Mutagenicity of herpes simplex virus type 1 in a shuttle vector

The shuttle vector plasmid pZ189 was used to find the kinds of mutations that are induced by herpes simplex virus type-1 (HSV-1). In cells infected by HSV-1 the frequency of mutation in supF gene, the mutagenesis marker, was increased over background by from two- to seven-fold, reaching 0.14-0.45%. No increase was induced by infection by vaccinia virus under the same conditions. Mutagenesis was an early event, showing a four-fold increase in mutation frequency at only two hours after infection, and peaking at a seven-fold increase at four hours after infection. DNA sequencing and gel electrophoresis analysis were performed on 105 HSV-1 induced mutants and 65 spontaneous mutants and provided the following information: (1) A change in plasmid size was seen in 54% of HSV-1 related mutants, compared with only 37% of spontaneous mutants. (2) Among point mutations, the predominant type was G:C to A:T transition, which accounted for 51% of point mutations in mutants isolated from cells infected with HSV-1, and 32% of point mutations in spontaneous mutants. (3) Deletions of DNA were seen in HSV-1 related mutants at a frequency of 40%, compared with 29% in spontaneous mutants. The HSV-1 related deletions were about half the length of spontaneous mutants and three contained short filler sequences. (4) Fifteen (15%) of HSV-1 induced mutants revealed the altered restriction patterns on agarose gel electrophoresis analysis and were due either to rearrangements of plasmid DNA, and/or to insertion of sequences derived from chromosomal DNA (seven plasmids). No insertions of DNA from HSV-1 were detected. Among spontaneous mutants, only 5 (7.7%) were rearrangements and none had inserted chromosomal DNA. (5) DNA sequence analysis of seven plasmids with inserted chromosomal DNA revealed that four cases had repetitive DNA sequences integrated and the other three were unidentified sequences from the GenBank database. Three repetitive DNA included $\alpha$ satellite, Alu and KpnI family sequences. The other sequence was identified as tRNA-like component. The observed mutations have implications for the mechanism of malignant transformation of cells by HSV-1. ^

Gene therapy of cancer: Mechanisms of ``bystander effect'' killing in cells expressing the herpes simplex virus-thymidine kinase gene and its potential clinical applications

At the fore-front of cancer research, gene therapy offers the potential to either promote cell death or alter the behavior of tumor-cells. One example makes use of a toxic phenotype generated by the prodrug metabolizing gene, thymidine kinase (HSVtk) from the Herpes Simplex Virus. This gene confers selective toxicity to a relatively nontoxic prodrug, ganciclovir (GCV). Tumor cells transduced with the HSVtk gene are sensitive to 1-50 $\mu$M GCV; normal tissue is insensitive up to 150-250 $\mu$M GCV. Utilizing these different sensitivities, it is possible to selectively ablate tumor cells expressing this gene. Interestingly, if a HSVtk$\sp+$ expressing population is mixed with a HSVtk$\sp-$ population at high density, all the cells are killed after GCV administration. This phenomenon for killing all neighboring cells is termed the "bystander effect", which is well documented in HSVtk$\sp-$ GCV systems, though its exact mechanism of action is unclear.^ Using the mouse colon carcinoma cell line CT26, data are presented supporting possible mechanisms of "bystander effect" killing of neighboring CT26-tk$\sp-$cells. A major requirement for bystander killing is the prodrug GCV: as dead or dying CT26tk$\sp+$ cells have no toxic effect on neighboring cells in its absence. In vitro, it appears the bystander effect is due to transfer of toxic GCV-metabolites, through verapamil sensitive intracellular-junctions. Additionally, possible transfer of the HSVtk enzyme to bystander cells after GCV addition, may play a role in bystander killing. A nude mouse model suggests that in a 50/50 (tk$\sp+$/tk$\sp-$) mixture of CT26 cells the bystander eradication of tumors does not involve an immune component. Additionally in a possible clinical application, the "bystander effect" can be directly exploited to eradicate preexisting CT26 colon carcinomas in mice by intratumoral implantation of viable or lethally irradiated CT26tk$\sp+$ cells and subsequent GCV administration. Lastly, an application of this toxic phenotype gene to a clinical marking protocol utilizing a recombinant adenoviral vector carrying the bifunctional protein GAL-TEK to eradicate spontaneously-arisen or vaccine-induced fibrosarcomas in cats is demonstrated. ^

Herpes simplex virus vector-mediated expression of Bcl-2 prevents 6-hydroxydopamine-induced degeneration of neurons in the substantia nigra in vivo

6-Hydroxydopamine (6-OHDA) is widely used to selectively lesion dopaminergic neurons of the substantia nigra (SN) in the creation of animal models of Parkinson’s disease. In vitro, the death of PC-12 cells caused by exposure to 6-OHDA occurs with characteristics consistent with an apoptotic mechanism of cell death. To test the hypothesis that apoptotic pathways are involved in the death of dopaminergic neurons of the SN caused by 6-OHDA, we created a replication-defective genomic herpes simplex virus-based vector containing the coding sequence for the antiapoptotic peptide Bcl-2 under the transcriptional control of the simian cytomegalovirus immediate early promoter. Transfection of primary cortical neurons in culture with the Bcl-2-producing vector protected those cells from naturally occurring cell death over 3 weeks. Injection of the Bcl-2-expressing vector into SN of rats 1 week before injection of 6-OHDA into the ipsilateral striatum increased the survival of neurons in the SN, detected either by retrograde labeling of those cells with fluorogold or by tyrosine hydroxylase immunocytochemistry, by 50%. These results, demonstrating that death of nigral neurons induced by 6-OHDA lesioning may be blocked by the expression of Bcl-2, are consistent with the notion that cell death in this model system is at least in part apoptotic in nature and suggest that a Bcl-2-expressing vector may have therapeutic potential in the treatment of Parkinson’s disease.

Social stress and the reactivation of latent herpes simplex virus type 1

Psychological stress is thought to contribute to reactivation of latent herpes simplex virus (HSV). Although several animal models have been developed in an effort to reproduce different pathogenic aspects of HSV keratitis or labialis, until now, no good animal model existed in which application of a psychological laboratory stressor results in reliable reactivation of the virus. Reported herein, disruption of the social hierarchy within colonies of mice increased aggression among cohorts, activated the hypothalamic-pituitary-adrenal axis, and caused reactivation of latent HSV type 1 in greater than 40% of latently infected animals. However, activation of the hypothalamic-pituitary-adrenal axis using restraint stress did not activate the latent virus. Thus, the use of social stress in mice provides a good model in which to investigate the neuroendocrine mechanisms that underlie behaviorally mediated reactivation of latent herpesviruses.

BAC-VAC, a novel generation of (DNA) vaccines: A bacterial artificial chromosome (BAC) containing a replication-competent, packaging-defective virus genome induces protective immunity against herpes simplex virus 1

This study aimed to exploit bacterial artificial chromosomes (BAC) as large antigen-capacity DNA vaccines (BAC-VAC) against complex pathogens, such as herpes simplex virus 1 (HSV-1). The 152-kbp HSV-1 genome recently has been cloned as an F-plasmid-based BAC in Escherichia coli (fHSV), which can efficiently produce infectious virus progeny upon transfection into mammalian cells. A safe modification of fHSV, fHSVΔpac, does not give rise to progeny virus because the signals necessary to package DNA into virions have been excluded. However, in mammalian cells fHSVΔpac DNA can still replicate, express the HSV-1 genes, cause cytotoxic effects, and produce virus-like particles. Because these functions mimic the lytic cycle of the HSV-1 infection, fHSVΔpac was expected to stimulate the immune system as efficiently as a modified live virus vaccine. To test this hypothesis, mice were immunized with fHSVΔpac DNA applied intradermally by gold-particle bombardment, and the immune responses were compared with those induced by infection with disabled infectious single cycle HSV-1. Immunization with either fHSVΔpac or disabled infectious single cycle HSV-1 induced the priming of HSV-1-specific cytotoxic T cells and the production of virus-specific antibodies and conferred protection against intracerebral injection of wild-type HSV-1 at a dose of 200 LD50. Protection probably was cell-mediated, as transfer of serum from immunized mice did not protect naive animals. We conclude that BAC-VACs per se, or in combination with genetic elements that support replicative amplification of the DNA in the cell nucleus, represent a useful new generation of DNA-based vaccination strategies for many viral and nonviral antigens.

Humoral response to herpes simplex virus is complement-dependent

The complement system represents a cascade of serum proteins, which provide a major effector function in innate immunity. Recent studies have revealed that complement links innate and adaptive immunity via complement receptors CD21/CD35 in that it enhances the B cell memory response to noninfectious protein antigens introduced i.v. To examine the importance of complement for immune responses to virus infection in a peripheral tissue, we compared the B cell memory response of mice deficient in complement C3, C4, or CD21/CD35 with wild-type controls. We found that the deficient mice failed to generate a normal memory response, which is characterized by a reduction in IgG antibody and germinal centers. Thus, complement is important not only in the effector function of innate immunity but also in the stimulation of memory B cell responses to viral-infected cell antigens in both blood and peripheral tissues.

Severe genital herpes infections in HIV-infected individuals with impaired herpes simplex virus-specific CD8+ cytotoxic T lymphocyte responses

The specific mechanisms underlying the varied susceptibility of HIV-infected (HIV+) individuals to opportunistic infections (OI) are still incompletely understood. One hypothesis is that quantitative differences in specific T cell responses to a colonizing organism determine the development of an AIDS-defining OI. We evaluated this hypothesis for herpes simplex virus (HSV) infection, a common OI in HIV+ patients. Using limiting dilution analyses, the frequency of HSV-specific CD8+ cytotoxic T lymphocyte precursors (pCTL) and proliferative precursors were quantitated in peripheral blood mononuclear cells from 20 patients coinfected with HIV and HSV-2. The frequency of HSV-specific CD8+ pCTL in HSV+HIV+ individuals was significantly lower than in HSV+HIV− individuals (1 in 77,000 vs. 1 in 6,000, P = .0005) and was not different than in HSV-HIV− individuals (1 in 100,000, P = .24). HIV+ patients who suffered more severe genital herpes recurrences had significantly lower HSV-specific CD8+ pCTL frequencies than those patients with mild recurrences (1 in 170,000 vs. 1 in 26,000, P = .03). In contrast, no significant difference was seen in proliferative precursor frequencies between those patients with mild vs. severe genital herpes (1 in 3,800 vs. 1 in 6,600, P > .5). Quantitative differences in pCTL frequency to HSV appear to be the most important host factor influencing the frequency and severity of HSV reactivation in HIV+ patients. Studies to reconstitute such immunity, especially in people with acyclovir-resistant HSV, appear warranted.

The product of ORF O located within the domain of herpes simplex virus 1 genome transcribed during latent infection binds to and inhibits in vitro binding of infected cell protein 4 to its cognate DNA site

The partially overlapping ORF P and ORF O are located within the domains of the herpes simplex virus 1 genome transcribed during latency. Earlier studies have shown that ORF P is repressed by infected cell protein 4 (ICP4), the major viral regulatory protein, binding to its cognate site at the transcription initiation site of ORF P. The ORF P protein binds to p32, a component of the ASF/SF2 alternate splicing factors; in cells infected with a recombinant virus in which ORF P was derepressed there was a significant decrease in the expression of products of key regulatory genes containing introns. We report that (i) the expression of ORF O is repressed during productive infection by the same mechanism as that determining the expression of ORF P; (ii) in cells infected at the nonpermissive temperature for ICP4, ORF O protein is made in significantly lower amounts than the ORF P protein; (iii) the results of insertion of a sequence encoding 20 amino acids between the putative initiator methionine codons of ORF O and ORF P suggest that ORF O initiates at the methionine codon of ORF P and that the synthesis of ORF O results from frameshift or editing of its RNA; and (iv) glutathione S-transferase–ORF O fusion protein bound specifically ICP4 and precluded its binding to its cognate site on DNA in vitro. These and earlier results indicate that ORF P and ORF O together have the capacity to reduce the synthesis or block the expression of regulatory proteins essential for viral replication in productive infection.

A herpes simplex virus 1 recombinant lacking the glycoprotein G coding sequences is defective in entry through apical surfaces of polarized epithelial cells in culture and in vivo

During infection of a new host, the first surfaces encountered by herpes simplex viruses are the apical membranes of epithelial cells of mucosal surfaces. These cells are highly polarized, and the protein composition of their apical and basolateral membranes are very different, so that different viral entry pathways have evolved for each surface. To determine whether the viral glycoprotein G (gG) is specifically required for efficient infection of a particular surface of polarized cells, apical and basal surfaces were infected with wild-type virus or a gG deletion mutant. After infection of polarized cells in culture, the gG− virus was deficient in infection of apical surfaces but was able to infect cells through basal membranes, replicate, and spread into surrounding cells. The gG-dependent step in apical infection was a stage beyond attachment. After in vivo infection of apical surfaces of epithelial cells of nonscarified mouse corneas, infection by glycoprotein C− or gG− virus was considerably reduced as compared with that observed after infection with wild-type virus. In contrast, when corneas were scarified, allowing virus access to other cell surfaces, the gG and glycoprotein C deletion mutants infected eyes as efficiently as wild-type viruses. A secondary mutation allowing infection of apical surfaces by gG− virus arose readily during passage of the virus in nonpolarized cells, indicating that either the gG-dependent step of apical infection can be bypassed or that another viral protein can acquire the same function.

Highly efficient electro-gene therapy of solid tumor by using an expression plasmid for the herpes simplex virus thymidine kinase gene

We report successful electro-gene therapy (EGT) by using plasmid DNA for tumor-bearing mice. Subcutaneously inoculated CT26 tumor was subjected to EGT, which consists of intratumoral injection of a naked plasmid encoding a marker gene or a therapeutic gene, followed by in vivo electroporation (EP). When this treatment modality is carried out with the plasmid DNA for the green fluorescent protein gene, followed by in vivo EP with the optimized pulse parameters, numerous intensely bright green fluorescent signals appeared within the tumor. EGT, by using the “A” fragment of the diphtheria toxin gene significantly inhibited the growth of tumors, by about 30%, on the flank of mice. With the herpes simplex virus thymidine kinase gene, followed by systemic injection of ganciclovir, EGT was far more effective in retarding tumor growth, varying between 50% and 90%, compared with the other controls. Based on these results, it appears that EGT can be used successfully for treating murine solid tumors.

The role of cdc2 in the expression of herpes simplex virus genes

Earlier reports have shown that cdc2 kinase is activated in cells infected with herpes simplex virus 1 and that the activation is mediated principally by two viral proteins, the infected cell protein 22 (ICP22) and the protein kinase encoded by UL13. The same proteins are required for optimal expression of a subset of late (γ2) genes exemplified by US11. In this study, we used a dominant-negative cdc2 protein to determine the role of cdc2 in viral gene expression. We report the following. (i) The cdc2 dominant-negative protein had no effect in the synthesis and accumulation of at least two α-regulatory proteins (ICP4 and ICP0), two β-proteins (ribonucleotide reductase major subunit and single-stranded DNA-binding protein), and two γ1-proteins (glycoprotein D and viral protease). US11, a γ2-protein, accumulated only in cells in which cdc2 dominant-negative protein could not be detected or was made in very small amounts. (ii) The sequence of amino acids predicted to be phosphorylated by cdc2 is present in at least 27 viral proteins inclusive of the regulatory proteins ICP4, ICP0, and ICP22. In in vitro assays, we demonstrated that cdc2 specifically phosphorylated a polypeptide consisting of the second exon of ICP0 but not a polypeptide containing the sequence of the third exon as would be predicted from the sequence analysis. We conclude that cdc2 is required for optimal expression of a subset of γ2-proteins whose expression is also regulated by the viral proteins (ICP22 and UL13) that mediate the activation of cdc2 kinase.

The essential protein encoded by the UL31 gene of herpes simplex virus 1 depends for its stability on the presence of UL34 protein

To pursue an earlier observation that the protein encoded by the UL34 gene binds to intermediate chain of dynein, we constructed a series of mutants from which sequences encoding the entire protein (ΔUL34) or amino-terminal [UL34Δ(3–119)] or carboxyl-terminal [UL34Δ(245–275)] domains were deleted. The mutant lacking the sequence encoding the carboxyl-terminal domain grew in all cell lines tested. The two other mutants replicated only in cell type-dependent manner and poorly. Rescue of ΔUL34 mutant with a fragment that does not encompass the UL31 ORF restored wild-type phenotype. UL34 protein interacts physically with UL31, and the UL31 deletion mutant appears to have a phenotype similar to that of UL34 deletion mutant. Experiments designed to determine whether the phenotypes of the deletion mutants have a common base revealed that cells infected with the ΔUL34 mutant accumulate UL31 RNA but not the corresponding protein. The UL31 protein accumulated, however, to near wild-type virus-infected cell levels in cells infected with ΔUL34 mutant and treated with the MG132 proteosomal inhibitor at 6 h after infection. This is evidence that the stability of an essential viral protein requires the presence of another protein. The observation raises the bar for identification of gene function on the basis of analyses of the phenotype of mutants in which the gene has been deleted or rendered inoperative.