 cpm of 32P-labeled oligonucleotide probe (1 ng) and 1.5 μg of poly (dA-dT) in DNA binding buffer (10% glycerol, 50 mM HEPES [pH 7.9], 100 mM NaCl, 0.5 mM dithiothreitol) in a final volume of 10 μl. Binding reaction mixtures were incubated for 30 min at room temperature. Protein-DNA complexes were resolved by electrophoresis on a 6% nondenaturing polyacrylamide (19:1, acrylamide/bisacrylamide ratio) gel (PAGE) at 4°C. Competition experiments were performed by adding a 100-fold excess of unlabeled DNA probe to the binding reaction. In some experiments, a double-stranded oligonucleotide containing the consensus binding site for nuclear factor 1 (NF-1) was used as a nonspecific competitor (6). Antibody supershift reactions were performed by adding 1 μl of antibody specific for OBP (R250; generously provided by Mark Challberg, National Institutes of Health, Bethesda, Md.), 1 μl of antibody specific for the glucocorticoid receptor (GR) (generously provided by Paul Farrell, Ludwig Institute for Cancer Research, London, United Kingdom), or 1 μl of antibody specific for ICP8 (generously provided by Martin Zweig, National Institutes of Health, Frederick, Md.) to the binding reaction after 5 min, and incubation was allowed to continue for 25 min. Gels were dried and exposed in a Phosphorlmager cassette (Molecular Dynamics, Sunnyvale, Calif.). To evaluate the mobility of OBPC alone bound to the site I probe, approximately 500 ng of bacterially expressed, six-histidine-tagged OBPC (HisOBPC [see below]) was incubated with 32P-labeled site I probe, using the binding reaction parameters described above.

FIG. 4
Mapping of the nucleotide binding sites of complexes A, B, and C. (A) Nucleotide sequence of the wild-type (wt) and mutant site I probes. Two-base-pair substitution mutations (indicated in bold) were introduced throughout the probe, yielding a total of ...
FIG. 5
Point mutations in the site I probe eliminate formation of specific protein-DNA complexes. (A) HSV-infected Nd-PC12 cell nuclear extract was incubated with wild-type (wt) or mutant [mutOBP or mutR(C-G); sequences shown in panel B] site ...

Bacterial production of HisOBPC.

The UL8.5 gene was cloned into the bacterial expression vector pTrcHisA (Invitrogen, Carlsbad, Calif.) such that a six-histidine tag was translated in frame with OBPC. Induction of HisOBPC expression was performed by addition of isopropyl-β-d-thiogalactopyranoside (1 mM) to XL1-Blue cells (Stratagene) as outlined in the Xpress system protein expression manual (Invitrogen). After 4 h, cells were lysed by addition of 1 mg of lysozyme per ml. Sarkosyl (1.5%) was added to cell lysates to improve protein solubility. HisOBPC was bound to a Ni2+ column containing a 50% slurry of ProBond resin (Invitrogen), and fractions were eluted by addition of 20 mM phosphate buffer (pH = 4) as outlined in the ProBond resin purification manual (Invitrogen). The purity of OBPC in each fraction was verified by sodium dodecyl sulfate-PAGE followed by Coomassie blue staining. The fraction in which HisOBPC was the only protein detectable by Coomassie blue staining was collected, and its protein concentration was measured by the method of Bradford (Bio-Rad, Hercules, Calif.), using a standard curve generated with known amounts of bovine serum albumin as the standard.

In vitro origin-dependent DNA replication assays.

In vitro oriS-dependent DNA replication assays were performed as described previously (10). Briefly, 3.5 × 106 PC12 cells were seeded in collagen-coated 100-mm-diameter plates. Twenty-four hours after plating, cells were transfected with 10 μg of wild-type or mutant pOS822 by the Lipofectin method (Gibco, Grand Island, N.Y.). After 5 h, cells were washed, and fresh medium containing NGF (100 ng/ml) was added. NGF differentiation proceeded for 6 days, with one medium change on day 3. After 6 days, cells were infected with KOS at a multiplicity of 10 PFU/cell. Cells were harvested at 18 hpi, and total cellular DNA was isolated. For Vero cells, 3.5 × 108 cells were plated, transfected 24 h later with the indicated plasmid, and infected 24 h after transfection. Five micrograms of total Nd-PC12 or Vero cell DNA was digested with HindIII (to linearize the vector) and either MboI (to cleave unmethylated DNA) or DpnI (to cleave methylated DNA). Digested DNA was resolved by electrophoresis on a 0.8% agarose gel and transferred to a nylon membrane. After UV cross-linking, the membrane was prehybridized for 1 h at 55°C in ExpressHyb solution (Clontech, San Francisco, Calif.) and hybridized for 3 h at 55°C with a 32P-labeled probe (3 × 106 cpm/ml) generated by nick translation of pGEM7Zf+ (vector backbone of pOS822). The membrane was then washed according to the ExpressHyb protocol (Clontech) and exposed in a Phosphorlmager cassette.

RESULTS

Three HSV-specific complexes bind to oriS site I when nuclear extracts of Vero or Nd-PC12 cells are used as the source of protein.

Previously published gel shift assays from this laboratory using total Vero cell extracts as the source of protein described binding of two HSV-specific complexes (complexes A and B) to oriS site I (5, 6, 9). In the present study, we used nuclear extracts of Vero, Nd-PC12 cells, or Nd-PC12 cells treated with DEX and observed binding of three HSV-specific complexes (complexes A, B, and C) to a double-stranded DNA probe containing oriS site I (Fig. (Fig.1).1). Specifically, to compare whole-cell versus nuclear extracts as the source of protein, we prepared extracts from KOS-infected Vero cells. Using the site I probe, the pattern produced using total Vero cell extract (Fig. (Fig.2A,2A, lane 1) was nearly identical to that observed in previous studies using total Vero cell extract (5, 6, 9). Several differences were observed, however, when total Vero cell extract was compared with nuclear extract as the source of protein (Fig. (Fig.2A).2A). Complex C was much less prominent in total cell extract (lane 1) than in nuclear extract (lane 2), suggesting that nuclear extracts are greatly enriched for the proteins that comprise complex C. The intensity of complex A was also reduced in total cell relative to nuclear extracts, whereas the intensity of complex B was considerably greater in total cell than in nuclear extracts. Moreover, the mobility of complex B in the two types of extract differed. The enhanced intensity of complex B formed from total cell extracts suggests that these extracts are enriched for components of complex B relative to nuclear extracts. A fourth complex that migrated slightly below complex B was prominent in gel shifts using total cell extracts but barely visible in shifts using nuclear extracts. Taken together, these observations indicate that the efficiency of formation of specific complexes at oriS site I is dependent on the presence of individual proteins within the cytoplasm or nucleus and the concentrations of the proteins within these two compartments.

FIG. 2
Identification and specificity of oriS binding complexes. (A) Vero cells were infected with KOS at a multiplicity of 10 PFU/cell. At 12 hpi, total cell or nuclear extracts were prepared and used in gel shift assays. (B) NGF-differentiated PC12 cells were ...

To address the possibility that protein complexes that form at oriS site I differ in Vero versus PC12 cells or as a function of NGF or DEX treatment of PC12 cells, we compared the profiles of site I-binding complexes that formed using nuclear extracts of Vero, untreated PC12, Nd-PC12, and DEX-treated Nd-PC12 cells infected with HSV (data not shown). All profiles closely resembled that shown in Fig. Fig.2A,2A, lane 2. Thus, as measured by gel shift analysis, the differential effects of cell-type-specific factors on oriS function do not appear to be mediated by protein-DNA complex formation at oriS site I. The results of gel shift assays using Nd-PC12 cell nuclear extracts are shown throughout the remainder of this report.

To determine the kinetics of protein-DNA complex formation at oriS site I during HSV infection, we performed a time course experiment in which Nd-PC12 cells were infected with KOS, nuclear extracts were prepared at 3-h intervals postinfection, and complex formation with the site I probe was evaluated by gel shift analysis. At 0 and 3 hpi, formation of two complexes with the probe was detected. As viral DNA replication begins at approximately 3 hpi, and the 0- and 3-hpi profiles were the same, it is likely that these complexes contain primarily cellular proteins. Efficient formation of three protein-DNA complexes, designated complexes A, B, and C, was first observed at 6 hpi. The intensity of these complexes increased throughout the course of infection (through 18 hpi), indicating that their formation was dependent on viral infection. Although the intensity of complex C was always greater than that of complex B, the increase in the intensities of the two complexes over time (i.e., the kinetics of their formation) paralleled each other at all times postinfection (Fig. (Fig.2C).2C). Specifically, for both complexes the most rapid increase in band intensity occurred between 3 and 9 hpi. In contrast, the most rapid increase in the intensity of complex A was evident slightly later, between 6 and 12 hpi. Notably, material that did not enter the gel is visible in this and other gels; it is not known if this material contains OBP or OBPC.

To determine whether binding of complexes A, B, and C to oriS site I was specific, complex formation was evaluated in the presence of unlabeled specific and nonspecific competitor DNA (Fig. (Fig.2D).2D). Whereas addition of a 100-fold molar excess of unlabeled oriS site I DNA abrogated formation of complexes A, B, and C (lane 2), addition of a 100-fold molar excess of nonspecific DNA (NF-1 binding site) had no effect on complex formation (lane 3), indicating that complexes A, B, and C bind specifically to the site I probe.

Do complexes A, B, and C contain OBP and/or OBPC?

To determine whether complexes A, B, and C contain OBP and/or OBPC, we performed antibody supershift experiments in which antibodies specific for OBP and OBPC were added to gel shift binding reactions (Fig. (Fig.3A).3A). Addition of antibody specific for OBP and OBPC shifted the mobility of all three complexes (lane 3, two supershifted bands are marked with asterisks; a third is frequently visible below the second supershifted band), indicating that complexes A, B, and C contain OBP and/or OBPC. Although addition of the OBP/OBPC-specific antibody also appears to shift the faint band which migrates slightly more slowly than complex C, this observation was not reproducible. Addition of antibody specific for ICP8 (provided by M. Zweig) had no effect on complex formation (data not shown).

FIG. 3
Characterization of HSV-specific complexes that form at oriS. (A) HSV-infected PC12 cell nuclear extracts were incubated with the site I probe. After 5 min of incubation, no antibody (lane 1), antibody specific for GR (lane 2), or antibody specific for ...

Based on previous studies by Hardwicke and Schaffer (10) demonstrating a functional role for GR binding to the consensus GRE in oriL, we investigated the possibility that GR may participate in complex formation at the degenerate GRE sequence present in oriS. For this purpose, antibody to GR was added to oriS site I binding reactions. Addition of antibody to GR had no discernible effect on complexes A and B but resulted reproducibly in the presence of an additional band above complex C and a slight decrease in the intensity of complex C, suggesting that GR may be a component of complex C (Fig. (Fig.3A,3A, lane 2).

To distinguish between complexes that contain OBP and those that contain OBPC, we took advantage of the fact that the synthesis of the delayed-early protein, OBPC, but not the early protein, OBP, is largely but not entirely dependent on HSV DNA synthesis. Thus, OBPC transcript and protein levels are greatly reduced in the presence of phosphonoacetic acid (PAA) (2). PAA used at 400 μg/ml inhibits HSV DNA synthesis by inhibiting the activity of the virus-encoded DNA polymerase. PAA was therefore used to reduce the synthesis of OBPC in an effort to distinguish between complexes containing OBP and OBPC. For this purpose, Nd-PC12 cells were infected with KOS in the absence (Fig. (Fig.3B,3B, lane 1) or presence (lane 2) of 400 μg of PAA per ml, and nuclear extracts were used in gel shift assays. As expected, addition of PAA decreased the intensities of all three complexes, as this drug inhibits viral DNA replication, resulting in fewer genomes from which viral proteins can be expressed. However, of the three complexes A, B, and C, addition of PAA decreased the intensity of complex A most markedly (12% of the no-PAA control), suggesting that the synthesis of the primary component of complex A is most dependent on HSV DNA synthesis and thus contains OBPC. Addition of PAA also reduced the intensity of complex B (34% of the no-PAA control), suggesting that in addition to OBP, this complex may also contain OBPC or another PAA-sensitive protein. Addition of PAA had the least effect on formation of complex C (56% of the no-PAA control), suggesting that this complex does not contain OBPC.

To determine whether PAA-sensitive complex A contained exclusively OBPC, we compared its mobility with that of a band produced by bacterially expressed OBPC bound to the site I probe (Fig. (Fig.3C).3C). HisOBPC was synthesized in bacteria and purified by metal chelate affinity chromatography. Incubation of HisOBPC with the site I probe resulted in the formation of a complex (lanes 2 and 3) that migrated with mobility identical to that of complex A from KOS-infected cells (lane 1; lane 3 is a longer exposure of lane 2). Together, these results suggest that complex A contains OBPC and no other viral or cellular proteins and that complexes B and C contain OBP. Notably, the presence of OBPC in complex B remains a possibility.

Nucleotide mapping of the binding sites of complexes A, B, and C.

To identify the precise nucleotides required for formation of complexes A, B, and C with the site I probe, we analyzed the ability of these complexes to form with probes that contained sequential two-base-pair substitution mutations (Fig. (Fig.4A,4A, mutations are indicated in bold). In contrast to complexes produced using the wild-type probe, complexes B and C were barely detectable upon incubation of nuclear extracts with probes Mut3 through Mut7, indicating that the mutations contained within these probes abrogate formation of these complexes. The intensity of complexes B and C was reduced to a lesser extent upon incubation of extracts with probes Mut8, -11, and -12 relative to the wild-type probe, implicating the wild-type nucleotides mutated in these probes as essential for efficient formation of complexes B and C with site I DNA. Formation of complex A was reduced significantly upon incubation of extracts with probes Mut4, -5, and -6 and slightly upon incubation with Mut12. The enhanced intensity of two bands that migrate below complex B in tests using probes Mut3 through -8 and Mut12 was notable. The composition of these bands (i.e., whether they contain viral or cellular proteins) is unknown. These results indicate that 10 nucleotides (represented by probes Mut3 through Mut7) are required for formation of complexes B and C, whereas only 6 nucleotides (represented by probes Mut4 through Mut6) are required for efficient formation of complex A. Interestingly, the dinucleotide represented by Mut12 appeared to be required for efficient formation of all three complexes. Whether this result is real or a consequence of the location of the mutated nucleotides at the 3′ end of the probe remains to be determined. Additional tests in which unlabeled mutant probes were used to compete for formation of complexes A, B, and C to the wild-type site I probe confirmed these results (data not shown). Based on these findings and as shown schematically in Fig. Fig.5B,5B, the binding site for complex A is contained entirely within the shared binding site for complexes B and C.

A point mutation eliminates formation of complexes B and C without affecting formation of complex A at oriS site I.

To further test the observation that the binding site for complex A is contained entirely within the shared binding site of complexes B and C, we generated a probe containing a single-nucleotide substitution mutation outside the binding site for complex A but within the binding site for complexes B and C, this mutant probe was designated mutOBP (Fig. (Fig.5B).5B). Similarly, we generated a probe that contained a single-nucleotide substitution mutation within the binding site for all three complexes; this probe was designated mutR(C-G). mutR(C-G) differs from mutR(C-A) described previously by Dabrowski et al. (5) in that in this study, C was changed to G rather than A (Fig. (Fig.5B).5B). Nuclear extracts from KOS-infected Nd-PC12 cells were incubated with the wild-type oriS site I, mutOBP, or mutR(C-G) probe, and protein-DNA complex formation was evaluated by gel shift analysis (Fig. (Fig.5A).5A). As expected, complexes A, B, and C formed efficiently with the wild-type probe; however, complex A, but not complex B or C, formed with mutOBP, and none of the three complexes formed with mutR(C-G). These findings confirm those presented in Fig. Fig.44 and demonstrate that a single-nucleotide substitution mutation is sufficient to eliminate binding of all three complexes. The nucleotide sequences of the wild-type and mutant probes and of the complexes that bind to each probe are summarized schematically in Fig. Fig.5B.5B.

Effect of mutOBP and mutR (C-G) on oriS-dependent HSV DNA replication.

To examine the effect of formation of complexes A, B, and C at site I on origin-dependent HSV DNA replication, the mutOBP and mutR(C-G) mutations were introduced into the wild-type oriS-containing plasmid, pOS822, and the effects of these mutations were evaluated in in vitro origin-dependent DNA replication assays (Fig. (Fig.6A).6A). Undifferentiated PC12 cells were transfected with pOS822 (lanes 1 to 4) or pOS822 containing the mutR(C-G) (lanes 5 to 8) or mutOBP (lanes 6 to 10) mutation. PC12 cells transfected with the pOS822 backbone (pGEM) which does not contain oriS were used as a negative control for plasmid amplification (data not shown). Following transfection, PC12 cells were differentiated with NGF for 6 days and then infected with KOS. At 18 hpi, total cell DNA was extracted, subjected to restriction enzyme digestion with MboI or DpnI to distinguish between input or newly replicated plasmid DNA, respectively, and analyzed by Southern blot. The probe used in these tests hybridizes to a DNA sequence contained in the vector component of all pOS822-derived plasmids. The blot in Fig. Fig.6A6A shows the results of each plasmid tested in duplicate, and the data are presented quantitatively in Fig. Fig.6B.6B. The intensity of the band corresponding to newly replicated pOS822 (DpnI resistant) (Fig. (Fig.6A,6A, lanes 2 and 4, arrow) was ~11-fold greater than that of input pOS822 (MboI resistant (lanes 1 and 3), indicating that the wild-type oriS-containing plasmid was amplified efficiently (Fig. (Fig.6B).6B). In contrast, amplification of plasmids containing the mutR(C-G) (lanes 6 and 8) or mutOBP (lanes 10 and 12) mutation was considerably less than that of the wild-type plasmid (lanes 1 and 3) (Fig. (Fig.6).6). Similar results to mutR(C-G) were obtained for mutR(C-A) by Dabrowski et al. (5). The levels of newly replicated mutant plasmids were only 1.4- and 2.0-fold, respectively, above the input plasmid levels. Therefore, the single-nucleotide substitution mutations in mutR(C-G), mutR(C-A), and mutOBP reduced the efficiency of plasmid replication to 10% [mutR(C-G); Fig. Fig.6B],6B], 4% [mutR(C-A)] (5), and 25% (mutOBP; Fig. Fig.6B)6B) of the wild-type level, indicating that mutations in the shared region of the binding sites of complexes A, B, and C [mutR(C-A), mutR(C-A)] or B and C (mutOBP) greatly reduce the ability of oriS to support efficient origin-dependent DNA replication.

FIG. 6
Effects of mutations mutOBP and mutR(C-G) on oriS-dependent HSV DNA replication. (A) PC12 cells were transfected with pOS822-derived plasmids containing wild-type (wt; lanes 1 to 4), mutR(C-G) (lanes 5 to 9), and mutOBP (lanes 9 to 12) or mutant [mutR(C-G) ...

DISCUSSION

OBP and OBPC-containing complexes that form at oriS site I.

The results presented here confirm and extend previous studies in which total Vero cell extracts were used to demonstrate formation of two protein complexes, complexes A and B, at oriS site I by gel shift analysis (5, 6, 9). Comparison of the site I-binding complexes from total cell and nuclear extracts revealed differences in the intensities of complexes A and B as well as the presence of an additional complex, complex C, whose formation appears to be dependent on nuclear factors (Fig. (Fig.2A).2A). The differences in complex formation noted when total cell versus nuclear extracts were used may reflect differences in the presence or concentration of nucleus- or cytoplasm-specific proteins present in these complexes. Alternatively, the conformation, oligomerization, or posttranslational modification of proteins contained within these complexes may differ in the cytoplasm versus the nucleus. That the interaction of OBP-containing complexes with oriS is dependent on factors present in the nucleus and/or cytoplasm likely has important implications for the functional role of OBP during HSV infection. Notably, however, no differences in complex formation were noted when proteins used in gel shift assays were derived from nuclei of Vero or Nd-PC12 cells or Nd-PC12 cells treated with DEX. Thus, the basis for the differential effects of NGF and DEX on oriS function noted previously (10) remains unclear.

Of the three HSV-specific complexes that form at site I, gel shift analysis using nuclear extracts of PAA-treated cells or bacterially expressed OBPC suggests that the sole protein component of complex A is OBPC. In contrast, several lines of evidence suggest that complexes B and C contain OBP: these complexes (i) supershift upon addition of antibody to OBP, (ii) are first detectable and increase in intensity at times consistent with E protein expression, and (iii) share a nucleotide binding site previously shown to be the binding site for OBP (6, 11).

The electrophoretic mobilities of complexes B and C differ, however, suggesting that they are composed of different oligomeric forms of OBP or that they contain additional viral or cellular proteins. Incubation of the site I probe with nuclear extracts of Vero cells infected with an OBP-expressing recombinant adenovirus resulted in the formation of two complexes that migrate with mobilities identical to those of complexes B and C (unpublished observations), indicating that other viral proteins are not required for formation of these complexes, and additional protein components of complexes B and C are therefore likely to be cellular in origin. This hypothesis is further supported by the finding that GR, a cellular protein known to affect the efficiency of HSV origin function (10), may be a component of complex C. Given that several members of the HSV DNA replication complex (i.e., UL8, UL42, and ICP8) have been shown to interact with OBP in assays using purified proteins (3, 16, 17), it is somewhat surprising that these proteins have not yet been detected in OBP-containing complexes bound to oriS by gel shift assay. Specifically, addition of antibody to ICP8 to binding reactions had no detectable affect on site I complex formation (data not shown), yet ICP8 has been shown to bind specifically to OBP (3), and the interaction site in OBP has been mapped. One possible explanation for our inability to detect ICP8 within these complexes is that the amounts of DNA replication proteins in HSV-infected cell nuclear extracts are limited. It is also possible that conditions compatible with formation of the replication complex at the origin are not duplicated in our in vitro assay. Identification of other protein components within the complexes that form at oriS will contribute to our understanding of initiation of HSV DNA replication at HSV-1 origins.

Roles of OBP and OBPC in HSV DNA replication.

Extensive mutagenesis of the oriS site I probe revealed that complexes B and C share a 10-bp binding site which was previously described as OBP binding site I (11). Despite the fact that OBP and OBPC share the same C-terminal DNA binding domain, the DNA binding site of complex A, which appears to consist solely of OBPC, was found to comprise only 6 bp which lie totally within the 10-bp binding site of complexes B and C. That the OBP-containing complexes require a larger binding site than OBPC may reflect the larger size of OBP relative to OBPC, dimer formation of OBP but not OBPC, or the presence of additional cellular proteins in complexes B and C but not A which are necessary for efficient contact with origin DNA. Notably, the sizes of complex A and B binding sites determined in this study are contrary to previous findings in which the nucleotide binding site of complex A was several nucleotides larger than that of complex B (6). These differences are likely due to differences in the composition of the cell extract used (total cell extract [6] versus nuclear extract [this study]) as well as differences in the mutant probes used for mapping studies (nucleotide deletions [6] versus dinucleotide substitutions [this study]).

Given that the binding site of complex A (OBPC) lies entirely within the binding site of OBP-containing complexes B and C, a single base pair was identified which, when mutated (mutOBP), eliminated binding of complexes B and C without affecting binding of complex A to site I. The mutOBP mutation reduced the replication efficiency of an oriS-containing plasmid to 25% of that of the wild-type plasmid, indicating that binding of OBPC alone is insufficient to support origin-dependent DNA replication. This finding is not unexpected, given that OBPC lacks multiple domains present in OBP that are thought to be necessary for HSV DNA replication. The location of the complex A binding site relative to that of complexes B and C does not allow us to make the reciprocal mutation in site I (i.e., eliminate binding of OBPC without affecting binding of OBP-containing complexes), and thus we cannot rule out the possibility that binding of OBPC is important for HSV DNA replication. Based on previous studies that describe a strong dominant-negative effect of OBPC on the replication of HSV (2), it has been postulated that OBPC may act as a repressor of origin-dependent DNA replication by competing with OBP for origin binding. As noted above, elucidation of the precise function of OBPC will require isolation and characterization of OBP+ OBPC plasmids and viruses.

ACKNOWLEDGMENTS

This study was funded by NIH grant R01-A128537 from the National Institute of Allergy and Infectious Diseases. J.A.I. was supported by NIH training grant T32-AI007325.

We gratefully acknowledge John Wagner for providing PC12 cells, Mark Challberg for providing antisera to OBP, Paul Farrel for providing antisera to GR, the University of Pennsylvania Nucleic Acid Facility for assistance in sequencing problematic regions of oriS, and members of the Schaffer laboratory for helpful discussions and ideas.

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