Skip to main page content

• HOME
• Current Issue
• Archive
• Alerts
• About ASM
• Contact us
• Tech Support
• Journals.ASM.org

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Link, M. A. Articles by Schaffer, P. A. Search for Related Content PubMed PubMed Citation Articles by Link, M. A. Articles by Schaffer, P. A.

 Previous Article  |  Next Article 

Journal of Virology, September 2007, p. 9175-9182, Vol. 81, No. 17
0022-538X/07/$08.00+0     doi:10.1128/JVI.00676-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Cathepsin B Mediates Cleavage of Herpes Simplex Virus Type 1 Origin Binding Protein (OBP) To Yield OBPC-1, and Cleavage Is Dependent upon Viral DNA Replication

Malen A. Link, Laurie A. Silva, and Priscilla A. Schaffer^*

Departments of Medicine and Microbiology and Molecular Genetics, Program in Virology, Harvard Medical School at Beth Israel Deaconess Medical Center, Boston, Massachusetts 02215

Received 29 March 2007/ Accepted 30 May 2007

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Although the seven viral proteins required for herpes simplex virus type 1 (HSV-1) DNA replication have been identified, the mechanism by which viral DNA synthesis is regulated is unclear. HSV-1 DNA replication is thought to occur in two stages: origin-dependent DNA replication (stage I) mediated by the origin binding protein (OBP), followed by origin- and OBP-independent DNA replication (stage II). The mechanism that facilitates the switch from stage I to stage II is unknown; however, it must involve the loss of OBP function or OBP itself from the replication initiation complex. Previous studies from this laboratory identified a transcript (UL8.5) and protein (OBPC) that are in frame with and comprise the C terminus of the gene specifying OBP. Because of its DNA binding ability, OBPC has been hypothesized to mediate the switch from stage I to stage II. Here, we identify a second protein (OBPC-2) that is also in frame with the C terminus of OBP but comprises a smaller portion of the protein. We demonstrate that the protein originally identified (OBPC-1) is a cathepsin B-mediated cleavage product of OBP, while OBPC-2 may be the product of the UL8.5 transcript. We further demonstrate that the cleavage of OBP to yield OBPC-1 is dependent upon viral DNA replication. These results suggest that cleavage may be a mechanism by which OBP levels and/or activity are regulated during infection.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The 152-kb herpes simplex virus type 1 (HSV-1) genome encodes at least 84 proteins, 7 of which are early genes essential for viral DNA replication in vitro (49). Functionally, the proteins specified by the UL5, UL8, and UL52 genes comprise the helicase/primase complex, which unwinds viral DNA prior to replication. The UL29 gene encodes infected cell protein 8 (ICP8), a single-stranded DNA (ssDNA) binding protein that stabilizes replicating ssDNA. The products of the UL30 and UL42 genes comprise the viral DNA polymerase and polymerase accessory protein, respectively, which together replicate the viral genome. The product of the UL9 gene is the origin binding protein (OBP), the viral DNA replication initiator protein, and the subject of this paper.

Initiator proteins are functionally similar in all life forms and are characterized by their ability to bind to specific sequences in origin DNA. This binding results in unwinding of the origin at a site that usually contains an AT-rich sequence. DNA binding and unwinding are followed by the recruitment of the DNA replication machinery. As a means of regulating the DNA replication process, the activities of initiator proteins themselves are highly regulated. For example, the activities of many viral initiator proteins including simian virus 40 large T antigen (44), polyomavirus large T antigen (59), human papilloma virus E1 (27), bovine papillomavirus E1 (65), and the NS1 protein of minute virus of mice (11) are regulated by differential phosphorylation. The activities of some initiator proteins such as the bacteriophage lambda O (60-62) and bovine papillomavirus E1 (28) are also regulated by ubiquitination. Still others, including the bacteriophage lambda oriC initiator protein, are regulated by a viral protein that inhibits their binding to origin sequences (10). Similarly, the binding of other viral initiator proteins to origin sequences is inhibited by the expression of C-terminal forms of the initiator protein itself, such as gene X of bacteriophage f1 (15) and gene A* of bacteriophage phiX174 (12).

HSV-1 OBP is a 95-kDa, 851-amino-acid nuclear phosphoprotein that forms homodimers and binds to specific sites within HSV-1 origin DNA via a domain in the C-terminal half of the protein (Fig. 1). The C terminus of the protein also contains a binding domain for ICP8 (5). The N-terminal half of OBP contains binding domains for at least two other viral proteins essential for viral DNA synthesis (UL8 and UL42) (5, 35, 36) as well as seven helicase domains that facilitate the unwinding of the double-stranded DNA at the AT-rich apices of origin palindromes.

View larger version (27K): [in this window] [in a new window]

FIG. 1. Diagrams of OBP, OBPC-1, and OBPC-2. The DNA binding domain is represented by the hatched region. Helicase domains are shown in gray and numbered I to VI. The ATPase domain, leucine zipper, and putative PEST sequence are shown in black. The N-terminal domain confers OBP dimerization ability and contains domains that interact with the proteins encoded by the UL8 (component of the helicase/primase complex) and UL42 (the polymerase accessory protein) genes. The nuclear localization signal (NLS) and ICP8 binding domain are indicated. Dotted lines at the beginning of OBPC-1 and OBPC-2 indicate that the translation start sites are unknown.

Based on work from other laboratories using temperature-sensitive mutants of UL9 (4, 51), a two-stage model of HSV DNA replication has been proposed (4, 51, 63). Stage I is thought to begin early in infection, followed by a switch to stage II. Stage I replication begins when OBP binds to one or more sites in origin DNA and recruits other essential viral replication proteins to these sites through direct protein-protein interactions (Fig. 2). As viral DNA circularizes upon infection (17, 42, 57), stage I replication likely proceeds by a theta-like mechanism. In contrast to initiation at early times postinfection (stage I), it has been demonstrated that OBP is not essential for viral DNA synthesis at later times postinfection or to elongate DNA using a preprimed template (4, 19, 51-53). Rather, overexpression of OBP following stage I initiation is detrimental to viral DNA synthesis and, by extension, viral replication (2, 4, 29, 32, 51). It has also been demonstrated that the inhibitory effect of OBP is dependent upon its ability to bind DNA (2, 30, 39, 56). These observations suggest that OBP is functionally altered and/or dissociates from origins and replication complexes following stage I initiation (32). This functional alteration/dissociation may result in a switch to stage II, origin-independent replication, which may proceed via a rolling circle and/or recombination-dependent mechanism.

View larger version (19K): [in this window] [in a new window]

FIG. 2. Model of initiation of origin-dependent viral DNA replication (after Boehmer and Lehman) (24). The model is similar for oriS and oriL. In step 1, OBP dimers (gray ovals) bind to site I in both arms of oriL or sites I and II in the arms of oriS. OBP binding initiates unwinding of the AT-rich apex of the origin (step 2). In step 3, unwound ssDNA is stabilized, and unwinding is enhanced by ICP8 binding (black ovals). Through interactions with the UL8 protein (step 4), the helicase/primase complex (the products of the UL5, UL8, and IL52 genes) (hatched hexagons) binds to OBP and origin DNA to further unwind DNA at the apex. The polymerase (UL30; black diamond) and polymerase accessory factor (UL42; white triangle) are recruited to the initiation complex in step 5. At this point, OBP may be released from the initiation complex. Following one or several rounds of origin-dependent replication, origin-independent replication may proceed (step 6) by a rolling circle mechanism and/or be initiated through a recombination-dependent mechanism (45-47). Stage I proceeds through step 5, and stage II begins with step 6 following the loss of OBP.

Based on these observations, it has been hypothesized that OBP functions and/or levels must be regulated during infection (8, 9). Specific posttranslational modifications of OBP may be used for this purpose. First, OBP is phosphorylated during infection (22), a common mechanism by which the functions of replication initiator proteins are regulated. Second, OBP contains a putative PEST motif (Fig. 1) (32). PEST motifs target proteins for degradation via the ubiquitin/proteasome pathway. Third, OBP interacts with and is degraded in the presence of FBX2, a neuron specific F-box protein (13, 14). To date, the functional consequences of these modifications in the context of lytic infection have not been determined. Although the dissociation of OBP from replication complexes has not been demonstrated directly, available evidence suggests that the nuclear localization of OBP may change following initiation of viral DNA synthesis (13, 14), suggesting the dissociation hypothesis. If OBP is, in fact, removed from the initiation complex, the mechanism underlying its removal and the mechanism of OBP-mediated interference in stage II viral DNA replication have yet to be determined.

Potential mechanisms for both of these events came to light when Baradaran et al. reported the identification of a transcript lying in frame with and comprising the C-terminal half of the UL9 transcript (1). This transcript, which is expressed with delayed early (DE) kinetics, was termed UL8.5. In in vitro transcription/translation assays using the UL8.5 transcript and an antibody specific for the C terminus of OBP, a protein of 53 kDa, similar in size to a protein observed in infected cell extracts, was observed (1). Based on these observations, it was assumed, though not tested, that the C-terminal protein, designated OBPC, was expressed from the UL8.5 transcript. OBPC expressed by in vitro transcription/translation assays using the UL8.5 transcript retained the ability to bind to HSV-1 origin DNA but, based on size, was assumed to lack the majority of the helicase domains as well as the N-terminal binding domains for UL42 and UL8 (2). Based on these properties, OBPC was hypothesized to be involved in mediating the switch from theta to rolling circle viral DNA replication by binding to origin sequences and blocking the binding of full-length OBP to origin sequences and, hence, formation of the initiation complex (2). If correct, this mechanism would be similar to that of gene X of bacteriophage f1 and gene A* of bacteriophage phiX174 (12, 16). This hypothesis is supported by several studies which have demonstrated that overexpression of C-terminal forms of OBP that are able to bind site I DNA are inhibitory to viral DNA synthesis and viral replication, while C-terminal forms that are not able to bind site I DNA are not inhibitory and at times are even potentiating (2, 9, 52, 56).

Many C-terminal overlapping transcripts have been reported in the HSV-1 genome including UL8.5, UL9.5, UL12.5, UL26.5, UL49.5, US1.5, US3.5, and US8.5 (1-3, 6, 7, 18, 21, 25, 34, 43, 45, 64, 66). Each of these overlapping transcripts encodes a protein that is in frame with and comprises the C terminus of the open reading frame (ORF) encoded by the larger transcript. Because these truncated C-terminal proteins share a subset of functional domains with their larger counterparts, the possibility that they play modulatory roles with regard to the proteins they overlap is intriguing. The discovery of a second protein of 35 kDa that is in frame with and comprises the C-terminal-most portion of OBP, designated OBPC-2 (Fig. 1), prompted us to ask whether the 53-kDa OBPC-1 (formerly designated OBPC) is indeed the product of the UL8.5 transcript.

We have shown that OBPC-1 is not the product of the UL8.5 transcript but, rather, is a cathepsin B-mediated cleavage product of OBP. Intriguingly, cleavage is dependent upon viral DNA replication. Cathepsin B-mediated cleavage of OBP may indicate a change in OBP structure and/or localization following the initiation of viral DNA replication and may serve to regulate OBP levels and/or function during infection.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Cells and viruses. Vero cells (ATCC CCL-81) were cultured in Dulbecco's modified Eagle medium supplemented with 5% fetal bovine serum, and all experiments in Vero cells were performed using Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum. Cells were propagated and maintained at 37°C in 5% CO₂.

The wild-type virus used in these studies was HSV-1 strain KOS and was propagated as previously described (50). Strain 17 was obtained from the Medical Research Council at the University of Glasgow, Scotland, United Kingdom. Strain F was obtained from Bernard Roizman, University of Chicago, Chicago, IL. Strains Seibert and Gayle were obtained from William Rawls, Baylor College of Medicine, Houston, TX. The hr94 virus, whose genome contains a ß-galactosidase expression cassette inserted at codon 534 of the OBP ORF, was isolated and kindly provided by Sandra Weller (University of Connecticut Health Sciences Center, Farmington, CT) (29).

Construction of the pUL9n24 plasmid. In order to construct a plasmid that would express the UL8.5 transcript and its protein product(s) but not OBP, a DNA fragment containing base pairs 20140 to 24136 of the HSV genome and containing the UL8.5 and UL9 genes in their entirety (see Fig. 4) was amplified by PCR using the following primers: 5'-CACAACGTAGTAGCCGTTGGTGTAATAGTG-3' and 5'-CATACAAAATACACCAGGGCGTGGAAGTAC-3'. The PCR product was cloned into the pCR-Blunt II-Topo vector using a Zero Blunt Topo PCR Cloning Kit (Invitrogen, Carlsbad, CA) following the manufacturer's protocols. The resulting plasmid (pUL9DE) was used to clone the DraIII-EcoRV fragment of the UL9 ORF into the pAlter (Promega, Madison, WI) backbone. This plasmid, pADE, was used for site-directed mutagenesis of the UL9 ORF. The UL9n24 nonsense mutation was introduced into the wild-type sequence 5'-CATTGGGGACGACGAGTGCGAACAGTACACGTCGAGCGTATC-3' of pADE by pAlter mutagenesis using the following mutagenic primer: 5'-CATTGGGGACGACGAGTGAGAACAGTACACGTAGAGCGTATC-3'. The mutations are shown in boldface. The pADE plasmid containing the UL9n24 mutation was digested with DraIII, and the resulting fragment was ligated into plasmid pUL9H (1) from which the wild-type DraIII fragment had been removed. Mutant plasmids were identified by restriction digest analysis, and the mutations were confirmed by sequencing (see Fig. 4).

View larger version (29K): [in this window] [in a new window]

FIG. 4. Expression of OBPC-1 requires OBP expression. (A) Diagram of the HSV-1 genome showing the UL region flanked by inverted repeat sequences, ab and b'a', and the US region flanked by inverted repeat sequences a'c' and ca. The location of oriL and both copies of oriS are indicated. Beneath the diagram of the genome is shown the region included in pUL9H. The locations and directions of the transcripts and protein products specified by the UL8, UL8.5, UL9, and UL10 genes are shown as are the DraIII sites used to clone from pADE. The segment of the HSV-1 genome included in pADE (nucleotides 24136 to 20140) is also shown (B). The wild-type sequences and the mutations introduced in codons 24 and 29 of the OBP ORF to create pUL9n24 are shown. The codons for amino acids 24 and 29 are underlined, and the mutations are shown in bold. (C) Western blot analysis of nuclear extracts of Vero cells transfected with pUL9n24 and infected with 10 PFU/cell of hr94 or KOS. The antibody used was anti-OPBCT. WT, wild type.

Antibodies. Two polyclonal rabbit antibodies specific for OBP were generated by Bethyl Laboratories (Montgomery, TX). Anti-OBPCT is directed against amino acids 831 to 851 of OBP, and anti-OBPNT is directed against amino acids 1 to 20.

Inhibitors. Acyclovir (Sigma, St. Louis, MO) (200 µM) was used to inhibit viral DNA replication. Ca074 methyl ester (Ca074Me) (Sigma, St. Louis, MO) (1 µM) was used to specifically inhibit cathepsin B, and calpain inhibitor peptide (American Peptide, Sunnyvale, CA) (1 µM) was used to specifically inhibit calpains I and II. To inhibit cathepsin B as well as calpains I and II, MDL 28170 (Sigma, St. Louis, MO) (100 µM) was used. Clasto-lactacystin ß-lactone (Boston Biochem, Cambridge, MA) (5 µM) was used as a specific inhibitor of the proteosome. MG132 (Z-Leu-Leu-Leu-CHO; 20 µM) inhibits the proteosome, cathepsin B, and calpains I and II (Boston Biochem, Cambridge, MA). Chloroquine (Sigma, St. Louis, MO) (200 µM), was used as an inhibitor of lysosomal proteases.

Transfections. Cells (5.5 x 10⁵) were seeded in 35-mm dishes. Twenty-four hours later, cells were transfected with 3 µg of the indicated plasmid DNA using Lipofectamine 2000 (Invitrogen, Carlsbad, CA). Cells were infected 18 h posttransfection as indicated in the figure legends.

Nuclear extracts. Nuclear extracts were prepared as previously described from 1 x 10⁶ cells infected with 10 PFU/cell of the indicated virus or mock-infected and harvested at 8 h postinfection (hpi) unless otherwise noted (20). Briefly, cells were harvested by scraping into medium. Cells were pelleted by low-speed centrifugation (5 min at 300 x g), washed twice with phosphate-buffered saline (PBS), and resuspended in resuspension buffer (10 mM Tris-HCl, ph 7.5, 10 mM NaCl, and 5 mM MgCl₂) supplemented with 0.5 mM dithiothreitol (DTT) (Sigma, St. Louis, MO) and 1 µg/ml of the following protease inhibitors: leupeptin (Sigma, St. Louis, MO), aprotinin (Roche, Indianapolis, IN), pepstatin (Sigma, St. Louis, MO), and phenylmethylsulfonyl fluoride (Sigma, St. Louis, MO). Cells were then pelleted and resuspended in resuspension buffer containing 0.5% NP-40 and protease inhibitors. Cells were again pelleted and were resuspended in 20 mM HEPES, pH 7.9, 25% glycerol, 0.42 M KCl, and 0.2 mM EDTA containing protease inhibitors and incubated at 4°C for 30 min. Cell debris was removed by centrifugation at 13,000 x g for 30 min, and the supernatant was dialyzed in 20 mM HEPES, pH 7.9, 20% glycerol, 0.1 M KCl, and 0.2 mM EDTA containing protease inhibitors and DTT. Nuclear extracts were aliquoted, snap frozen in liquid nitrogen, and stored at –80°C.

Whole-cell extracts. Cells (3.1 x 10⁶) were seeded in 100-mm dishes and infected 24 h later at a multiplicity of infection (MOI) of 10 PFU/cell with the virus indicated in the figure legend. At 8 hpi, cells were washed twice with cold PBS and harvested by scraping into 5 ml of cold PBS. Cells were pelleted and resuspended in 100 µl of NET buffer (50 mM Tris, pH 7.8, 100 mM NaCl, 1 mM EDTA) supplemented with leupeptin, aprotinin, pepstatin, phenylmethylsulfonyl fluoride, and DTT. Cells were snap frozen in liquid nitrogen and quickly thawed at 37°C. The supernatant was then sonicated twice for 30 s, cellular debris was pelleted at 13,000 x g, and the supernatant was retained.

Western blot analysis. Laemmli buffer (2.5 µl of 4x) (23) was added to 10 µl of each whole-cell extract or 10 µg of each nuclear extract. Samples were boiled for 5 min, and proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on 8% gels and transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA) using a Mini Trans-Blot cell (Bio-Rad Laboratories, Hercules, CA). Membranes were blocked in Tris-buffered saline ([TBS] 0.1 M Tris, pH 5, 150 mM NaCl) containing 0.05% Tween-20 (TBS-T) and 5% nonfat dry milk overnight at 4°C. Primary antibody was incubated with the membrane at a 1:500 dilution in TBS-T containing 5% milk and allowed to incubate for 1 h at room temperature. Membranes were washed three times for 20 min per wash with TBS-T. Goat anti-rabbit secondary antibody conjugated to horseradish peroxidase (Jackson ImmunoResearch Laboratories, West Grove, PA) was added to the blot at a 1:10,000 dilution in block for 1 h at room temperature. The membrane was washed as described above. Membranes were incubated with Immobilon Western chemiluminescent horseradish peroxidase substrate (Millipore, Billerica, MA) according to the manufacturer's instructions and exposed on CL-X Posure film (Pierce, Rockford, IL).

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Two proteins in frame with and comprising the C terminus of OBP are expressed in HSV-1-infected cells. OBPC was first identified by Baradaran et al. during mapping studies of the UL8, UL9, and UL10 regions of the HSV-1 genome (1). To determine whether OBPC was present in extracts of cells infected with other strains of HSV-1, Western blot analysis of nuclear extracts of Vero cells infected with each of three lab strains (KOS, F, and 17), two clinical isolates (Siebert and Gayle), or mock-infected cells was performed using an affinity-purified antibody directed against the 20 C-terminal amino acids of OBP (anti-OBPCT). This analysis revealed the presence of two C-terminal forms of OBP in nuclear extracts of all viral strains tested but not in mock-infected cells. The two forms have been designated OBPC-1 (53 kDa) and OBPC-2 (35 kDa) (Fig. 3). This unexpected finding represents the first evidence that a second N-terminally truncated form of OBP (OBPC-2) of uniform size is expressed in cells infected with multiple strains of HSV-1.

View larger version (41K): [in this window] [in a new window]

FIG. 3. OBPC-1 and OBPC-2 are expressed in cells infected with multiple strains of HSV-1. Nuclear extracts of the KOS, F, 17, Siebert, and Gayle (10 PFU/cell) strains or mock-infected Vero cells were prepared at 8 hpi and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Bands in gels were probed for OBP, OBPC-1, and OBPC-2 by Western blotting with anti-OBPCT. OBP (95 kDa) and the two C-terminal forms of OBP, OBPC-1 (53 kDa) and OBPC-2 (35 kDa), are indicated.

OBPC-1 is a cleavage product of OBP. Although the UL8.5 transcript was shown by Baradaran et al. (1) to specify a protein product of the approximate size of OBPC-1 in in vitro transcription/translation assays, expression of OBPC-1 from the UL8.5 gene has never been directly demonstrated in cell culture. Therefore, the possibility remained that OBPC-1 is a specific cleavage or degradation product of OBP. To test this possibility, a plasmid with nonsense mutations 24 and 29 amino acids downstream of the OBP translational initiation codon (pUL9n24) was constructed (Fig. 4A and B) (see Materials and Methods). These mutations were designed to eliminate OBP translation while preserving transcription and translation of the UL8.5 gene. Because the UL8.5 transcript is expressed with DE kinetics, cells were transfected with the wild-type or mutant plasmid and then infected 24 h later with the OBP null virus, hr94, in order to induce maximum expression of the UL8.5 transcript. Cell extracts were harvested at 18 hpi.

When Vero cells were transfected with pUL9n24 and infected with hr94, Western blot analysis using anti-OBPCT demonstrated that neither OBP nor OBPC-1 was detected whereas OBPC-2 was detected. As expected, infection with the wild-type virus, KOS, as well as transfection with the wild-type plasmid expressed all three proteins while the vector-only and UL9 null virus (hr94) controls did not express OBP, OBPC-1, or OBPC-2 (Fig. 4C). These observations demonstrate that OBPC-1 expression is dependent upon expression of OBP, whereas expression of OBPC-2 is independent of OBP expression. This finding suggests either that OBP facilitates the expression of OBPC-1 or that OBPC-1 is a cleavage product of OBP. In contrast, OBPC-2 expression is independent of OBP and therefore not a degradation product of OBP. Rather, OBPC-2 is possibly the product of the UL8.5 transcript.

Cathepsin B mediates the cleavage of OBP to produce OBPC-1. To test the hypothesis that OBPC-1 is a cleavage or degradation product of OBP, cells were infected with KOS in the presence or absence of MG132, an inhibitor of the proteasome, cathepsin B, and calpains I and II; and cell lysates were examined for the presence of OBPC-1 by Western blot analysis. Addition of MG132 at 1 hpi inhibited the expression of OBPC-1 but not OBP or OBPC-2 (Fig. 5A). Because MG132 is able to inhibit the proteasome as well as cathepsin B and calpains I and II, a more specific proteasome inhibitor was tested for the ability to affect OBPC-1 expression to determine if OBP was degraded or specifically cleaved by a protease. Addition of clasto-lactasystin ß-lactone, a more specific inhibitor of the proteasome, did not affect expression of OBPC-1 (Fig. 5A). Together, these observations indicate that OBPC-1 is not produced by the proteasome but, rather, by either cathepsin B or calpains I or II. This observation was confirmed by the addition of MDL 28170, which inhibits calpain I and II and cathepsin B but not the proteasome. The addition of MDL 28170 inhibited production of OBPC-1 but not OBP or OBPC-2 (Fig. 5A), confirming that OBP is cleaved by either calpain or cathepsin B. Addition of chloroquine, an inhibitor of lysosomal enzymes, inhibited production of OBPC-1 (Fig. 5A). Although OBPC-2 levels appear reduced in the presence of chloroquine in the experiment shown in Fig. 5A, this was not seen consistently from experiment to experiment and is likely an artifact of this particular experiment. Cathepsin B is a lysosomal enzyme, whereas calpains are not associated with lysosomes. Therefore, these data indicate that cathepsin B, not calpains I or II, cleaves OBP to produce OBPC-1. This conclusion is supported by the following experiments. Addition of calpain inhibitor peptide, which specifically inhibits calpains I and II, did not block cleavage of OBP (Fig. 5A). However, addition of Ca074Me, a highly specific cell-permeable cathepsin B inhibitor, did prevent OBP cleavage, demonstrating that OBP is cleaved in a cathepsin B-dependent manner to produce OBPC-1 (Fig. 5A). The addition of protease inhibitors did not affect the expression of OBP or OBPC-2.

View larger version (43K): [in this window] [in a new window]

FIG. 5. Cathepsin B-mediated cleavage of OBP yields OBPC-1. (A) Vero cells were mock-infected (M) or infected with KOS at an MOI of 10 PFU/cell. Cells were either mock treated (NT) or treated with vehicle (dimethyl sulfoxide [DMSO]) or with the indicated protease inhibitors at 1 hpi. Whole-cell extracts were prepared at 8 hpi, and Western blot analysis using anti-OBPCT was performed. OBP (95 kDa), OBPC-1 (53 kDa), OBPC-2 (35 kDa), and nonspecific (NS) bands are indicated. Molecular size markers are shown on the left. (B) Vero cells were infected and harvested as described in panel A. Western blot analysis of whole-cell extracts was performed using anti-OBPNT. OBPN (42 kDa) is indicated. CIP, calpain inhibitor peptide.

Importantly, expression of an N-terminal fragment (OBPN) (42 kDa) of nearly the same size as OBPC-1 can also be detected following cathepsin B-mediated cleavage of OBP, and its expression is inhibited by Ca074Me (Fig. 5B). The sizes of OBPC-1 (53 kDa) and OBPN (42 kDa) suggest that these proteins are the product of a single cleavage of OBP mediated by cathepsin B. Levels of OBPN are reduced in the nucleus compared to OBPC-1 (data not shown); however, OBPN is abundant in whole-cell extracts, suggesting that it is abundant in the cytoplasm. These findings indicate that both products of OBP cleavage by cathepsin B, OBPC-1 and OBPN, are stably expressed in infected cells.

OBP cleavage is dependent upon viral DNA replication. In order to determine whether OBP cleavage is dependent upon viral DNA replication, acyclovir was added to cells at 1 hpi, and the expression of OBP, OBPC-1, and OBPC-2 was monitored by Western blot analysis (Fig. 6). Acyclovir inhibits viral DNA replication by inhibiting elongation of the replicating strand. In the absence of acyclovir, i.e., during DNA synthesis, OBP, OBPC-1, and OBPC-2 were detected in KOS-infected nuclear extracts but not in mock- or hr94-infected nuclear extracts. The addition of acyclovir at 1 hpi inhibited the production of OBPC-1 (Fig. 6) but not OBP, indicating that OBP cleavage is dependent upon either DNA replication or some event following DNA replication. The addition of acyclovir also reduced levels of OBPC-2 (Fig. 6), suggesting that OBPC-2 is expressed with DE kinetics, similar to the UL8.5 transcript (1).

View larger version (34K): [in this window] [in a new window]

FIG. 6. OBP cleavage is dependent upon viral DNA replication. Vero cells were infected with KOS or hr94 or mock infected at an MOI of 10 PFU/cell in the absence or presence of 200 µM acyclovir (Acyc). Nuclear extracts were prepared at 8 hpi, and Western blot analysis was performed using anti-OBPCT.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The identification of two proteins that are in frame with and comprise different lengths of the C terminus of OBP led us to investigate the origins of these two proteins. While we have demonstrated that OBPC-1 is a product of cathepsin B-mediated cleavage of OBP, OBPC-2 may well be a product of the UL8.5 transcript as (i) it is expressed, like the UL8.5 transcript, with DE kinetics, and (ii) its expression is not dependent upon OBP expression. It should be noted that previous evidence suggesting that OBPC-1 is the product of the UL8.5 transcript relied solely on in vitro transcription/translation assays (1). When the UL8.5 transcript was used in these assays, a protein product of 53 kDa was observed. This product is similar but not identical in size to the protein detected by Baradaran et al. (1) in infected cell extracts and is the predicted size of a protein transcribed from the first in-frame translational start codon in the UL8.5 transcript. Therefore, the conclusion that OBPC-1 is a product of the UL8.5 transcript was reasonable, albeit incorrect (1). Evidence presented in this paper indicates that in the context of viral infection, the first in-frame translational start codon of the UL8.5 transcript is not utilized, as the molecular mass of OBPC-2 (35 kDa) is much smaller than would be predicted (64 kDa) if the first start codon of the UL8.5 transcript were used. In fact, the small size of the protein suggests that it was not detected by Baradaran et al. because the protein ran off the gels used for Western blotting. It is also possible that the antibody used by Baradaran et al. and obtained from Olivo et al. (37, 38), which is directed against the last 10 amino acids of OBP, does not detect OBPC-2, and only upon generation of a new antibody specific for the C-terminal 20 amino acids of OBP has OBPC-2 been detectable. These data suggest that in systems with complex transcriptional and translational strategies such as HSV-1, in vitro transcription/translation data should be supported by evidence from infected cells. We have recently completed a more detailed investigation of the source of OBPC-2 with the following results: we have demonstrated that OBPC-2 is a product of the UL8.5 transcript, and we have mapped its translational start site (unpublished observations).

As noted above, the activities of DNA replication initiator proteins can be regulated in many ways. One mode of regulation is through the activities of a protein that is able to compete with or inhibit the activities of the initiator protein. As OBPC-1 shares the DNA binding domain of OBP but lacks the domains necessary to unwind DNA or to recruit other viral factors essential for the initiation of viral DNA replication, OBPC-1 was considered a prime candidate for an inhibitor of OBP function. Indeed, overexpression of a protein similar in size to OBPC-1 (50 kDa), which presumably contains amino acids 365 to 851 of OBP, is inhibitory to origin-dependent DNA replication (2). Based on these considerations, we hypothesized that OBPC-1 may play a role in the negative regulation of OBP activity by inhibiting its binding to origin sequences and thereby facilitating the switch from origin-dependent DNA replication to origin-independent DNA replication (2). It has recently been demonstrated, however, that C-terminal forms of OBP with various amounts of N-terminal sequence behave differently during infection, with some acting as inhibitors of viral replication and others actually increasing viral replication (9). Indeed, C-terminal fragments of OBP of similar size to OBPC-1 were not able to bind origin sequences in this study (9). Therefore, it is unclear if OBPC-1 is able to bind to origins. We are in the process of determining if OBPC-1 is able to bind origin sequences to begin to understand what role OBPC-1 may play during viral infection.

In the course of attempts to express OBPC-1 in the absence of OBP, we have demonstrated that OBPC-1 is a cathepsin B-mediated cleavage product of OBP. As OBP is a nuclear protein while cathepsin B is most commonly a lysosomal protein, it is difficult to imagine how cathepsin B directly cleaves OBP. However, cathepsin B and cathepsin B activity have been identified in the nuclei of multiple cell types (40, 41, 48, 54). Overexpression of OBP is detrimental to viral DNA synthesis and viral plaque formation (2, 29, 32, 52, 55). Therefore, this cleavage event may play a role in regulating the levels of OBP in the nucleus during viral infection, possibly by down-regulating the initiator function of OBP and thus down-regulating viral DNA replication. Consequently, it will be of interest to study the replication kinetics of a virus that does not express OBPC-1.

To characterize an OBPC-1 null virus in cell culture and in mice, identification of the cathepsin B cleavage site of OBP will be necessary. Unfortunately, cathepsin B cleavage sites are very poorly defined. To date, only six known sites have been mapped (26), and attempts to identify a consensus sequence for cleavage have been unsuccessful. This may be because cleavage likely occurs at a site distinct from the cathepsin B binding site and may be more dependent on the secondary structure of the protein than on a specific amino acid sequence (26). Indeed, while the cathepsin B cleavage site of sphingosine kinase 1 has been identified, mutagenesis of the P1, P2, and P1' amino acids did not eliminate cathepsin B cleavage of the protein (58). These complications will make identification of the cathepsin B cleavage site in OBP and elimination of cathepsin B cleavage of OBP very difficult, even using protein-sequencing techniques. An available cathepsin B cleavage site prediction program (26) predicted a cathepsin B cleavage site at amino acids 479 to 484 of OBP. However, mutagenesis of this region did not eliminate OBP cleavage (data not shown). Furthermore, the N- and C-terminal products of cathepsin B cleavage of OBP tend to coimmunoprecipitate (data not shown). Due to the similarities in sizes of these N- and C-terminal fragments, it has been difficult to separate them by gel electrophoresis. This has complicated attempts to analyze the start site of OBPC-1 by mass spectrometry and N-terminal sequencing.

The aim of this study was to begin to study the two C-terminal forms of OBP, OBPC-1 and OBPC-2, separately; however, the possibility remains that these two proteins share similar roles during viral replication. It will be necessary to eliminate the expression of both proteins to further test this hypothesis. These studies are the subject of a forthcoming manuscript.

What, then, may be the function of the cleavage of OBP to yield OBPC-1 and OBPN? As noted above, in order for stage II origin-independent DNA replication to begin, OBP's initiator function must be eliminated, or OBP must be removed from viral origins and the initiator complex degraded or otherwise inactivated in order to block the initiation of origin-dependent viral DNA replication (stage I) (31, 33). Intriguingly, cathepsin B cleavage of OBP is dependent upon viral DNA synthesis. We hypothesize, therefore, that the process of viral DNA replication itself, or a late function of the virus, results in a change in the conformation or modification of OBP (possibly by phosphorylation or ubiquitination). This change may result in the release of OBP from the origins, rendering it available for cleavage by cathepsin B. Alternatively, cleavage of OBP may occur while OBP is still bound to the origin. This cleavage event may have two distinct effects on the regulation of OBP activity by (i) rendering the initiator protein inactive and (ii) producing a protein that might bind to origins, inhibiting new rounds of OBP-dependent viral DNA replication. Further study of the regulation of this cleavage event should shed light on the regulation of OBP function during viral DNA replication.

 
We thank Sandra Weller for the kind gift of the hr94 virus and 2B.11 cells and David Knipe for the gift of the 39S antibody. We thank members of the Schaffer laboratory for valuable ideas and thoughtful discussions.

This work was supported by research grant RO1 AI28537 from the National Institute of Allergy and Infectious Diseases, National Institutes of Health.

 
* Corresponding author. Mailing address: Department of Medicine, Harvard Medical School at the Beth Israel Deaconess Medical Center, 330 Brookline Avenue, RN 123, Boston, MA 02215. Phone: (617) 667-2958. Fax: (617) 667-8540. E-mail: pschaffe{at}bidmc.harvard.edu

Published ahead of print on 6 June 2007.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

1
1. Baradaran, K., C. E. Dabrowski, and P. A. Schaffer. 1994. Transcriptional analysis of the region of the herpes simplex virus type 1 genome containing the UL8, UL9, and UL10 genes and identification of a novel delayed-early gene product, OBPC. J. Virol. 68:4251-4261.[Abstract/Free Full Text]
2
2. Baradaran, K., M. A. Hardwicke, C. E. Dabrowski, and P. A. Schaffer. 1996. Properties of the novel herpes simplex virus type 1 origin binding protein, OBPC. J. Virol. 70:5673-5679.[Abstract/Free Full Text]
3
3. Barker, D. E., and B. Roizman. 1992. The unique sequence of the herpes simplex virus 1 L component contains an additional translated open reading frame designated UL49.5. J. Virol. 66:562-566.[Abstract/Free Full Text]
4
4. Blumel, J., and B. Matz. 1995. Thermosensitive UL9 gene function is required for early stages of herpes simplex virus type 1 DNA synthesis. J. Gen. Virol. 76:3119-3124.[Abstract/Free Full Text]
5
5. Boehmer, P. E., M. C. Craigie, N. D. Stow, and I. R. Lehman. 1994. Association of origin binding protein and single strand DNA-binding protein, ICP8, during herpes simplex virus type 1 DNA replication in vivo. J. Biol. Chem. 269:29329-29334.[Abstract/Free Full Text]
6
6. Bronstein, J. C., S. K. Weller, and P. C. Weber. 1997. The product of the UL12.5 gene of herpes simplex virus type 1 is a capsid-associated nuclease. J. Virol. 71:3039-3047.[Abstract]
7
7. Carter, K. L., and B. Roizman. 1996. The promoter and transcriptional unit of a novel herpes simplex virus 1 alpha gene are contained in, and encode a protein in frame with, the open reading frame of the alpha 22 gene. J. Virol. 70:172-178.[Abstract]
8
8. Chattopadhyay, S., Y. Chen, and S. K. Weller. 2006. The two helicases of herpes simplex virus type 1 (HSV-1). Front Biosci. 11:2213-2223.[Medline]
9
9. Chattopadhyay, S., and S. K. Weller. 2006. DNA binding activity of the herpes simplex virus type 1 origin binding protein, UL9, can be modulated by sequences in the N terminus: correlation between transdominance and DNA binding. J. Virol. 80:4491-4500.[Abstract/Free Full Text]
10
10. Datta, I., S. Sau, A. K. Sil, and N. C. Mandal. 2005. The bacteriophage lambda DNA replication protein P inhibits the oriC DNA- and ATP-binding functions of the DNA replication initiator protein DnaA of Escherichia coli. J Biochem. Mol. Biol. 38:97-103.[Medline]
11
11. Dettwiler, S., J. Rommelaere, and J. P. Nuesch. 1999. DNA unwinding functions of minute virus of mice NS1 protein are modulated specifically by the lambda isoform of protein kinase C. J. Virol. 73:7410-7420.[Abstract/Free Full Text]
12
12. Eisenberg, S., and R. Ascarelli. 1981. The A* protein of phi X174 is an inhibitor of DNA replication. Nucleic Acids Res. 9:1991-2001.[Abstract/Free Full Text]
13
13. Eom, C. Y., W. D. Heo, M. L. Craske, T. Meyer, and I. R. Lehman. 2004. The neural F-box protein NFB42 mediates the nuclear export of the herpes simplex virus type 1 replication initiator protein (UL9 protein) after viral infection. Proc. Natl. Acad. Sci. USA 101:4036-4040.[Abstract/Free Full Text]
14
14. Eom, C. Y., and I. R. Lehman. 2003. Replication-initiator protein (UL9) of the herpes simplex virus 1 binds NFB42 and is degraded via the ubiquitin-proteasome pathway. Proc. Natl. Acad. Sci. USA 100:9803-9807.[Abstract/Free Full Text]
15
15. Fulford, W., and P. Model. 1988. Bacteriophage f1 DNA replication genes. II. The roles of gene V protein and gene II protein in complementary strand synthesis. J. Mol. Biol. 203:39-48.[CrossRef][Medline]
16
16. Fulford, W., and P. Model. 1988. Regulation of bacteriophage f1 DNA replication. I. New functions for genes II and X. J. Mol. Biol. 203:49-62.[CrossRef][Medline]
17
17. Garber, D. A., S. M. Beverley, and D. M. Coen. 1993. Demonstration of circularization of herpes simplex virus DNA following infection using pulsed-field gel electrophoresis. Virology 197:459-462.[CrossRef][Medline]
18
18. Georgopoulou, U., A. Michaelidou, B. Roizman, and P. Mavromara-Nazos. 1993. Identification of a new transcriptional unit that yields a gene product within the unique sequences of the short component of the herpes simplex virus 1 genome. J. Virol. 67:3961-3968.[Abstract/Free Full Text]
19
19. Gottlieb, J., A. I. Marcy, D. M. Coen, and M. D. Challberg. 1990. The herpes simplex virus type 1 UL42 gene product: a subunit of DNA polymerase that functions to increase processivity. J. Virol. 64:5976-5987.[Abstract/Free Full Text]
20
20. Hardwicke, M. A., and P. A. Schaffer. 1997. Differential effects of nerve growth factor and dexamethasone on herpes simplex virus type 1 oriL- and oriS-dependent DNA replication in PC12 cells. J. Virol. 71:3580-3587.[Abstract]
21
21. Isler, J. A., and P. A. Schaffer. 2001. Origin binding protein-containing protein-DNA complex formation at herpes simplex virus type 1 oriS: role in oriS-dependent DNA replication. J. Virol. 75:6808-6816.[Abstract/Free Full Text]
22
22. Isler, J. A., and P. A. Schaffer. 2001. Phosphorylation of the herpes simplex virus type 1 origin binding protein. J. Virol. 75:628-637.[Abstract/Free Full Text]
23
23. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685.[CrossRef][Medline]
24
24. Lehman, I. R., and P. E. Boehmer. 1999. Replication of herpes simplex virus DNA. J. Biol. Chem. 274:28059-28062.[Free Full Text]
25
25. Liu, F. Y., and B. Roizman. 1991. The promoter, transcriptional unit, and coding sequence of herpes simplex virus 1 family 35 proteins are contained within and in frame with the UL26 open reading frame. J. Virol. 65:206-212.[Abstract/Free Full Text]
26
26. Lohmuller, T., D. Wenzler, S. Hagemann, W. Kiess, C. Peters, T. Dandekar, and T. Reinheckel. 2003. Toward computer-based cleavage site prediction of cysteine endopeptidases. Biol. Chem. 384:899-909.[CrossRef][Medline]
27
27. Ma, T., N. Zou, B. Y. Lin, L. T. Chow, and J. W. Harper. 1999. Interaction between cyclin-dependent kinases and human papillomavirus replication-initiation protein E1 is required for efficient viral replication. Proc. Natl. Acad. Sci. USA 96:382-387.[Abstract/Free Full Text]
28
28. Malcles, M. H., N. Cueille, F. Mechali, O. Coux, and C. Bonne-Andrea. 2002. Regulation of bovine papillomavirus replicative helicase E1 by the ubiquitin-proteasome pathway. J. Virol. 76:11350-11358.[Abstract/Free Full Text]
29
29. Malik, A. K., R. Martinez, L. Muncy, E. P. Carmichael, and S. K. Weller. 1992. Genetic analysis of the herpes simplex virus type 1 UL9 gene: isolation of a LacZ insertion mutant and expression in eukaryotic cells. Virology 190:702-715.[CrossRef][Medline]
30
30. Malik, A. K., and S. K. Weller. 1996. Use of transdominant mutants of the origin-binding protein (UL9) of herpes simplex virus type 1 to define functional domains. J. Virol. 70:7859-7866.[Abstract]
31
31. Marintcheva, B., and S. K. Weller. 2001. A tale of two HSV-1 helicases: roles of phage and animal virus helicases in DNA replication and recombination. Prog. Nucleic Acid Res. Mol. Biol. 70:77-118.[Medline]
32
32. Marintcheva, B., and S. K. Weller. 2003. Existence of transdominant and potentiating mutants of UL9, the herpes simplex virus type 1 origin-binding protein, suggests that levels of UL9 protein may be regulated during infection. J. Virol. 77:9639-9651.[Abstract/Free Full Text]
33
33. Marintcheva, B., and S. K. Weller. 2001. Residues within the conserved helicase motifs of UL9, the origin-binding protein of herpes simplex virus-1, are essential for helicase activity but not for dimerization or origin binding activity. J. Biol. Chem. 276:6605-6615.[Abstract/Free Full Text]
34
34. Martinez, R., J. N. Goldstein, and S. K. Weller. 2002. The product of the UL12.5 gene of herpes simplex virus type 1 is not essential for lytic viral growth and is not specifically associated with capsids. Virology 298:248-257.[CrossRef][Medline]
35
35. McLean, G. W., A. P. Abbotts, M. E. Parry, H. S. Marsden, and N. D. Stow. 1994. The herpes simplex virus type 1 origin-binding protein interacts specifically with the viral UL8 protein. J. Gen. Virol. 75:2699-2706.[Abstract/Free Full Text]
36
36. Monahan, S. J., L. A. Grinstead, W. Olivieri, and D. S. Parris. 1998. Interaction between the herpes simplex virus type 1 origin-binding and DNA polymerase accessory proteins. Virology 241:122-130.[CrossRef][Medline]
37
37. Olivo, P. D., N. J. Nelson, and M. D. Challberg. 1988. Herpes simplex virus DNA replication: the UL9 gene encodes an origin-binding protein. Proc. Natl. Acad. Sci. USA 85:5414-5418.[Abstract/Free Full Text]
38
38. Olivo, P. D., N. J. Nelson, and M. D. Challberg. 1989. Herpes simplex virus type 1 gene products required for DNA replication: identification and overexpression. J. Virol. 63:196-204.[Abstract/Free Full Text]
39
39. Perry, H. C., D. J. Hazuda, and W. L. McClements. 1993. The DNA binding domain of herpes simplex virus type 1 origin binding protein is a transdominant inhibitor of virus replication. Virology 193:73-79.[CrossRef][Medline]
40
40. Pietras, R. J., and J. A. Roberts. 1981. Cathepsin B-like enzymes. Subcellular distribution and properties in neoplastic and control cells from human ectocervix. J. Biol. Chem. 256:8536-8544.[Free Full Text]
41
41. Pizzorno, M. C. 2001. Nuclear cathepsin B-like protease cleaves transcription factor YY1 in differentiated cells. Biochim. Biophys. Acta 1536:31-42.[Medline]
42
42. Poffenberger, K. L., and B. Roizman. 1985. A noninverting genome of a viable herpes simplex virus 1: presence of head-to-tail linkages in packaged genomes and requirements for circularization after infection. J. Virol. 53:587-595.[Abstract/Free Full Text]
43
43. Poon, A. P., and B. Roizman. 2005. Herpes simplex virus 1 ICP22 regulates the accumulation of a shorter mRNA and of a truncated US3 protein kinase that exhibits altered functions. J. Virol. 79:8470-8479.[Abstract/Free Full Text]
44
44. Prives, C. 1990. The replication functions of SV40 T antigen are regulated by phosphorylation. Cell 61:735-738.[CrossRef][Medline]
45
45. Reuven, N. B., S. Antoku, and S. K. Weller. 2004. The UL12.5 gene product of herpes simplex virus type 1 exhibits nuclease and strand exchange activities but does not localize to the nucleus. J. Virol. 78:4599-4608.[Abstract/Free Full Text]
46
46. Reuven, N. B., A. E. Staire, R. S. Myers, and S. K. Weller. 2003. The herpes simplex virus type 1 alkaline nuclease and single-stranded DNA binding protein mediate strand exchange in vitro. J. Virol. 77:7425-7433.[Abstract/Free Full Text]
47
47. Reuven, N. B., and S. K. Weller. 2005. Herpes simplex virus type 1 single-strand DNA binding protein ICP8 enhances the nuclease activity of the UL12 alkaline nuclease by increasing its processivity. J. Virol. 79:9356-9358.[Abstract/Free Full Text]
48
48. Riccio, M., R. Di Giaimo, S. Pianetti, P. P. Palmieri, M. Melli, and S. Santi. 2001. Nuclear localization of cystatin B, the cathepsin inhibitor implicated in myoclonus epilepsy (EPM1). Exp. Cell Res. 262:84-94.[CrossRef][Medline]
49
49. Roizman, B., and D. M. Knipe. 2001. Herpes simplex viruses and their replication, p. 1123-1184. In D. M. Knipe and P. M. Howley (ed.), Fundamental virology, 4th ed. Lippincott Williams & Wilkins, Philadelphia, PA.
50
50. Sacks, W. R., C. C. Greene, D. P. Aschman, and P. A. Schaffer. 1985. Herpes simplex virus type 1 ICP27 is an essential regulatory protein. J. Virol. 55:796-805.[Abstract/Free Full Text]
51
51. Schildgen, O., S. Graper, J. Blumel, and B. Matz. 2005. Genome replication and progeny virion production of herpes simplex virus type 1 mutants with temperature-sensitive lesions in the origin-binding protein. J. Virol. 79:7273-7278.[Abstract/Free Full Text]
52
52. Skaliter, R., and I. R. Lehman. 1994. Rolling circle DNA replication in vitro by a complex of herpes simplex virus type 1-encoded enzymes. Proc. Natl. Acad. Sci. USA 91:10665-10669.[Abstract/Free Full Text]
53
53. Skaliter, R., A. M. Makhov, J. D. Griffith, and I. R. Lehman. 1996. Rolling circle DNA replication by extracts of herpes simplex virus type 1-infected human cells. J. Virol. 70:1132-1136.[Abstract]
54
54. Spiess, E., A. Bruning, S. Gack, B. Ulbricht, H. Spring, G. Trefz, and W. Ebert. 1994. Cathepsin B activity in human lung tumor cell lines: ultrastructural localization, pH sensitivity, and inhibitor status at the cellular level. J. Histochem. Cytochem. 42:917-929.[Abstract]
55
55. Stow, N. D. 1992. Herpes simplex virus type 1 origin-dependent DNA replication in insect cells using recombinant baculoviruses. J. Gen. Virol. 73:313-321.[Abstract/Free Full Text]
56
56. Stow, N. D., O. Hammarsten, M. I. Arbuckle, and P. Elias. 1993. Inhibition of herpes simplex virus type 1 DNA replication by mutant forms of the origin-binding protein. Virology 196:413-418.[CrossRef][Medline]
57
57. Strang, B. L., and N. D. Stow. 2005. Circularization of the herpes simplex virus type 1 genome upon lytic infection. J. Virol. 79:12487-12494.[Abstract/Free Full Text]
58
58. Taha, T. A., M. El-Alwani, Y. A. Hannun, and L. M. Obeid. 2006. Sphingosine kinase-1 is cleaved by cathepsin B in vitro: identification of the initial cleavage sites for the protease. FEBS Lett. 580:6047-6054.[CrossRef][Medline]
59
59. Wang, E. H., S. Bhattacharyya, and C. Prives. 1993. The replication functions of polyomavirus large tumor antigen are regulated by phosphorylation. J. Virol. 67:6788-6796.[Abstract/Free Full Text]
60
60. Wegrzyn, A., A. Czyz, M. Gabig, and G. Wegrzyn. 2000. ClpP/ClpX-mediated degradation of the bacteriophage lambda O protein and regulation of lambda phage and lambda plasmid replication. Arch. Microbiol. 174:89-96.[CrossRef][Medline]
61
61. Wegrzyn, G., A. Pawlowicz, and K. Taylor. 1992. Stability of coliphage lambda DNA replication initiator, the lambda O protein. J. Mol. Biol. 226:675-680.[CrossRef][Medline]
62
62. Wegrzyn, G., and K. Taylor. 1992. Inheritance of the replication complex by one of two daughter copies during lambda plasmid replication in Escherichia coli. J. Mol. Biol. 226:681-688.[CrossRef][Medline]
63
63. Weller, S. K. 1995. The DNA provirus: Howard Temin's scientific legacy. American Society for Microbiology, Washington, DC.
64
64. Yu, D., and S. K. Weller. 1998. Genetic analysis of the UL 15 gene locus for the putative terminase of herpes simplex virus type 1. Virology 243:32-44.[CrossRef][Medline]
65
65. Zanardi, T. A., C. M. Stanley, B. M. Saville, S. M. Spacek, and M. R. Lentz. 1997. Modulation of bovine papillomavirus DNA replication by phosphorylation of the viral E1 protein. Virology 228:1-10.[CrossRef][Medline]
66
66. Zhang, X., S. Efstathiou, and A. Simmons. 1994. Identification of novel herpes simplex virus replicative intermediates by field inversion gel electrophoresis: implications for viral DNA amplification strategies. Virology 202:530-539.[CrossRef][Medline]

---

Journal of Virology, September 2007, p. 9175-9182, Vol. 81, No. 17
0022-538X/07/$08.00+0     doi:10.1128/JVI.00676-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

This article has been cited by other articles:

• Chattopadhyay, S., Weller, S. K. (2007). Direct Interaction between the N- and C-Terminal Portions of the Herpes Simplex Virus Type 1 Origin Binding Protein UL9 Implies the Formation of a Head-to-Tail Dimer. J. Virol. 81: 13659-13667 [Abstract] [Full Text]  
• Link, M. A., Schaffer, P. A. (2007). Herpes Simplex Virus Type 1 C-Terminal Variants of the Origin Binding Protein (OBP), OBPC-1 and OBPC-2, Cooperatively Regulate Viral DNA Levels In Vitro, and OBPC-2 Affects Mortality in Mice. J. Virol. 81: 10699-10711 [Abstract] [Full Text]  

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Link, M. A. Articles by Schaffer, P. A. Search for Related Content PubMed PubMed Citation Articles by Link, M. A. Articles by Schaffer, P. A.