Unwinding of the box I element of a herpes simplex virus type 1 origin by a complex of the viral origin binding protein, single-strand DNA binding protein, and single-stranded DNA

Single-stranded (ssDNA) DNA binding proteins (SSBs) bind preferentially ssDNA in stoichiometric quantities with respect to their substrate, displaying little sequence preference and no associated ATPase activity (11). The binding is typically cooperative, though the level of cooperativity varies widely. Much effort has been spent on elucidation of the structural mechanism accounting for the cooperativity and its functional implications in the case of the filamentous phage GVP (6, 68), the phage T4 gp32 (10, 33), the adenovirus DNA binding protein (DBP) (34), the Escherichia coli SSB (19,42), and the eukaryotic replication protein A (30, 31), while little is known about the SSBs of the Herpesviridae.The herpes simplex virus type 1 (HSV-1) SSB, infected cell polypeptide 8 (ICP8), is a 128-kDa nuclear zinc metalloprotein encoded by the UL29 gene (22). By virtue of its ability to bind oligonucleotide as well as to mediate specific protein-protein interactions, it was shown to have essential functions in DNA metabolism during the viral lytic cycle. ICP8 is one of the seven ␤, or delayed-early, genes required for viral genome replication, which proceeds via a rolling circle mechanism (63, 65). Replication occurs in globular nuclear domains termed replication compartments (55) whose location is defined by the preexisting host cell nuclear architecture, most probably at the periphery of the nuclear matrix-associated ND10 domains, where the viral transactivator ICP0 and the viral input genome migrate in the early stages of infection (43, 47). Evidence has been provided that the seven essential proteins, the origin binding protein (OBP), the polymerase with its processivity factor (UL30 and UL42), the trimeric primase-helicase complex (UL52, UL5, and UL8), and ICP8, accumulate in punctuate prereplicative sites for the assembly of the multiprotein complex, or primosome, which promotes efficient genomic replication (40,71,76). During initiation, ICP8 associates with the carboxy-terminal domain of the OBP at the origin of replication to assist the ATP-dependent bidirectional origin unwinding (3,36,37,45). It enhances the polymerase processivity (29, 50, 61) and stimulates the helicase-primase activity via interaction with the UL8 subunit (4,17,21). In accord with its ability to destabilize DNA helices (5) and promote Mg-dependent complementary-strand renaturation (16), ICP8 can catalyze homologous pairing and strand transfer (7), and hence it participates in the frequent DNA recombination events. Genetic evidence implies a role for ICP8 in the regulation of viral gene expression (12,26), in agreement with the reported ICP8 affinity for polyriboadenylate (61).The DNA binding properties of ICP8 have been extensively investigated. ICP8 binds ssDNA rapidly and cooperatively, with a modest preference for the HSV genomic GC-rich sequences, holding the nucleotide filaments in an extended conformation (35,58). Optimal binding occurs at neutral pH and 150 mM salt concentration (61). Based on filter binding assays (58), nuclease prote...

mentioning

confidence: 99%

The 60-Residue C-Terminal Region of the Single-Stranded DNA Binding Protein of Herpes Simplex Virus Type 1 Is Required for Cooperative DNA Binding

Mapelli

Mühleisen²,

Persico³

et al. 2000

“…Earlier studies with the box I substrate had led to a model in which destabilization of the AϩT sequence linking boxes I and II to provide a binding site for ICP8 was the first step in the unwinding of Ori S by the UL9 protein (8). Unwinding of Ori S is required to provide an entry site for the replication machinery needed to initiate DNA replication.…”

mentioning

confidence: 99%

“…Previous studies have shown that a complex of the UL9 protein and the HSV-1-encoded single-stranded DNA binding protein, ICP8, can efficiently unwind a duplex box I if it possesses a 3Ј single-stranded tail at least 18 nucleotides in length, positioned downstream of box I (8). These findings suggested a model for the unwinding of Ori S in which a complex of the UL9 protein bound to boxes I and II and ICP8 bound to single-stranded DNA generated at the AϩT-rich linker, possibly as a consequence of transcription (4), unwinds the origin of DNA replication to provide access to the replication machinery, thereby permitting the initiation of DNA replication.…”

mentioning

confidence: 99%

“…To measure the unwinding of the duplex oligonucleotides, reaction mixtures (25 l) containing 50 mM HEPES-KOH (pH 8.15), 10 mM NaCl, 10 MgCl 2 , 2.0 mM dithiothreitol, 10% (vol/vol) glycerol, 10 g of bovine serum albumin, 2 pmol of box I substrate, 1.0 pmol of Ori S , 1.8 pmol of Ori S T 18 1.8 pmol of mutant Ori S , 1.6 pmol ICP8 (8), and 1.6 pmol of UL9 protein (8) were incubated on ice for 5 min. ATP (50 M) was added, and the reaction mixtures were incubated at 37°C for 60 min.…”

mentioning

confidence: 99%

See 1 more Smart Citation

Unwinding of a Herpes Simplex Virus Type 1 Origin of Replication (Ori _S ) by a Complex of the Viral Origin Binding Protein and the Single-Stranded DNA Binding Protein

Lehman

2000

Self Cite

A herpes simplex virus type 1 (HSV-1) Ori S analogue in which the A؉T sequence linking the box I and II elements was replaced by two single-stranded oligo(dT)s is unwound by the UL9 protein-ICP8 complex. Unwinding of wild-type Ori S by the UL9 protein-ICP8 complex was also observed under conditions which destabilize the A؉T sequence. These experiments support a model for the unwinding of Ori S in which destabilization of the A؉T sequence can generate a single-stranded DNA binding site for ICP8, which then associates with the UL9 protein bound to boxes I and II to promote the bidirectional unwinding of Ori S .Herpes simplex virus type 1 (HSV-1) encodes a 94-kDa origin binding protein, the product of the UL9 gene, which is essential for HSV-1 DNA replication (5, 10). The origin binding protein (UL9 protein) is a homodimer that binds the two inverted pentanucleotide repeats, boxes I and II of the HSV-1 origin of replication, Ori S . Boxes I and II are linked by an AϩT-rich sequence of 18 nucleotide residues (5). In addition to its origin binding activity, the UL9 protein possesses DNAdependent ATPase and 3Ј-5Ј helicase activities (3, 6). Despite its helicase activity, we have been unable to detect the unwinding of duplex DNA containing Ori S (9).Previous studies have shown that a complex of the UL9 protein and the HSV-1-encoded single-stranded DNA binding protein, ICP8, can efficiently unwind a duplex box I if it possesses a 3Ј single-stranded tail at least 18 nucleotides in length, positioned downstream of box I (8). These findings suggested a model for the unwinding of Ori S in which a complex of the UL9 protein bound to boxes I and II and ICP8 bound to single-stranded DNA generated at the AϩT-rich linker, possibly as a consequence of transcription (4), unwinds the origin of DNA replication to provide access to the replication machinery, thereby permitting the initiation of DNA replication.To test this model, we have investigated the ability of the UL9 protein-ICP8 complex to unwind an Ori S analogue in which the AϩT-rich linker has been replaced by two single strands each consisting of 18 deoxythymidylate residues. We have found this Ori S analogue to be unwound by the complex. In addition, we have found that at temperatures that would be expected to destabilize the AϩT-rich linker, some unwinding of wild-type Ori S can be observed. Ori S bearing mutations in boxes I and II which inhibit binding of UL9 protein could not be unwound under these conditions. These findings support the model in which destabilization of the AϩT-rich linker to provide a binding site for ICP8 represents the initial event in the UL9 protein-promoted unwinding of Ori S .The structure of the duplex oligonucleotides used are shown in Fig. 1. The single-stranded oligonucleotides were purchased from Operon Technologies, Inc. They were purified prior to use by 16% polyacrylamide electrophoresis under denaturing conditions. The concentration of single-stranded oligonucleotides was determined spectrophotometrically assuming that 1 absorbance uni...

“…The functional equivalent of Zta in herpes simplex virus 1 (HSV1) is the origin binding protein (OBP) encoded by the HSV1 UL9 gene. The HSV1 OBP is an ATP-dependent DNA helicase that appears to work together with the single-stranded DNA (ssDNA) binding protein ICP8 to accomplish DNA strand separation at the HSV1 origin OriS (25,32). The EBV genome contains an ICP8 orthologue, referred to as BALF2, and a processive helicase (encoded by the BBLF4 gene) but lacks a replication initiator helicase like UL9.…”

mentioning

confidence: 99%

Initiation of Epstein-Barr Virus Lytic Replication Requires Transcription and the Formation of a Stable RNA-DNA Hybrid Molecule at OriLyt

Rennekamp

Lieberman

2011

The genetic elements of herpesvirus origins of lytic replication have been characterized in detail; however, much remains to be elucidated concerning their functional role in replication initiation. In the case of the Epstein-Barr virus (EBV), we have found that in addition to the two well-defined critical elements required for lytic replication (the upstream and downstream essential elements, UEE and DEE), the origin of lytic replication (OriLyt) also requires the presence of a GC-rich RNA in cis. The BHLF1 transcript is similar to the essential K5 transcript identified at the Kaposi's sarcoma-associated herpesvirus OriLyt. We have found that truncation of the BHLF1 transcript or deletion of the TATA box, but not the putative ATG initiation codon, reduce OriLyt function to background levels. By using an antibody specific for RNA-DNA hybrid molecules, we found the BHLF1 RNA stably annealed to its DNA template during the early steps of lytic reactivation. Furthermore, expression of human RNase H1, which degrades RNA in RNA-DNA hybrids, drastically reduces OriLyt-dependent DNA replication as well as recruitment of the viral single-stranded DNA binding protein BALF2 to OriLyt. These studies suggest that a GC-rich OriLyt transcript is an important component of gammaherpesvirus lytic origins and is required for initial strand separation and loading of core replication proteins. Epstein-Barr virus (EBV) is a human gammaherpesvirus 1 (also known as human herpesvirus 4 [HHV4]) and the etiological agent responsible for infectious mononucleosis, oral hairy leukoplakia, AIDS immunoblastic lymphomas, posttransplant lymphoproliferative disease, 50% of Hodgkin's lymphomas, and the endemic forms of nasopharyngeal carcinoma and Burkitt's lymphoma (56, 79). Successful infection and viral spread, within and between individual hosts, are necessary prerequisites for EBV pathogenesis. Each of these requires productive lytic replication of the virus, which includes the duplication of its 170-to 175-kbp double-stranded DNA genome. To date, no antiviral drug has been approved, nor shown to be highly effective, in blocking EBV lytic replication, suggesting that mechanisms controlling viral DNA replication are sufficiently diverged from related members of the herpesvirus family (2).Upon host cell infection and establishment of latency, herpesvirus genomes, including EBV, adopt a closed circular conformation (11, 64). During lytic reactivation from viral latency, DNA replication must initiate from this circular template. The viral basic leucine zipper (b-ZIP) protein Zta (encoded by the immediate-early BZLF1 gene, and also known as Z, ZEBRA, and EB1) governs this process (10, 13, 17, 57, 69). Zta has been described as both a transcription factor and an origin binding protein, activating both virus early gene transcription and the EBV origin of lytic replication (OriLyt) (18,24,36,38,65). Zta's ability to activate OriLyt is thought to be at least partially due to its ability to bind viral replication proteins, perhaps recruiting them to th...