Herpes Simplex Virus Type 1 DNA Research Articles

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.

The herpes simplex virus type 1 (HSV-1) genome contains three origins of replication: oriL and two copies of oriS. These origins contain specific sequences, box I and box II, linked by an AT-rich segment, that are recognized by an HSV-1-encoded origin binding protein (UL9 protein) which also possesses DNA helicase activity. Despite its intrinsic helicase activity, the UL9 protein is unable to unwind oriS or the box I element of oriS, either in the presence or absence of the HSV-1-encoded single-strand DNA binding protein, ICP8. However, a complex of the UL9 protein and ICP8 can unwind box I if it contains a 3' single-stranded tail at least 18 nt in length positioned downstream of box I. These findings suggest a model for the initiation of HSV-1 DNA replication in which a complex consisting of the UL9 protein bound to box I, and ICP8 bound to single-stranded DNA generated at the A+T rich linker, perhaps as a consequence of transcription, unwinds an HSV-1 origin of replication to provide access to the replication machinery with the consequent initiation of viral DNA replication. This mode of unwinding is distinct from that observed for other animal viruses--e.g., simian virus 40 or bovine papilloma virus--in which the initiator protein, T antigen, or E1 protein alone, unwinds elements of the origin sequence, and the single-strand DNA binding protein serves only to keep the separated strands apart.

Toxicity studies in thymidine kinase-deficient herpes simplex virus therapy for malignant astrocytoma.

Previous studies have shown that genetically engineered thymidine kinase (tk)-defective herpes simplex virus type 1 (HSV-1) can effectively and selectively destroy gliomas in animal models. The consequences of viral infection and tumor regression must be characterized before this therapy can be applied in human trials. To study the potential for long-term toxicity, immunocompetent rats harboring 9L gliosarcomas were injected intratumorally with a tk-defective HSV-1, KOS-SB, at titers that previously have been demonstrated to cause tumor regression. In animals surviving 3 months or longer following viral treatment, there was no evidence of persistent infection or inflammation in peritumoral brain tissue or in remote systemic organs studied with routine histological and immunocytochemical analyses. Polymerase chain reaction using primers specific for HSV-1 detected HSV-1 DNA in peritumoral tissue only in animals sacrificed within 3 months of viral injection. There was no evidence of HSV-1 DNA in systemic tissues at any time after treatment. We conclude that stereotactic intratumoral injection of tk-deficient HSV can be attempted for the treatment of brain tumors without risk of systemic infection or significant toxicity to normal brain or remote proliferating tissues.

Replication of herpes simplex virus type 1 DNA is inhibited in a temperature-sensitive mutant of BHK-21 cells lacking RCC1 (regulator of chromosome condensation) and virus DNA remains linear.

tsBN2, a temperature-sensitive (ts) growth mutant of the hamster cell line BHK-21, has a point mutation in the RCC1 (regulator of chromosome condensation) gene, and prematurely enters mitosis at 39.5 degrees C, a nonpermissive temperature. In this mutant at 39.5 degrees C infectious progeny of herpes simplex virus type 1 (HSV-1) was not produced and replication of HSV-1 DNA was inhibited. HSV-1 DNA from virus particles is normally circularized upon infection, and circularized HSV-1 DNA molecules can serve as template for DNA replication. In tsBN2 at 39.5 degrees C, HSV-1 DNA appeared to remain linear after infection, suggesting the obstruction of HSV-1 DNA circularization, which could account for failure of HSV-1 DNA replication. In transient replication assays performed in tsBN2 at 39.5 degrees C, through superinfection with HSV-1 helper virus, there was no evidence of replication of circular DNA of the hybrid plasmid containing the HSV-1 replication origin. Production of mRNAs of HSV-1 early genes required for HSV-1 DNA replication was decreased in tsBN2 at 39.5 degrees C. Therefore, RCC1 was assumed to be involved in the formation of an HSV-1 DNA configuration suitable for replication (that is circularization) and the supply of proteins required for replication of the circularized HSV-1 DNAs.

Suitability and clinical application of a multiplex nested PCR assay for the diagnosis of herpes simplex virus infections.

A novel multiplex nested polymerase chain reaction (PCR) assay was designed and evaluated for routine diagnosis of herpes simplex virus (HSV) infections in patients with either putative HSV infection of the central nervous system or suspected HSV keratitis. Single-tube amplification of HSV type 1 (HSV-1) or type 2 (HSV-2) DNA extracted from cerebrospinal fluid (CSF) or from keratectomy specimens was followed by differentiation of the virus type-specific PCR products either by agarose gel analysis or by DNA enzyme immunoassay. Among 417 CSF specimens obtained from 395 consecutive patients with clinically suspected HSV infection, 11 (2.6%) were positive for HSV-1 DNA and four (1.0%) probes were positive for HSV-2 DNA. None of the specimens was positive for both HSV-1 and HSV-2 DNA. The genome of HSV-2 was detected in a CSF sample obtained from a woman with meningoencephalitis and genital herpes. The presence of PCR inhibitors was detected in six of 111 (5.4%) reconstructed CSF samples. Inhibition could be removed following extraction with a commercial kit. HSV-1 DNA, but no HSV-2 DNA, was detected in corneal buttons obtained from patients with suspected herpetic keratitis. No contamination has been recorded during the 2-year routine use of this test, which has met the specific requirements of a diagnostic laboratory.

The UL8 Subunit of the Herpes Simplex Virus Type-1 DNA Helicase-Primase Optimizes Utilization of DNA Templates Covered by the Homologous Single-strand DNA-binding Protein ICP8

The herpes simplex virus type-1 DNA helicase-primase is a heterotrimer encoded by the UL5, UL8, and UL52 genes. The core enzyme, specified by the UL5 and UL52 genes, retains DNA helicase, DNA-dependent nucleoside triphosphatase, and primase activities. The UL8 subunit has previously been implicated in increasing primer stability and in stimulating primer synthesis by the core enzyme. To further characterize the function of the UL8 subunit, we have examined its effect on the activities of the UL5/52 core enzyme using DNA templates covered by the herpes simplex virus type-1 single-strand DNA-binding protein ICP8. We found that while ICP8 stimulated the DNA helicase activity of the UL5/52 proteins up to 3-fold, maximum stimulation by ICP8 required the presence of UL8 protein. Moreover, UL8 protein was required to reverse the inhibitory effect of ICP8 on the DNA-dependent ATPase and primase activities of the UL5/52 proteins. These observations were specific for ICP8 since the heterologous Escherichia coli single-strand DNA-binding protein could not substitute for ICP8. These data suggest that UL8 protein mediates an interaction between the UL5/52 core enzyme and ICP8 that optimizes the utilization of ICP8-covered DNA templates during DNA replication.

Detection of a transforming fragment of herpes simplex virus type 2 in clinical specimens by PCR. The Canadian Women's HIV Study Group.

A PCR assay for the sensitive detection of a transforming fragment of herpes simplex virus type 2 (HSV-2) was developed. Oligonucleotide primers were selected in Xho-2, a transforming region of the BglII N fragment of HSV-2. The assay reached a sensitivity endpoint of 10 copies of the Xho-2 subfragment and did not show cross-reactivity with other herpesviruses, including HSV-1. All 42 HSV-2 isolates scored positive in the assay. The Xho-2 PCR assay was evaluated with 216 clinical specimens and the results were compared with those of cell culture. The best protocol for processing specimens contained in viral transport medium included a centrifugation step followed by cell lysis. Of the 107 specimens positive for HSV-2 by culture, 105 were PCR positive (sensitivity, 98.1%). For one of the two falsely negative samples, beta-globin as well as sequences from the HSV-2 DNA polymerase gene could not be amplified. The other sample scored positive in both of these reactions but was indeterminate in duplicate tests by Xho-2 PCR. Two of 109 HSV-2 culture-negative specimens tested positive in the PCR assay (specificity, 98.2%). The latter two samples tested positive in a PCR test for the HSV-2 DNA polymerase gene. This novel tool was shown to be sensitive and specific for HSV-2 sequences and should allow for the investigation of the role of HSV-2 in genital cancers.

Detection of herpes simplex virus (types 1 and 2) and human herpesvirus 6 DNA in human brain tissue by polymerase chain reaction

Background: Previous studies, using a variety of techniques to determine whether herpes simplex virus type 1 (HSV-1) and/or type 2 (HSV-2) are present in normal brains or have a higher incidence in either multiple sclerosis (MS) or psychiatric disorders have yielded conflicting results. Similarly, studies to examine human brain tissue for human herpes virus 6 (HHV-6) have also proved inconsistent. These discrepancies may be partially due to differences in sensitivity of the methods used. Objectives: To determine whether: (i) Herpesvirus latency is a normal occurrence in the human central nervous system (CNS), (ii) the incidence of latency is higher in either demyelinating diseases or schizophrenia (iii) significant virus reactivation occurs in demyelinating diseases. Study Design: Frozen brain tissue from 7 cases of MS/demyelinating disease, 6 cases of schizophrenia and 27 non-neurological and 3 neurological controls were examined by polymerase chain reaction (PCR) for the presence of HSV-1 DNA. Tissue from the above catagories (except schizophrenia) were also examined for HSV-2 and HHV-6 DNA. In situ hybridization (ISH) and immunocytochemistry (ICC) were carried out in formalin-fixed paraffin sections from selected HSV PCR positive cases, including a case of HSV encephalitis (HSE). Results: Cases from all groups were found to be positive for HSV-1 by PCR. Only one case (MS) was found positive for HSV-2, whereas HHV-6 DNA was present in 18 of 23 brains (MS and controls). Only the HSE case gave positive results with ISH and ICC techniques. Conclusions: These results suggest that herpesvirus latency in the human CNS is a common occurrence but there is no obvious correlation with increased incidence in either demyelinating disease or schizophrenia. Furthermore, failure to detect virus by ISH or ICC (except in a case of HSE) indicates lack of any significant virus reactivation in demyelinating diseases.

Branched structures in the intracellular DNA of herpes simplex virus type 1.

Herpes simplex virus type 1 (HSV-1) replication produces large intracellular DNA molecules that appear to be in a head-to-tail concatemeric arrangement. We have previously suggested (A. Severini, A.R. Morgan, D.R. Tovell, and D.L.J. Tyrrell, Virology 200:428-435, 1994) that these DNA species may have a complex branched structure. We now provide direct evidence for the presence of branches in the high-molecular-weight DNA produced during HSV-1 replication. On neutral agarose two-dimensional gel electrophoresis, a technique that allows separation of branched restriction fragments from linear fragments, intracellular HSV-1 DNA produces arches characteristic of Y junctions (such as replication forks) and X junctions (such as merging replication forks or recombination intermediates). Branched structures were resolved by T7 phage endonuclease I (gene 3 endonuclease), an enzyme that specifically linearizes Y and X structures. Resolution was detected by the disappearance of the arches on two-dimensional gel electrophoresis. Branched structures were also visualized by electron microscopy. Molecules with a single Y junction were observed, as well as large tangles containing two or more consecutive Y junctions. We had previously shown that a restriction enzyme which cuts the HSV-1 genome once does not resolve the large structure of HSV-1 intracellular DNA on pulsed-field gel electrophoresis. We have confirmed that result by using sucrose gradient sedimentation, in which both undigested and digested replicative intermediates sediment to the bottom of the gradient. Taken together, our experiments show that the intracellular HSV-1 DNA is held together in a large complex by frequent branches that create a network of replicating molecules. The fact that most of these branches are Y structures suggests that the network is held together by frequent replication forks and that it resembles the replicative intermediates of bacteriophage T4. Our findings add complexity to the simple model of rolling-circle DNA replication, and they pose interesting questions as to how the network is formed and how it is resolved for packaging into progeny virions.

Quantitation of herpes simplex virus type 1 DNA and latency-associated transcripts in rabbit trigeminal ganglia demonstrates a stable reservoir of viral nucleic acids during latency

In this investigation we determined the dynamics of herpes simplex virus type 1 (HSV-1) DNA and latency-associated transcripts (LAT) in the latently infected rabbit trigeminal ganglion. Rabbit eyes were infected with either the McKrae strain or the l7Syn+ strain of HSV-1. Rabbits were sacrificed between 5 and 360 days after infection and their trigeminal ganglia were analyzed for the number of HSV DNA genomes and the number of neuronal cells expressing LAT. There was no statistically significant change in the number of HSV genomes or the number of neuronal cells expressing LAT in these ganglia between 20 and 360 days after infection. For both strains, the amount of HSV DNA averaged 16.8 genomes per 100 cells, and 9.2% of the neurons expressed LAT. There were 17 to 34 HSV genomes per LAT-expressing neuronal cell. The number of LAT-expressing neurons did not change over the 360 days. Spontaneous reactivation (HSV-1 recovery in tear film) and recurrence (HSV-1-specific epithelial lesions) occurred during the period of this study; however, these events did not alter the quantity of HSV-1 DNA or the number of LAT-expressing cells. These results suggest that after the latent infection is established, the viral DNA in the ganglia does not replicate to any measurable extent over long periods of latency, since no significant change in the number of HSV genomes occurs. The results also suggest that only a very small number of latently infected neuronal cells are needed to produce infectious HSV-1 during reactivation.

Nuclear Domain 10 as Preexisting Potential Replication Start Sites of Herpes Simplex Virus Type-1

The herpes simplex virus type 1 (HSV-1) nuclear replication cycle begins at localized sites, but it has remained unclear whether these sites are associated with any defined nuclear structure. We have previously shown that during infection, the HSV-1 immediate-early protein ICP0 dispersed proteins associated with ND10, nuclear sites that contain high concentrations of PML and other potentially regulatory proteins and correspond to the ultrastructurally defined nuclear bodies. Usingin situhybridization and immunohistochemical techniques, we found that ICP0 mutants of HSV-1 replicate in the close proximity with ND10, but increasing replication sites develop away from these nuclear structures. Input wild-type HSV-1 DNA was found preferentially adjacent to ND10 before ICP0 modified these nuclear structures and did not colocalize with ICP8 containing so-called prereplication sites. The sites where HSV-1 can begin replication then need to be redefined as preexisting potential replication sites. Viral RNA was also found associated with ND10 before early protein synthesis (ICP8), suggesting that input virus genomes are deposited at ND10 before they start replication. The deposition of input viral DNA at ND10 is virus gene expression independent, probably indicating cell regulation of this process. Taken together, these data demonstrate that some very early processes of the nuclear viral replication cycle happen in close proximity or at the periphery of ND10. The localization of input HSV-1 to ND10 represents a new host–virus interaction and provides an unexpected functional property for this large nuclear site.

2] Expression, purification, and characterization of the herpes simplex virus type-1 DNA polymerase

2] Expression, purification, and characterization of the herpes simplex virus type-1 DNA polymerase

Mode of action of (R)-9-[4-hydroxy-2-(hydroxymethyl)butyl]guanine against herpesviruses.

The activity, metabolism, and mode of action of (R)-9-[4-hydroxy-2-(hydroxymethyl)butyl]guanine (H2G) against herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) and varicella-zoster virus (VZV) were studied. Compared to acyclovir (ACV), H2G has superior activity against VZV (50% inhibitory concentration of 2.3 microM) and Epstein-Barr virus (50% inhibitory concentration of 0.9 microM), comparable activity against HSV-1, and weaker activity against HSV-2. The antiviral effect on HSV-1 showed persistence after removal of compound. H2G was metabolized to its mono-, di- and triphosphate derivatives in virus-infected cells, with H2G-triphosphate being the predominant product. Only small amounts of H2G-triphosphate were detected in uninfected cells (1 to 10 pmol/10(6) cells), whereas the level in HSV-1-infected cells reached 1,900 pmol/10(6) cells. H2G was a substrate for all three viral thymidine kinases and could also be phosphorylated by mitochondrial deoxyguanosine kinase. The intracellular half-life of H2G-triphosphate varied in uninfected (2.5 h) and infected (HSV-1, 14 h; VZV, 3.7 h) cells but was always longer than the half-life of ACV-triphosphate (1 to 2 h). H2G-triphosphate inhibited HSV-1, HSV-2, and VZV DNA polymerases competitively with dGTP (Ki of 2.8, 2.2, and 0.3 microM, respectively) but could not replace dGTP as a substrate in a polymerase assay. H2G was not an obligate chain terminator but would only support limited DNA chain extension. Only very small amounts of radioactivity, which were too low to be identified by high-performance liquid chromatography analysis of the digested DNA, could be detected in purified DNA from uninfected cells incubated with [3H]H2G. Thus, H2G acts as an anti-herpesvirus agent, particularly potent against VZV, by formation of high concentrations of relatively stable H2G-triphosphate, which is a potent inhibitor of the viral DNA polymerases.

Herpes simplex Virus dna in normal corneas: Persistence without viral shedding from ganglia

Herpes simplex virus type 1 (HSV-1) DNA has been shown to persist in the cornea not only after inoculation of experimental animals but also in surgical samples from patients with herpes keratitis. The further observation of corneal HSV-1 DNA in subjects without known HSV eye disease prompted the present study of the presence and distribution of HSV-1 in eye bank corneas. Prior to DNA extraction, the corneas were trephined, separating the central and peripheral cornea. With polymerase chain reaction (PCR) for HSV-1 thymidine kinase (TK) and glycoprotein D (gD) gene sequences, we found HSV-1 in 10 of 24 eye bank corneas, from the 4 mm wide corneal rim in 8 eyes and from the 8 mm diameter central cornea in 2 eyes. In 9 subjects, both eyes were assayed, and HSV-1 was detected in 6 subjects. In only one subject was HSV-1 detected in both eyes and in only one subject was HSV-1 detected in the central and peripheral cornea of the same eye. The biological role of HSV-1 DNA corneal sequences is unknown. To investigate this, a rabbit animal model was established by transplantation of corneas containing viral DNA sequences in HSV-1 naive recipients. Followed for 5 months, there was no evidence of sheeding of HSV-1 in the tear film or seroconversion of the recipient rabbits. At the end of this time, HSV-1 DNA was detected in the corneal graft at a similar intensity to the PCR signal from the donor rims.(ABSTRACT TRUNCATED AT 250 WORDS)

Stereospecificity of human DNA polymerases alpha, beta, gamma, delta and epsilon, HIV-reverse transcriptase, HSV-1 DNA polymerase, calf thymus terminal transferase and Escherichia coli DNA polymerase I in recognizing D- and L-thymidine 5'-triphosphate as substrate.

L-beta-Deoxythymidine (L-dT), the optical enantiomer of D-beta-deoxythymidine (D-dT), and L-enantiomers of nucleoside analogs, such as 5-iodo-2'-deoxy-L-uridine (L-IdU) and E-5-(2-bromovinyl)-2'-deoxy-L-uridine (L-BVdU), are not recognized in vitro by human cytosolic thymidine kinase (TK), but are phosphorylated by herpes simplex virus type 1 (HSV-1) TK and inhibit HSV-1 proliferation in infected cells. Here we report that: (i) L-dT is selectively phosphorylated in vivo to L-dTMP by HSV-1 TK and L-dTMP is further phosphorylated to the di- and triphosphate forms by non-stereospecific cellular kinases; (ii) L-dTTP not only inhibits HSV-1 DNA polymerase in vitro, but also human DNA polymerase alpha, gamma, delta and epsilon, human immunodeficiency virus reverse transcriptase (HIV-1 RT), Escherichia coli DNA polymerase 1 and calf thymus terminal transferase, although DNA polymerase beta was resistant; (iii) whereas DNA polymerase beta, gamma, delta and epsilon are unable to utilize L-dTTP as a substrate, the other DNA polymerases clearly incorporate at least one L-dTMP residue, with DNA polymerase alpha and HIV-1 RT able to further elongate the DNA chain by catalyzing the formation of the phosphodiester bond between the incorporated L-dTMP and an incoming L-dTTP; (iv) incorporated L-nucleotides at the 3'-OH terminus make DNA more resistant to 3'-->5' exonucleases. In conclusion, our results suggest a possible mechanism for the inhibition of viral proliferation by L-nucleosides.

The Varicella-Zoster virus origin-binding protein can substitute for the herpes simplex virus origin-binding protein in a transient origin-dependent DNA replication assay in insect cells

We isolated two recombinant baculoviruses each of which expresses a varicella-zoster virus (VZV) homolog of one ofthe seven herpes simplex virus type 1 (HSV-1) genes required for DNA replication. We performed transient origin-dependent DNA replication assays in insect cells in which we substituted a baculovirus which expresses a VZV protein for a baculovirus which expresses its HSV homolog. VZV gene 51 protein was found to be able to support origin-dependent DNA synthesis when it was substituted for UL9, the HSV-1 origin-binding protein (01313). This occurred whether an HSV-1 or a VZV origin-containing plasmid was used in the assay. These results suggest that VZV gene 51 protein is able to interact with the HSV replication machinery, and in light of the extensive structural divergence of these proteins, it suggests that initiation of VZV and HSV-1 DNA synthesis may involve a limited number of interactions between the OBP and other replication factors. Substitution of infected-cell protein 8 (ICP8), the major single-stranded DNA-binding protein of HSV-1, with VZV gene 29 protein, however, did not result in amplification of plasmids containing either an HSV-1 or a VZV origin. In the absence of ICP8, addition of both VZV gene 51 protein and gene 29 protein was also negative for origin-dependent replication whether or not UL9 was present. Although demonstration that our baculovirus-expressed VZV gene 29 protein is functional for DNA replication will await development of a VZV replication system, our results suggest that VZV gene 29 protein is unable to interact functionally with one or more of the HSV replication proteins. This approach should contribute to efforts to define the interactions among the alphaherpesvirus DNA replication proteins.

The replicating intermediates of herpes simplex virus type 1 DNA are relatively short.

Herpes simplex virus type 1 (HSV-1) replication is thought to occur via a rolling circle type of mechanism, generating large DNA concatemers from which unit length genomes are subsequently cleaved. In this report, we have employed field inversion gel electrophoresis (FIGE), Southern blot hybridization, and endonuclease digestion, to identify and characterize these DNAs. Two species of HSV-1 DNA: (1) genome-length and (2) DNA that remained at the electrophoresis origin (referred to as well-associated DNA) were detected. To ascertain that the latter was large in size and not virion DNA trapped at the origin with high molecular weight cellular DNA, the infected cell DNA was digested with a restriction enzyme that does not cut the viral DNA. In order to do this HSV-1 strain 1702, lacking any XbaI sites in its genome, was utilized. After digestion of samples with XbaI, and FIGE, cellular DNA was seen to migrate into the gel; however, the viral DNA remained in the sample wells. Pulse labeling experiments showed that this large DNA was processed to 150 kb genome lengths. Endonuclease digestion of the well-associated DNA revealed that it contained a greater ratio of joint to terminal fragments than virion DNA-a characteristic of long concatemers. Quantitation of the terminal fragments revealed mainly L termini. Surprisingly, the ratio of joint to terminal fragments was 2.5 suggesting that the lengths of concatemers were short (in the order of 1-2 genome lengths) and that the well association was due to conformation rather than concatemeric length. Because one of these genome lengths is present as the replication intermediate, the growing tail must be less than genome length. Thus genome lengths must be processed from the replication intermediate soon after they are completed.

Long term herpes simplex virus type 1 infection of nerve growth factor-treated PC12 cells

The behaviour of herpes simplex virus type 1 (HSV-1) strain 17 in tissue cultures of PC12 cells treated with nerve growth factor (NGF) was studied. PC12 cells respond to NGF by ceasing to proliferate and extending long neurites. After differentiation with NGF, cultures were infected with HSV-1 and maintained in the presence of the hormone for several weeks. These long-term infected cultures were tested for HSV DNA, transcripts and the ability to produce virus, before and after NGF removal. Before NGF removal, the cultures were characterized by little or no virus production and the presence of HSV-1 DNA in a predominantly endless form. In situ analysis of long-term infected cultures revealed latency-associated transcript expression in only a portion of the cells. However, as shown by an infectious centre assay, virus was present in almost all cells in the population. Moreover, removal of NGF from long-term cultures resulted in the appearance of significantly increased amounts of virus in the media. The degree to which this system resembles HSV latency in vivo is discussed.

Interaction of herpes simplex virus type 1 DNA polymerase and the UL42 accessory protein with a model primer template.

Genetic and biochemical studies have shown that the products of the herpes simplex virus type 1 (HSV-1) DNA polymerase (UL30) and UL42 genes are both required for viral DNA replication. A number of studies have previously suggested that these two proteins specifically interact, and more recent studies have confirmed that the viral DNA polymerase from HSV-1-infected cells consists of a heterodimer of the UL30 (Pol; the catalytic subunit) and UL42 polypeptides. A comparison of the catalytic properties of the Pol-UL42 complex with those of the isolated subunits of the enzyme purified from recombinant baculovirus-infected insect cells indicated that the Pol-UL42 complex is more highly processive than Pol alone on singly primed M13 single-stranded substrates. The results of these studies are consistent with the idea that the UL42 polypeptide is an accessory subunit of the HSV-1 DNA polymerase that acts to increase the processivity of polymerization. Preliminary experiments suggested that the increase in processivity was accompanied by an increase in the affinity of the polymerase for the ends of linear duplex DNA. We have further characterized the effect of the UL42 polypeptide on a defined hairpin primer template substrate. Gel shift and filter binding studies show that the affinity of the Pol catalytic subunit for the 3' terminus of the primer template increases 10-fold in the presence of UL42. DNase I footprinting experiments indicate that the Pol catalytic subunit binds to the primer template at a position that protects 14 bp of the 3' duplex region and an adjacent 18 bases of the single-stranded template. The presence of the UL42 polypeptide results in the additional protection of a contiguous 5 to 14 bp in the duplex region but does not affect the 5' position of the Pol subunit. Free UL42 protects the entire duplex region of the substrate but does not bind to the single-stranded region. Taken together, these results suggest that the increase in processivity in the presence of UL42 is related to the double-stranded DNA-binding activity of free UL42 and that the role of UL42 in the DNA polymerase complex is to act as a clamp, decreasing the probability that the polymerase will dissociate from the template after each cycle of catalysis.

Excision of DNA fragments corresponding to the unit-length a sequence of herpes simplex virus type 1 and terminus variation predominate on one side of the excised fragment

DNA fragments corresponding to the unit-length a sequence of herpes simplex virus type 1 (HSV-1) were identified in HSV-1 DNA preparations extracted by the method of Hirt. The DNA fragments were molecularly cloned, and nucleotide sequences were determined. Most termini of the fragments were at sites on DR1 corresponding to the termini of linear HSV-1 DNA generated by the cleavage-packaging system. In one-step growth experiments, DNA fragments of the unit-length a sequence appeared simultaneously with the termini of linear HSV-1 DNAs produced by cleavage of circular and concatemeric DNAs. Therefore, excision of the unit-length a sequence appeared closely related to the cleavage-packaging system. Termini of the excised DNA fragments of the variant a sequence with two DR2 arrays varied on the L-component side, while termini on the S-component side were at the site on DR1 corresponding to the authentic cleavage site. It is thus assumed that the cleavage-packaging system functions adequately on the DR1 second distal from the S component, and cleavages of other DR1 are rare and less accurate. If this notion is tenable, then most termini on the S-component side of the excised DNA fragments are derived from the second DR1 properly cleaved and should be constant, while termini on the L-component side are from regions on and around the DR1 third distal from the S component and may be variable. Cleavage of DR1 is likely to be affected by the topological relationship with the S component.

Herpes simplex virus type 1 DNA persistence, progressive disease and transgenic immediate early gene promoter activity in chronic corneal infections in mice.

We have used a mouse model system and the corneal route of inoculation to examine the issue of extra-neuronal persistence of herpes simplex virus type 1 (HSV-1). HSV-1 strain F DNA and inflammatory lesions were detected in corneal tissue of mice at 5, 11, 23, 37 and 60 days post-infection (p.i.) Viral DNA was localized by in situ PCR to epithelial cells and less frequently to cells in the stroma of the cornea. Viral proteins were not detected in the cornea and virus could not be isolated from tissue homogenates after 11 days p.i. even though histopathological lesions became progressively more severe at 37 and 60 days p.i. The DNA-containing cells were usually adjacent to the sites of inflammation or within these sites in the chronic stage (23, 37 and 60 days p.i.). In contrast to strain F, persistence of HSV-1 strain KOS DNA and inflammatory lesions were not detected after 11 days p.i.; this result suggests that the long-term persistence of HSV-1 DNA and the development of inflammatory lesions are virus strain-dependent. We tested for the possibility of transgenic HSV-1 immediate early gene (ICP4) promoter activity in chronically infected corneas of transgenic mice containing the ICP4 promoter fused to the bacterial beta-galactosidase coding sequence. Our results indicated that the chimeric transgene was expressed in the cornea at 5, 11, 23, 37 and 41 days p.i. Possible explanations for these results and mechanisms for the generation of the chronic inflammatory lesions are discussed. The properties of chronic HSV infections in the cornea may be similar to those which have been described for persistent or defective viral infections in other systems.