Point mutations in herpes simplex virus type 1 oriL, but not in oriS, reduce pathogenesis during acute infection of mice and impair reactivation from latency

In vitro studies of herpes simplex virus type 1 (HSV-1) viruses containing mutations in core sequences of the viral origins of DNA replication, oriL and oriS, that eliminate the ability of these origins to initiate viral-DNA synthesis have demonstrated little or no effect on viral replication in cultured cells, leading to the conclusion that the two types of origins are functionally redundant. It remains unclear, therefore, why origins that appear to be redundant are maintained evolutionarily in HSV-1 and other neurotropic alphaherpesviruses. To test the hypothesis that oriL and oriS have distinct functions in the HSV-1 life cycle in vivo, we determined the in vivo phenotypes of two mutant viruses, DoriL-I(LR) and DoriS-I, containing point mutations in oriL and oriS site I, respectively, that eliminate origin DNA initiation function. Following corneal inoculation of mice, tear film titers of DoriS-I were reduced relative to wild-type virus. In all other tests, however, DoriS-I behaved like wild-type virus. In contrast, titers of DoriL-I(LR) in tear film, trigeminal ganglia (TG), and hindbrain were reduced and mice infected with DoriL-I(LR) exhibited greatly reduced mortality relative to wild-type virus. In the TG explant and TG cell culture models of reactivation, DoriL-I(LR) reactivated with delayed kinetics and, in the latter model, with reduced efficiency relative to wild-type virus. Rescuant viruses DoriL-I(LR)-R and DoriS-I-R behaved like wild-type virus in all tests. These findings demonstrate that functional differences exist between oriL and oriS and reveal a prominent role for oriL in HSV-1 pathogenesis.     

Cloning and characterization of herpes simplex virus type 1 oriL: comparison of replication and protein-DNA complex formation by oriL and oriS.

The herpes simplex virus type 1 genome contains three origins of DNA replication: two copies of oriS and one copy of oriL. Although oriS has been characterized extensively, characterization of oriL has been severely limited by the inability to amplify oriL sequences in an undeleted form in Escherichia coli. We report the successful cloning of intact oriL sequences in an E. coli strain, SURE, which contains mutations in a series of genes involved in independent DNA repair pathways shown to be important in the rearrangement and deletion of DNA containing irregular structures such as palindromes. The oriL-containing clones propagated in SURE cells contained no deletions, as determined by Southern blot hybridization and DNA sequence analysis, and were replication competent in transient DNA replication assays. Deletion of 400 bp of flanking sequences decreased the replication efficiency of oriL twofold in transient assays, demonstrating a role for flanking sequences in enhancing replication efficiency. Comparison of the replication efficiencies of an 822-bp oriS-containing plasmid and an 833-bp oriL-containing plasmid demonstrated that the kinetics of replication of the two plasmids were similar but that the oriL-containing plasmid replicated 60 to 70% as efficiently as the oriS-containing plasmid at both early and late times after infection with herpes simplex virus type 1. The virus-specified origin-binding protein (OBP) and a cellular factor(s) (OF-1) have been shown in gel mobility shift experiments to bind specific sequences in oriS (C.E. Dabrowski, P. Carmillo, and P.A. Schaffer, Mol. Cell. Biol. 14:2545-2555, 1994; C.E. Dabrowski and P.A. Schaffer, J. Virol. 65:3140-3150, 1991). Although the nucleotides required for the binding of OBP to OBP binding site I in oriL and oriS are the same, a single nucleotide difference distinguishes OBP binding site III in the two origins. The nucleotides adjacent to oriS sites I and III have been shown to be important for the binding of OF-1 to oriS site I. Several nucleotide differences exist in these sequences in oriL and oriS. Despite these minor nucleotide differences, the protein-DNA complexes that formed with oriL and oriS sites I and III were indistinguishable when extracts of infected and uninfected cells were used as the source of protein. Furthermore, the results of competition analysis suggest that the proteins involved in protein-DNA complex formation with sites I and III of the two origins are likely the same.     

A novel conformation of the herpes simplex virus origin of DNA replication recognized by the origin binding protein

The Herpes simplex virus type I origin binding protein (OBP) is a sequence-specific DNA-binding protein and a dimeric DNA helicase encoded by the UL9 gene. It is required for the activation of the viral origin of DNA replication oriS. Here we demonstrate that the linear double-stranded form of oriS can be converted by heat treatment to a stable novel conformation referred to as oriS*. Studies using S1 nuclease suggest that oriS* consists of a central hairpin with an AT-rich sequence in the loop. Single-stranded oligonucleotides corresponding to the upper strand of oriS can adopt the same structure. OBP forms a stable complex with oriS*. We have identified structural features of oriS* recognized by OBP. The central oriS palindrome as well as sequences at the 5' side of the oriS palindrome were required for complex formation. Importantly, we found that mutations that have been shown to reduce oriS-dependent DNA replication also reduce the formation of the OBP-oriS* complex. We suggest that oriS* serves as an intermediate in the initiation of DNA replication providing the initiator protein with structural information for a selective and efficient assembly of the viral replication machinery.     

Sequence and structural requirements of a herpes simplex viral DNA replication origin.

Herpes simplex virus (HSV) types 1 and 2 contain two classes of origins of DNA replication, oriS and oriL, which are closely related. A series of plasmids was constructed which contained specifically altered versions of the HSV type 2 oriS replication origin. Their ability to replicate in an in vivo replicon assay allowed a core origin of 75 base pairs (bp) to be defined. It included both arms of a 56-bp palindrome and from 13 to 20 bp of sequence leftward of the palindrome. The AT-rich sequence at the center of the palindrome was essential. Sequences on either side of the core origin enhanced replication. When additional copies of the -AT-dinucleotide were introduced progressively into the center of the palindrome, an oscillating effect on origin function was observed. These and other data implicate a linear rather than a cruciform conformation of the oriS palindrome in the initiation of HSV replication.     

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.     

Identification of herpes simplex virus type 1 genes required for origin-dependent DNA synthesis.

The herpes simplex virus (HSV) genome contains both cis- and trans-acting elements which are important in viral DNA replication. The cis-acting elements consist of three origins of replication: two copies of oriS and one copy of oriL. It has previously been shown that five cloned restriction fragments of HSV-1 DNA together can supply all of the trans-acting functions required for the replication of plasmids containing oriS or oriL when cotransfected into Vero cells (M. D. Challberg, Proc. Natl. Acad. Sci. USA, 83:9094-9098, 1986). These observations provide the basis for a complementation assay with which to locate all of the HSV sequences which encode trans-acting functions necessary for origin-dependent DNA replication. Using this assay in combination with the data from large-scale sequence analysis of the HSV-1 genome, we have now identified seven HSV genes which are necessary for transient replication of plasmids containing either oriS or oriL. As shown previously, two of these genes encode the viral DNA polymerase and single-stranded DNA-binding protein, which are known from conventional genetic analysis to be essential for viral DNA replication in infected cells. The functions of the products of the remaining five genes are unknown. We propose that the seven genes essential for plasmid replication comprise a set of genes whose products are directly involved in viral DNA synthesis.     

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.     

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*.     

The DNA replication origins of herpes simplex virus type 1 strain Angelotti

The nucleotide sequences of the origins of DNA replication (ori) of the S- and L-component (oriS, oriL) of the herpes simplex virus type 1 (HSV-1) standard genome were determined from HSV-1 strain Angelotti (ANG). In contrast to other HSV-1 strains, the ANG oriS sequence revealed an insertion of an TA-dinucleotide in an otherwise very conserved but imperfect palindromic sequence of 47 bp. The oriL sequence of the standard ANG genome was found to be identical to that of an ANG class II defective genome which exhibits a duplication of a 144 bp palindrome. A model is presented to explain the origination of the amplified ANG oriL sequences from the parental genome with a single copy of oriL via illegitimate recombination. Alignment of the ori sequences of HSV, adeno- and papovaviruses unveiled that the HSV ori region can be subdivided into two distinct sites of homology to the DNA initiation signals of papova- and adenoviruses, suggesting that the HSV origins of replication comprise elements for DNA replication by both, cellular and virus-encoded DNA polymerases.     

ATP-dependent unwinding of a minimal origin of DNA replication by the origin-binding protein and the single-strand DNA-binding protein ICP8 from herpes simplex virus type I

The Herpes simplex virus type I origin-binding protein, OBP, is encoded by the UL9 gene. OBP binds the origin of DNA replication, oriS, in a cooperative and sequence-specific manner. OBP is also an ATP-dependent DNA helicase. We have recently shown that single-stranded oriS folds into a unique and evolutionarily conserved conformation, oriS*, which is stably bound by OBP. OriS* contains a stable hairpin formed by complementary base pairing between box I and box III in oriS. Here we show that OBP, in the presence of the single-stranded DNA-binding protein ICP8, can convert an 80-base pair double-stranded minimal oriS fragment to oriS* and form an OBP-oriS* complex. The formation of an OBP-oriS* complex requires hydrolysable ATP. We also demonstrate that OBP in the presence of ICP8 and ATP promotes slow but specific and complete unwinding of duplex minimal oriS. The possibility that the OBP-oriS* complex may serve as an assembly site for the herpes virus replisome is discussed.     

Cloning, sequencing, and functional analysis of oriL, a herpes simplex virus type 1 origin of DNA synthesis.

The herpes simplex virus type 1 genome (160 kilobases) contains three origins of DNA synthesis: two copies of oriS located within the repeated sequences flanking the short unique arm (US), and one copy of oriL located within the long unique arm (UL). Precise localization and characterization of oriL have been severely hampered by the inability to clone sequences which contain it (coordinates 0.398 to 0.413) in an undeleted form in bacteria. We report herein the successful cloning of sequences between 0.398 to 0.413 in an undeleted form, using a yeast cloning vector. Sequence analysis of a 425-base pair fragment spanning the deletion-prone region has revealed a perfect 144-base pair palindrome with striking homology to oriS. In a functional assay, the undeleted clone was amplified when functions from herpes simplex virus type 1 were supplied in trans, whereas clones with deletions of 55 base pairs or more were not amplified.     

Differential effects of nerve growth factor and dexamethasone on herpes simplex virus type 1 oriL- and oriS-dependent DNA replication in PC12 cells.

The herpes simplex virus type 1 (HSV-1) genome contains three origins of DNA replication, one copy of oriL and two copies of oriS. Although oriL and oriS are structurally different, they have extensive nucleotide sequence similarity and can substitute for each other to initiate viral DNA replication. A fundamental question that remains to be answered is why the HSV-1 genome contains two types of origin. We have recently identified a novel glucocorticoid response element (GRE) within oriL that is not present in oriS and have shown by gel mobility shift assays that purified glucocorticoid receptor (GR), as well as GR present in cellular extracts, can bind to the GRE in oriL. To determine whether glucocorticoids and the GRE affect the efficiency of oriL-dependent DNA replication, we performed transient DNA replication assays in the presence and absence of dexamethasone (DEX). Because HSV-1 is a neurotropic virus and establishes latency in cells of neural origin, these tests were conducted in PC12 cells, which assume the properties of sympathetic neurons when differentiated with nerve growth factor (NGF). In NGF-differentiated PC12 cells, oriL-dependent DNA replication was enhanced 5-fold by DEX, whereas in undifferentiated cells, DEX enhanced replication approximately 2-fold. Notably, the enhancement of oriL function by DEX was abolished when the GRE was mutated. NGF-induced differentiation alone had no effect. In contrast to oriL, oriS-dependent DNA replication was reduced approximately 5-fold in NGF-differentiated PC12 cells and an additional 4-fold in differentiated cells treated with DEX. In undifferentiated PC12 cells, DEX had only a minor inhibitory effect (approximately 2-fold) on oriS function. Although the cis-acting elements that mediate the NGF- and DEX-specific repression of oriS-dependent DNA replication are unknown, a functional GRE is critical for the DEX-induced enhancement of oriL function in NGF-differentiated PC12 cells. The enhancement of oriL-dependent DNA replication by DEX in differentiated PC12 cells suggests the possibility that glucocorticoids, agents long recognized to enhance reactivation of latent herpesvirus infections, act through the GRE in oriL to stimulate viral DNA replication and reactivation in terminally differentiated neurons in vivo.     

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.     

Mutational analysis of simian virus 40 large T antigen DNA binding sites.

We have tested the effects of various mutations within SV40 T antigen DNA recognition sites I and II on specific T antigen binding using the DNase footprint technique. In addition, the replication of plasmid DNA templates carrying these T antigen binding site mutations was monitored by Southern analysis of transfected DNA in COS cells. Deletion mapping of site I sequences defined a central core of approximately 18 bp that is both necessary and sufficient for T antigen recognition; this region contains the site I contact nucleotides that were previously mapped using methylation-interference and methylation-protection experiments. A similar deletion analysis delineated sequences that impart specificity of binding to site II. We find that T antigen is capable of specific recognition of site II in the absence of site I sequences, indicating that binding to site II in vitro is not dependent on binding of T antigen at site I. Site II binding was not diminished by small deletion or substitution mutations that perturb the 27-bp palindrome central to binding site II, whereas extensive substitution of site II sequences completely eliminated specific site II binding. Analysis of the replication in COS7 cells of plasmids that contain these mutant origins revealed that sequences both at the late side of binding site I and within the site II palindrome are crucial for viral DNA replication, but are not involved in binding T antigen.     

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.     

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.