Binding of the herpes simplex virus type 1 UL9 gene product to an origin of viral DNA replication.

The binding of a herpes simplex virus type 1 (HSV-1) encoded polypeptide to a viral origin of DNA replication has been studied by using a gel retardation assay. Incubation of nuclear extract from HSV-1 infected cells with a labelled origin-containing fragment resulted in the formation of a specific retarded complex, the migration of which was further reduced in the presence of an antibody reactive with the UL9 gene product. Introduction of an additional copy of the UL9 gene, under the control of an immediate early (IE) promoter, conferred the ability to express origin binding activity at the non-permissive temperature upon an HSV-1 ts mutant blocked at the IE stage of infection. Endogenous or exogenous proteolytic activity revealed the presence of a relatively protease-resistant domain which retained sequence-specific DNA binding activity. The C-terminal 317 amino acids of the UL9 gene expressed as a fusion protein in Escherichia coli also bound to the origin. Our results demonstrate that the UL9 gene product binds to a viral origin and that sequence specific recognition and binding are specified by the C-terminal 37% of the polypeptide.     

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.     

Fine-structure mapping of herpes simplex virus type 1 temperature-sensitive mutations within the short repeat region of the genome.

Cloned herpes simplex virus type 1 (HSV-1) DNA fragments were used to fine-structure map the temperature-sensitive (ts) lesions from four mutants, ts T, D, c75, and K, by marker rescue. These mutants all overproduced immediate-early viral polypeptides at the nonpermissive temperature. Although one of these viruses, ts K, gave a more restricted infected-cell polypeptide profile under these conditions than the other three, no complementation was detected between pairwise crosses of these mutants in the yield test. Recombination, however, was obtained between all mutant pairs except ts T and D. In physical mapping experiments, ts+ virus was recovered from cells coinfected with DNA of ts T, D, or c75 and BamHI fragment k from wild-type strain 17 HSV-1 DNA cloned in pAT153, whereas ts K was rescued by cloned HSV-1 BamHI-y. Both of these cloned DNA fragments contained sequences from the short repeat region of the HSV-1 genome. The ts mutations were more precisely mapped by marker rescue, using restriction enzyme fragments within BamHI-k and -y from cloned DNA. The smallest fragment able to rescue a mutant was 320 base pairs long. The order of the four mutations derived from these studies was consistent with the assignment by genetic recombination. All four lesions mapped within the coding sequences of the immediate-early polypeptide Vmw IE 175 (ICP4) which lie outside the "a" sequence. The results showed that mutations in different regions of the gene encoding Vmw IE 175 could produce similar phenotype effects at the nonpermissive temperature.     

A subset of herpes simplex virus replication genes induces DNA amplification within the host cell genome.

Herpes simplex virus (HSV) induces DNA amplification of target genes within the host cell chromosome. To characterize the HSV genes that mediate the amplification effect, combinations of cloned DNA fragments covering the entire HSV genome were transiently transfected into simian virus 40 (SV40)-transformed hamster cells. This led to amplification of the integrated SV40 DNA sequences to a degree comparable to that observed after transfection of intact virion DNA. Transfection of combinations of subclones and of human cytomegalovirus immediate-early promoter-driven expression constructs for individual open reading frames led to the identification of six HSV genes which together were necessary and sufficient for the induction of DNA amplification: UL30 (DNA polymerase), UL29 (major DNA-binding protein), UL5, UL8, UL42, and UL52. All of these genes encode proteins necessary for HSV DNA replication. However, an additional gene coding for an HSV origin-binding protein (UL9) was required for origin-dependent HSV DNA replication but was dispensible for SV40 DNA amplification. Our results show that a subset of HSV replication genes is sufficient for the induction of DNA amplification. This opens the possibility that HSV expresses functions sufficient for DNA amplification but separate from those responsible for lytic viral growth. HSV infection may thereby induce DNA amplification within the host cell genome without killing the host by lytic viral growth. This may lead to persistence of a cell with a new genetic phenotype, which would have implications for the pathogenicity of the virus in vivo.     

Human cytomegalovirus TRS1 and IRS1 gene products block the double-stranded-RNA-activated host protein shutoff response induced by herpes simplex virus type 1 infection

Human cytomegalovirus (HCMV) attachment and entry stimulates the expression of cellular interferon-inducible genes, many of which target important cellular functions necessary for viral replication. Double-stranded RNA-dependent host protein kinase (PKR) is an interferon-inducible gene product that limits viral replication by inhibiting protein translation in the infected cell. It was anticipated that HCMV encodes gene products that facilitate the evasion of this PKR-mediated antiviral response. Using a deltagamma1 34.5 herpes simplex virus type 1 (HSV-1) recombinant that triggers PKR-mediated protein synthesis shutoff, experiments identified an HCMV gene product expressed in the initial hours of infection that allows continued protein synthesis in the infected cell. Recombinant HSV-1 viruses expressing either the HCMV TRS1 or IRS1 protein demonstrate that either of these HCMV gene products allows the deltagamma1 34.5 recombinant viruses to evade PKR-mediated protein shutoff and maintain late viral protein synthesis.     

Physical map of the origin of defective DNA in herpes simplex virus type 1 DNA.

The origin of defective DNA (dDNA) of the Patton strain of herpes simplex virus type 1 (HSV-1) was physically mapped with BamHI in the parental DNA. The dDNA obtained from virus passaged at high multiplicities of infection was resistant to cleavage with HindIII, whereas digestion with EcoRI yielded a cluster of fragments 5.4 to 5.7 megadaltons (Mdal) in size. Cleavage with BamHI gave a cluster of fragments 2.6 to 3.2 Mdal in size, plus two homogeneous, comigrating 1-Mdal fragments. One of the latter fragments contained the single EcoRI site approximately 65 base pairs from one end. Hybridization of in vitro labeled dDNA probe to EcoRI, HindIII, BamHI, and Hpa I digests of nondefective HSV-1 DNA demonstrated that, in addition to the S-region terminal repeat, only one end of the S region was involved in the generation of this class of dDNA. Thus, the dDNA probe did not hybridize to either the S region 3.0-Mdal HindIIIN fragment or a 3.0-Mdal BamHI fragment of the adjacent 8.7-Mdal HindIIIG fragment, but did hybridize to four BamHI fragments of HindIII G (approximately 5.7 Mdal). The cluster of 2.6- to 3.2-Mdal fragments obtained with BamHI digestion of dDNA appears to represent a novel junction between the termination of dDNA adjacent to the 3.0-Mdal BamHI fragment in HindIII G and the 2.0- to 2.3-Mdal BamHI fragment terminal in HSV-1 DNA.     

Nuclear delivery mechanism of herpes simplex virus type 1 genome

Herpes simplex virus type 1 (HSV-1) is a widespread human pathogen infecting more than 80% of the population worldwide. Its replication involves an essential, poorly understood multistep process, referred to as uncoating. Uncoating steps are as follows: (1) The incoming capsid pinpoints the nuclear pore complex (NPC). (2) It opens up at the NPC and releases the highly pressurized viral genome. (3) The viral genome translocates through the NPC. In the present review, we highlight recent advances in this field and propose mechanisms underlying the individual steps of uncoating. We presume that the incoming HSV-1 capsid pinpoints the NPC by hydrophobic interactions and opens up upon binding to NPC proteins. Genome translocation is initially pressure-driven.     

A point mutation within conserved region VI of herpes simplex virus type 1 DNA polymerase confers altered drug sensitivity and enhances replication fidelity

Herpes simplex virus type 1 (HSV-1) DNA polymerase contains several conserved regions within the polymerase domain. The conserved regions I, II, III, V, and VII have been shown to have functional roles in the interaction with deoxynucleoside triphosphates (dNTPs) and DNA. However, the role of conserved region VI in DNA replication has remained unclear due, in part, to the lack of a well-characterized region VI mutant. In this report, recombinant viruses containing a point mutation (L774F) within the conserved region VI were constructed. These recombinant viruses were more susceptible to aphidicolin and resistant to both foscarnet and acyclovir, compared to the wild-type KOS strain. Marker transfer experiments demonstrated that the L774F mutation conferred the altered drug sensitivities. Furthermore, mutagenesis assays demonstrated that L774F recombinant viruses containing the supF marker gene, which was integrated within the thymidine kinase locus (tk), exhibited increased fidelity of DNA replication. These data indicate that conserved region VI, together with other conserved regions, forms the polymerase active site, has a role in the interaction with deoxyribonucleotides, and regulates DNA replication fidelity. The possible effect of the L774F mutation in altering the polymerase structure and activity is discussed.     

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.     

Mutations in a herpes simplex virus type 1 origin that inhibit interaction with origin-binding protein also inhibit DNA replication.

The herpes simplex virus type 1 genome contains three origins of replication: OriL and a diploid OriS. The origin-binding protein, the product of the UL9 gene, interacts with two sites within OriS, box I and box II. A third site, box III, which is homologous to boxes I and II, may also be a binding site for the origin-binding protein. Mutations in these three sites significantly reduce OriS-directed plasmid replication measured in transient replication assays. The reduction in replication efficiency of the mutants correlates well with the decrease in the ability to bind to the origin-binding protein, as determined by Elias et al. (P. Elias, C. M. Gustafsson, and O. Hammarsten, J. Biol. Chem. 265: 17167-17173, 1990). The effect of multiple mutations in boxes I, II, and III on plasmid replication suggests that there are multiple binding sites in OriS for the origin-binding protein. These studies indicate that proper interaction of the origin-binding protein with the OriS sequence is essential for OriS-directed DNA replication.     

Topoisomerase II cleavage of herpes simplex virus type 1 DNA in vivo is replication dependent

The genome of herpes simplex virus type 1 contains a large number of recognition sites for eucaryotic DNA type II topoisomerase. Topoisomerase II sites were identified by means of the consensus sequence described previously (J.R. Spitzner and M.T. Muller, Nucleic Acids Res. 16:5553-5556, 1988) and then confirmed by sequencing DNA cleavages introduced by purified topoisomerase II. In vivo, host topoisomerase II also introduced double-stranded DNA breaks in the viral genome at sites predicted by the consensus sequence. Host topoisomerase II acted on all immediate-early genes as well as on genes from other temporal classes; however, cleavages were not detected until 4 to 5 h postinfection and were most intense at 10 h postinfection. Topoisomerase II cleavages were not detected when viral DNA replication was prevented with phosphonoacetic acid. These data indicate that, although progeny viral genomes are acted upon by host topoisomerase II, this enzyme either does not act on parental viral genomes before DNA replication or acts on them with such low efficiency that cleavages are beyond our limit of detection. The findings suggest that host topoisomerase II is involved in aspects of viral replication at late times in the infectious cycle.     

Latent herpes simplex virus type 1 DNA contains two copies of the virion DNA joint region.

Southern blot analysis of latent herpes simplex virus DNA detected in mouse brain and digested with a restriction enzyme revealed two copies of the virion DNA joint fragment. Thus, the absence of free ends noted previously in latent herpes simplex virus type 1 DNA is due to joining of the termini.     

Rep-dependent initiation of adeno-associated virus type 2 DNA replication by a herpes simplex virus type 1 replication complex in a reconstituted system

Productive infection by adeno-associated virus type 2 (AAV) requires coinfection with a helper virus, e.g., adenovirus or herpesviruses. In the case of adenovirus coinfection, the replication machinery of the host cell performs AAV DNA replication. In contrast, it has been proposed that the herpesvirus replication machinery might replicate AAV DNA. To investigate this question, we have attempted to reconstitute AAV DNA replication in vitro using purified herpes simplex virus type 1 (HSV-1) replication proteins. We show that the HSV-1 UL5, UL8, UL29, UL30, UL42, and UL52 gene products along with the AAV Rep68 protein are sufficient to initiate replication on duplex DNA containing the AAV origins of replication, resulting in products several hundred nucleotides in length. Initiation can occur also on templates containing only a Rep binding site and a terminal resolution site. We further demonstrate that initiation of DNA synthesis can take place with a subset of these factors: Rep68 and the UL29, UL30, and UL42 gene products. Since the HSV polymerase and its accessory factor (the products of the UL30 and UL42 genes) are unable to efficiently perform synthesis by strand displacement, it is likely that in addition to creating a hairpin primer, the AAV Rep protein also acts as a helicase for DNA synthesis. The single-strand DNA binding protein (the UL29 gene product) presumably prevents reannealing of complementary strands. These results suggest that AAV can use the HSV replication apparatus to replicate its DNA. In addition, they may provide a first step for the development of a fully reconstituted AAV replication assay.     

DNA sequence of the region in the genome of herpes simplex virus type 1 containing the exonuclease gene and neighbouring genes.

We report the sequence of a 7800 base pair region of herpes simplex virus type 1 DNA, representing approximately 0.16 to 0.20 map units in the genome. This contains sequences transcribed into a leftward oriented set of five 3' coterminal mRNAs, together with two rightward transcribed flanking genes. One of the leftward genes encodes the virus's alkaline exonuclease, but the other gene products are uncharacterized. The amino acid sequence of one encoded protein suggested that it is a membrane embedded species. The DNA sequence is densely utilised, with two predicted out-of-frame overlaps of coding sequences, and probably six occurrences of promoter elements within coding sequences. Homologues of five of the genes were found for the distantly related Epstein-Barr virus, with a similar overall relative arrangement.     

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.     

Rolling circle DNA replication by extracts of herpes simplex virus type 1-infected human cells.

Whole-cell extracts of herpes simplex virus type 1-infected human cells (293 cells) can promote the rolling circle replication of circular duplex DNA molecules. The products of the reaction are longer than monomer unit length and are the result of semiconservative DNA replication by the following criteria: (i) resistance to DpnI and susceptibility to MboI restriction enzymes, (ii) shift in density on a CsCl gradient of the products synthesized in the presence of bromo-dUTP to a position on the gradient consistent with those of molecules composed mainly of one parental DNA strand and one newly synthesized DNA strand, and (iii) the appearance in the electron microscope of molecules consisting of duplex circles with multiunit linear appendages, a characteristic of a rolling circle mode of DNA replication. The reaction requires ATP and is dependent on herpes simplex virus type 1-encoded DNA polymerase.     

Role of protein-protein interactions during herpes simplex virus type 1 recombination-dependent replication

Recombination-dependent replication is an integral part of the process by which double-strand DNA breaks are repaired to maintain genome integrity. It also serves as a means to replicate genomic termini. We reported previously on the reconstitution of a recombination-dependent replication system using purified herpes simplex virus type 1 proteins (Nimonkar A. V., and Boehmer, P. E. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 10201-10206). In this system, homologous pairing by the viral single-strand DNA-binding protein (ICP8) is coupled to DNA synthesis by the viral DNA polymerase and helicase-primase in the presence of a DNA-relaxing enzyme. Here we show that DNA synthesis in this system is dependent on the viral polymerase processivity factor (UL42). Moreover, although DNA synthesis is strictly dependent on topoisomerase I, it is only stimulated by the viral helicase in a manner that requires the helicase-loading protein (UL8). Furthermore, we have examined the dependence of DNA synthesis in the viral system on species-specific protein-protein interactions. Optimal DNA synthesis was observed with the herpes simplex virus type 1 replication proteins, ICP8, DNA polymerase (UL30/UL42), and helicase-primase (UL5/UL52/UL8). Interestingly, substitution of each component with functional homologues from other systems for the most part did not drastically impede DNA synthesis. In contrast, recombination-dependent replication promoted by the bacteriophage T7 replisome was disrupted by substitution with the replication proteins from herpes simplex virus type 1. These results show that although DNA synthesis performed by the T7 replisome is dependent on cognate protein-protein interactions, such interactions are less important in the herpes simplex virus replisome.     

Localization of epitopes of herpes simplex virus type 1 glycoprotein D.

We previously defined eight groups of monoclonal antibodies which react with distinct epitopes of herpes simplex virus glycoprotein D (gD). One of these, group VII antibody, was shown to react with a type-common continuous epitope within residues 11 to 19 of the mature glycoprotein (residues 36 to 44 of the predicted sequence of gD). In the current investigation, we have localized the sites of binding of two additional antibody groups which recognize continuous epitopes of gD. The use of truncated forms of gD as well as computer predictions of secondary structure and hydrophilicity were instrumental in locating these epitopes and choosing synthetic peptides to mimic their reactivity. Group II antibodies, which are type common, react with an epitope within residues 268 to 287 of the mature glycoprotein (residues 293 to 312 of the predicted sequence). Group V antibodies, which are gD-1 specific, react with an epitope within residues 340 to 356 of the mature protein (residues 365 to 381 of the predicted sequence). Four additional groups of monoclonal antibodies appear to react with discontinuous epitopes of gD-1, since the reactivity of these antibodies was lost when the glycoprotein was denatured by reduction and alkylation. Truncated forms of gD were used to localize these four epitopes to the first 260 amino acids of the mature protein. Competition experiments were used to assess the relative positions of binding of various pairs of monoclonal antibodies. In several cases, when one antibody was bound, there was no interference with the binding of an antibody from another group, indicating that the epitopes were distinct. However, in other cases, there was competition, indicating that these epitopes might share some common amino acids.