Characterization of the herpes simplex virus origin binding protein interaction with OriS.

The origin binding protein (OBP) of herpes simplex virus (HSV), which is essential for viral DNA replication, binds specifically to sequences within the viral replication origin(s) (for a review, see Challberg, M.D., and Kelly, T. J. (1989) Annu. Rev. Biochem. 58, 671-717). Using either a COOH-terminal OBP protein A fusion or the full-length protein, each expressed in Escherichia coli, we investigated the interaction of OBP with one HSV origin, OriS. Binding of OBP to a set of binding site variant sequences demonstrates that the 10-base pair sequence, 5' CGTTCGCACT 3', comprises the OBP-binding site. This sequence must be presented in the context of at least 15 total base pairs for high affinity binding, Ka = approximately 0.3 nM. Single base pair mutations in the central CGC sequence lower the affinity by several orders of magnitude, whereas a substitution at any of the other seven positions reduces the affinity by 10-fold or less. OBP binds with high affinity to duplex DNA containing mismatched base pairs. This property is exploited to analyze OBP binding to DNA heteroduplexes containing singly substituted mutant and wild-type DNA strands. For positions 2, 3, 5, 6, 7, 8, and 9, substitutions are tolerated on one or the other DNA strand, indicating that base-mediated interactions are limited to one base of each pair. For both Boxes I and II, these interactions are localized to one face of the DNA helix, forming a recognition surface in the major groove. In OriS, the 31 base pairs which separate Boxes I and II orient the two interaction surfaces to the same side of the DNA.     

Expression of the varicella-zoster virus origin-binding protein and analysis of its site-specific DNA-binding properties.

The varicella-zoster virus (VZV) genome contains homologs to each of the seven herpes simplex virus (HSV) genes that are required for viral DNA synthesis. VZV gene 51 is homologous to HSV UL9, which encodes an origin of DNA replication binding protein (OBP). It was previously shown, by using a protein A fusion protein, that the product of gene 51 is a site-specific DNA-binding protein which binds to sequences within the VZV origin (Stow et al., Virology 177:570-577, 1990). In this report, gene 51 was expressed in an in vitro translation system. Rabbit antiserum raised against the carboxyl-terminal 20 amino acids was used to confirm expression of the full-length gene 51 protein, and site-specific DNA-binding activity was demonstrated in a gel retardation assay. The origin-binding domain was located within a 263-amino-acid region of the carboxyl terminus by using a series of deletion mutants. The affinity of binding of the VZV OBP to the three binding sites in the VZV origin was found to be similar. In addition, as with UL9, a CGC triplet within a 10-bp consensus sequence is critical to the interaction between the OBP and the origin. The HSV and VZV OBPs, therefore, appear to have virtually identical recognition sequences despite only 33% identity and 44% similarity in the primary structure of their site-specific DNA-binding domains.     

Sequence requirements for interaction of human herpesvirus 7 origin binding protein with the origin of lytic replication

As do human herpesvirus 6 variants A and B (HHV-6A and -6B), HHV-7 encodes a homolog of the alphaherpesvirus origin binding protein (OBP), which binds at sites in the origin of lytic replication (oriLyt) to initiate DNA replication. In this study, we sought to characterize the interaction of the HHV-7 OBP (OBP(H7)) with its cognate sites in the 600-bp HHV-7 oriLyt. We expressed the carboxyl-terminal domain of OBP(H7) and found that amino acids 484 to 787 of OBP(H7) were sufficient for DNA binding activity by electrophoretic mobility shift analysis. OBP(H7) has one high-affinity binding site (OBP-2) located on one flank of an AT-rich spacer element and a low-affinity site (OBP-1) on the other. This is in contrast to the HHV-6B OBP (OBP(H6B)), which binds with similar affinity to its two cognate OBP sites in the HHV-6B oriLyt. The minimal recognition element of the OBP-2 site was mapped to a 14-bp sequence. The OBP(H7) consensus recognition sequence of the 9-bp core, BRTYCWCCT (where B is a T, G, or C; R is a G or A; Y is a T or C; and W is a T or A), overlaps with the OBP(H6B) consensus YGWYCWCCY and establishes YCWCC as the roseolovirus OBP core recognition sequence. Heteroduplex analysis suggests that OBP(H7) interacts along one face of the DNA helix, with the major groove, as do OBP(H6B) and herpes simplex virus type 1 OBP. Together, these results illustrate both conserved and divergent DNA binding properties between OBP(H7) and OBP(H6B).     

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

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.     

Structural elements required for the cooperative binding of the herpes simplex virus origin binding protein to oriS reside in the N-terminal part of the protein.

The origin binding protein (OBP) of herpes simplex virus type 1 is required to activate a viral origin of replication in vivo. We have used intact OBP as well as a truncated form of the protein expressed in Escherichia coli to investigate the protein-protein interactions, as well as the protein-DNA interactions involved in the formation of a nucleoprotein complex at a viral origin of replication (oriS) in vitro. The salient findings demonstrate that the N-terminal part of OBP is required for the cooperative binding of OBP to three sites (boxes I, II, and III) within oriS. A detailed model for the interaction of OBP with the viral origins of replication oriS and oriL is presented.     

Analysis of the herpes simplex virus type 1 OriS sequence: mapping of functional domains.

The herpes simplex virus type 1 (HSV-1) OriS region resides within a 90-bp sequence that contains two binding sites for the origin-binding protein (OBP), designated sites I and II. A third presumptive OBP-binding site (III) within OriS has strong sequence similarity to sites I and II, but no sequence-specific OBP binding has yet been demonstrated at this site. We have generated mutations in sites I, II, and III and determined their replication efficiencies in a transient in vivo assay in the presence of a helper virus. Mutations in any one of the sites reduced DNA replication significantly. To study the role of OriS sequence elements in site I and the presumptive site III in DNA replication, we have also generated a series of mutations that span from site I across the presumptive binding site III. These mutants were tested for their ability to replicate and for the ability to bind OBP by using gel shift analyses. The results indicate that mutations across site I drastically reduce DNA replication. Triple-base-pair substitution mutations that fall within the crucial OBP-binding domain, 5'-YGYTCGCACT-3' (where Y represents C or T), show a reduced level of OBP binding and DNA replication. Substitution mutations in site I that are outside this crucial binding sequence show a more detrimental effect on DNA replication than on OBP binding. This suggests that these sequences are required for initiation of DNA replication but are not critical for OBP binding. Mutations across the presumptive OBP-binding site III also resulted in a loss in efficiency of DNA replication. These mutations influenced OBP binding to OriS in gel shift assays, even though the mutated sequences are not contained within known OBP-binding sites. Replacement of the wild-type site III with a perfect OBP-binding site I results in a drastic reduction of DNA replication. Thus, our DNA replication assays and in vitro DNA-binding studies suggest that the binding of the origin sequence by OBP is not the only determining factor for initiation of DNA replication in vivo.     

The origin binding protein of herpes simplex virus 1 binds cooperatively to the viral origin of replication oris

The origin binding protein (OBP) or herpes simplex virus 1 has been expressed in Escherichia coli and used to study the role of multiple OBP binding sites in the herpes simplex virus #1 origin of replication, oris. Our results showed that the sequence CGTTCGCACTT was required for the binding of OBP to duplex DNA with high affinity. The minimal oris contains three repeats of this sequence or close derivatives thereof. Filter binding experiments have demonstrated that specific binding occurs to two of these repeats, box I and box II. An investigation using the DNase I footprinting technique revealed that the binding of OBP to box I and box II was cooperative and led to the formation of a highly organized complex in which the entire oris sequence was induced. We observed furthermore that the AT-rich sequence of the oris dyad was readily accessible to macromolecules even in the OBP.oris complex. The DNase I cleavage pattern of this sequence was, however, altered radically, indicating that a significant conformational change had occurred. A tentative structural model for the OBP-oris interaction is discussed on the basis of these observations.     

Phosphorylation of the herpes simplex virus type 1 origin binding protein

The herpes simplex virus type 1 (HSV-1) origin binding protein (OBP), the product of the UL9 gene, is one of seven HSV-encoded proteins required for viral DNA replication. OBP performs multiple functions characteristic of a DNA replication initiator protein, including origin-specific DNA binding and ATPase and helicase activities, as well as the ability to interact with viral and cellular proteins involved in DNA replication. Replication initiator proteins in other systems, including those of other DNA viruses, are known to be regulated by phosphorylation; however, the role of phosphorylation in OBP function has been difficult to assess due to the low level of OBP expression in HSV-infected cells. Using a metabolic labeling and immunoprecipitation approach, we obtained evidence that OBP is phosphorylated during HSV-1 infection. Kinetic analysis of metabolically labeled cells indicated that the levels of OBP expression and phosphorylation increased at approximately 4 h postinfection. Notably, when expressed from a transfected plasmid, a recombinant baculovirus, or a recombinant adenovirus (AdOBP), OBP was phosphorylated minimally, if at all. In contrast, superinfection of AdOBP-infected cells with an OBP-null mutant virus increased the level of OBP phosphorylation approximately threefold, suggesting that HSV-encoded viral or HSV-induced cellular factors enhance the level of OBP phosphorylation. Using HSV mutants inhibited at sequential stages of the viral life cycle, we demonstrated that this increase in OBP phosphorylation is dependent on early protein synthesis and is independent of viral DNA replication. Based on gel mobility shift assays, phosphorylation does not appear to affect the ability of OBP to bind to the HSV origins.     

Functional properties of the herpes simplex virus type I origin-binding protein are controlled by precise interactions with the activated form of the origin of DNA replication

The herpes simplex virus, type I origin-binding protein, OBP, is a superfamily II DNA helicase encoded by the UL9 gene. OBP binds in a sequence-specific and cooperative way to the viral origin of replication oriS. OBP may unwind partially and introduce a hairpin into the double-stranded origin of replication. The formation of the novel conformation referred to as oriS* also requires the single-stranded DNA-binding protein, ICP8, and ATP hydrolysis. OBP forms a stable complex with oriS*. The hairpin in oriS* provides a site for sequence-specific attachment, and a single-stranded region triggers ATP hydrolysis. Here we use Escherichia coli exonuclease I to map the binding of the C-terminal domain of OBP to the hairpin and the helicase domains to the single-stranded tail. The helicase domains cover a stretch of 23 nucleotides of single-stranded DNA. Using streptavidin-coated magnetic beads, we show that OBP may bind two copies of double-stranded DNA (one biotin-labeled and the other one radioactively labeled) but only one copy of oriS*. It is the length of the single-stranded tail that determines the stoichiometry of OBP.DNA complexes. OBP interacts with the bases of the single-stranded tail, and ATP hydrolysis is triggered by position-specific interactions between OBP and bases in the single-stranded tail of oriS*.     

Constitutive binding of EBNA1 protein to the Epstein-Barr virus replication origin, oriP, with distortion of DNA structure during latent infection.

Replication of the circular, 170 kb genome of Epstein-Barr virus (EBV) during latent infection is performed by the cellular replication machinery under cell-cycle control. A single viral protein, EBNA1, directs the cellular replication apparatus to initiate replication within the genetically defined replication origin, oriP, at a cluster of four EBNA1 binding sites, referred to here as the physical origin of bidirectional replication, or OBR. A second cluster of EBNA1 binding sites within oriP, the 30 bp repeats, serves an essential role as a replication enhancer and also provides a distinct episome maintenance function that is unrelated to replication. We examined the functional elements of oriP for binding by EBNA1 and possibly other proteins in proliferating Raji cells by generating in vivo footprints using two reagents, dimethylsulfate (DMS) and KMnO4. We also employed deoxyribonuclease I (DNase I) with permeabilized cells. The in vivo and permeabilized cell footprints at the EBNA1 binding sites, particularly those obtained using DMS, gave strong evidence that all of these sites are bound by EBNA1 in asynchronously dividing cells. No consistent evidence was found to suggest binding by other proteins at any other sites within the functional regions of oriP. Thymines at symmetrical positions of the OBR within oriP were oxidized when cells were treated with permanganate, suggestive of bends or other distortions of DNA structure at these positions; binding of EBNA1 in vitro to total DNA from Raji cells induced reactivity to permanganate at identical positions. The simplest interpretation of the results, which were obtained using asynchronously dividing cells, is that EBNA1 binds to its sites at oriP and holds the OBR in a distorted conformation throughout most of the cell cycle, implying that replication is initiated by a cellular mechanism and is not limited by an availability of EBNA1 for binding to oriP.     

Cellular protein interactions with herpes simplex virus type 1 oriS.

The herpes simplex virus type 1 (HSV-1) origin of DNA replication, oriS, contains an AT-rich region and three highly homologous sequences, sites I, II, and III, identified as binding sites for the HSV-1 origin-binding protein (OBP). In the present study, interactions between specific oriS DNA sequences and proteins in uninfected cell extracts were characterized. The formation of one predominant protein-DNA complex, M, was demonstrated in gel shift assays following incubation of uninfected cell extracts with site I DNA. The cellular protein(s) that comprises complex M has been designated origin factor I (OF-I). The OF-I binding site was shown to partially overlap the OBP binding site within site I. Complexes with mobilities indistinguishable from that of complex M also formed with site II and III DNAs in gel shift assays. oriS-containing plasmid DNA mutated in the OF-I binding site exhibited reduced replication efficiency in transient assays, demonstrating a role for this site in oriS function. The OF-I binding site is highly homologous to binding sites for the cellular CCAAT DNA-binding proteins. The binding site for the CCAAT protein CP2 was found to compete for OF-I binding to site I DNA. These studies support a model involving the participation of cellular proteins in the initiation of HSV-1 DNA synthesis at oriS.     

Targeting Holliday junctions by origin DNA-binding protein of herpes simplex virus type 1

In the present paper, the interactions of the origin binding protein (OBP) of herpes simplex virus type 1 (HSV1) with synthetic four-way Holliday junctions (HJs) were studied using electrophoresis mobility shift assay and the FRET method and compared with the interactions of the protein with duplex and single-stranded DNAs. It has been found that OBP exhibits a strong preference for binding to four-way and three-way DNA junctions and possesses much lower affinities to duplex and single-stranded DNAs. The protein forms three types of complexes with HJs. It forms complexes I and II which are reminiscent of the tetramer and octamer complexes with four-way junction of HJ-specific protein RuvA of Escherichia coli. The binding approaches saturation level when two OBP dimers are bound per junction. In the presence of Mg²⁺ ions (≥2 mM) OBP also interacts with HJ in the stacked arm form (complex III). In the presence of 5 mM ATP and 10 mM Mg²⁺ ions OBP catalyzes processing of the HJ in which one of the annealed oligonucleotides has a 3′-terminal tail containing 20 unpaired thymine residues. The observed preference of OBP for binding to the four-way DNA junctions provides a basis for suggestion that OBP induces large DNA structural changes upon binding to Box I and Box II sites in OriS. These changes involve the bending and partial melting of the DNA at A+T-rich spacer and also include the formation of HJ containing Box I and Box II inverted repeats and flanking DNA sequences.     

Kaposi's sarcoma-associated herpesvirus lytic origin (ori-Lyt)-dependent DNA replication: identification of the ori-Lyt and association of K8 bZip protein with the origin

Herpesviruses utilize different origins of replication during lytic versus latent infection. Latent DNA replication depends on host cellular DNA replication machinery, whereas lytic cycle DNA replication requires virally encoded replication proteins. In lytic DNA replication, the lytic origin (ori-Lyt) is bound by a virus-specified origin binding protein (OBP) that recruits the core replication machinery. In this report, we demonstrated that DNA sequences in two noncoding regions of the Kaposi's sarcoma-associated herpesvirus (KSHV) genome, between open reading frames (ORFs) K4.2 and K5 and between K12 and ORF71, are able to serve as origins for lytic cycle-specific DNA replication. The two ori-Lyt domains share an almost identical 1,153-bp sequence and a 600-bp downstream GC-rich repeat sequence, and the 1.7-kb DNA sequences are sufficient to act as a cis signal for replication. We also showed that an AT-palindromic sequence in the ori-Lyt domain is essential for the DNA replication. In addition, a virally encoded bZip protein, namely K8, was found to bind to a DNA sequence within the ori-Lyt by using a DNA binding site selection assay. The binding of K8 to this region was confirmed in cells by using a chromatin immunoprecipitation method. Further analysis revealed that K8 binds to an extended region, and the entire region is 100% conserved between two KSHV ori-Lyt's. K8 protein displays significant similarity to the Zta protein of Epstein-Barr virus (EBV), which is a known OBP of EBV. This notion, together with the ability of K8 to bind to the KSHV ori-Lyt, suggests that K8 may function as an OBP in KSHV.     

Specific interaction between the initiator protein (Rep) and origin of plasmid ColE2-P9

The replication initiator protein (Rep) of plasmid ColE2-P9 (ColE2) is multifunctional. We are interested in how Rep binds to the origin (Ori) to perform various functions. We used the wild type and variants of Rep to study the Rep-Ori interaction by both in vitro and in vivo approaches, including biochemical analyses of protein-DNA interactions and an in vivo replication assay. We identified three regions (I, II, and III) of Rep, located in the C-terminal half, and three corresponding binding sites (I, II, and III) in Ori which are important for Rep-Ori interaction. We showed that region I, containing a putative helix-turn-helix motif, is necessary and sufficient for specific Ori recognition, interacting with site I of the origin DNA from the major groove. Region II interacts with site II of the origin DNA, from the adjacent minor groove in the left half of Ori, and region III interacts with site III, next to the template sequence for primer synthesis, which is one and one-half turn apart from site I on the opposite surface of the origin DNA. A putative linker region located between the two DNA binding domains (regions II and III) was identified, which might provide Rep an extended conformation suitable for binding to the two separate sites in Ori. Based on the results presented in this paper, we propose a model for Rep-Ori interaction in which Rep binds to Ori as a monomer.     

Universal minicircle sequence-binding protein, a sequence-specific DNA-binding protein that recognizes the two replication origins of the kinetoplast DNA minicircle

Replication of the kinetoplast DNA minicircle lagging (heavy (H))-strand initiates at, or near, a unique hexameric sequence (5'-ACGCCC-3') that is conserved in the minicircles of trypanosomatid species. A protein from the trypanosomatid Crithidia fasciculata binds specifically a 14-mer sequence, consisting of the complementary strand hexamer and eight flanking nucleotides at the H-strand replication origin. This protein was identified as the previously described universal minicircle sequence (UMS)-binding protein (UMSBP) (Tzfati, Y., Abeliovich, H., Avrahami, D., and Shlomai, J. (1995) J. Biol. Chem. 270, 21339-21345). This CCHC-type zinc finger protein binds the single-stranded form of both the 12-mer (UMS) and 14-mer sequences, at the replication origins of the minicircle L-strand and H-strand, respectively. The attribution of the two different DNA binding activities to the same protein relies on their co-purification from C. fasciculata cell extracts and on the high affinity of recombinant UMSBP to the two origin-associated sequences. Both the conserved H-strand hexamer and its flanking nucleotides at the replication origin are required for binding. Neither the hexameric sequence per se nor this sequence flanked by different sequences could support the generation of specific nucleoprotein complexes. Stoichiometry analysis indicates that each UMSBP molecule binds either of the two origin-associated sequences in the nucleoprotein complex but not both simultaneously.     

Identification of the replication-associated protein binding domain within the intergenic region of tomato leaf curl geminivirus.

The geminiviral replication-associated protein (Rep) is the only viral protein required for viral DNA replication. Tomato leaf curl virus (TLCV) Rep was expressed in Escherichia coli as a histidine-tagged fusion protein and purified to homogeneity in non-denaturing form. The fusion protein was used in in vitro binding experiments to identify the Rep-binding elements within the origin of replication of TLCV. Electrophoretic mobility shift assays demonstrated that the Rep binds specifically to a 120 bp fragment within the TLCV intergenic region. Fine resolution of the binding regions within the 120 bp fragment, using DNase I footprinting, demonstrated two footprints covering the sequences GCAATTGGTGTCTCTCAA and TGAATCGGTGTCTGGGG containing a direct repeat of the motif GGTGTCT (underlined). Our results suggest that the repeated motif is involved in virus-specific Rep-binding, but may not constitute the entire binding element. This is the first demonstration of geminivirus sequence elements involved in Rep-binding by direct protein-DNA interaction assays.     

Replication enhancer-independent mutation increases the co-operativity with which an initiator protein binds its origin

The plus-strand replication origin of bacteriophage fl has a bipartite structure consisting of a required core origin region and an adjacent A + T-rich enhancer sequence that potentiates replication approximately 100-fold. The core origin binds the initiator protein (gpII) and the enhancer binds the Escherichia coli integration host factor (IHF). gpII binds the core origin in two steps, forming a binding intermediate (complex I) and a functional complex for nicking (complex II). We have used a double-label gel binding assay to determine the stoichiometry of the gpII-origin interaction. The results indicate that complex I contains two gpII molecules per origin, and complex II contains four gpII molecules per origin. Enhancer-independent mutations in gpII allow wild-type levels of replication in the absence of either the enhancer or IHF. We have examined the binding of an enhancer-independent gpII mutant (mp1) protein to the replication origin. The mp1 mutation in gpII (Met40----Ile) increases the co-operativity with which the protein binds to form complex II. In addition, the mutant gpII forms both complexes with a DNA fragment containing only two (beta-gamma) of the three repeats from the core origin sequence, while the wild-type protein forms only complex I with this fragment. We discuss how a mutation that increases the co-operativity of binding of an initiator protein might stimulate DNA replication.