Herpes simplex virus type 1-induced hydrocephalus in mice.

Adult ICR/Slc or BALB/c mice developed hydrocephalus when attenuated herpes simplex virus type 1 (HSV-1) (strain Ska) was injected intracerebrally 2 to 4 weeks earlier and then after mice were challenged with the same virus or virulent HSV-1. Initial inoculation of the Ska strain elicited acute meningitis and ependymitis with transient mild hydrocephalus. Viral antigen was seen in the meninges and subependymal areas, and the virus was titrated during the acute phase of infection. After the second virus inoculation, more prominent inflammation was evoked in the same area, and the animals developed hydrocephalus, although viral antigen and infectious virus were hardly detected. When the mice were immunosuppressed with cyclophosphamide, they ceased to develop hydrocephalus. BALB/c nude mice did not show the same pathology, even though they were treated in the same way. When irradiated mice, which had been infected with the Ska strain intracerebrally 2 weeks earlier, received syngeneic immune spleen cells, they developed hydrocephalus. The T-cell nature of the effector cells was confirmed by the elimination of the pathology after treatment of the donor cells with anti-Thy-1.2 plus complement. No hydrocephalic mice were observed after treatment of the donor cells with anti-Lyt-1.2 plus complement, which gave further evidence of the T-cell nature of the effector cells as the Lyt-1+.2+ antigen-bearing subsets. Intervals between priming and challenge virus inoculation could be more than 18 months. The presence of purified HSV-1 envelope protein was feasible for the development of the hydrocephalic animals.     

Herpes simplex virus infection in normal and mutant human fibroblast cultures

Reproductive efficiency of herpes simplex virus type 1 has been determined in several normal and mutant human skin fibroblast lines. The mutant cell lines were derived from individuals diagnosed as having Duchenne muscular dystrophy, a disease thought to involve genetically determined membrane defects. Yields of infectious virus, determined by plaque assay on rabbit kidney cell monolayers, were consistently lower from dystrophic cells than from normal cells. The yield from dystrophic lines was 3–20% of the normal. The time course of production of infectious particles did not appear to vary between dystrophic and normal host cells. Also, the initiation of replication of the viral genome did not appear to be altered in the dystrophic lines. It is proposed that a late maturation function is involved in the lower virus productivity in dystrophic cells.     

Temperature sensitive mutant of Herpes simplex virus type 1. II. Neurovirulence and latency]

The course of acute infection of mice with ts mutant or the native strain DNA and the antigens of HSV in brain nerve cells were determined. Virus DNA was detected in brains of all mice in both animal groups while the virus antigens--only in cells of mice infected with the native strain. It can be suggested, therefore, that the ability of ts mutant to replicate in central nervous system of the infected mice is lacking or much lower. The detection of virus nucleic acid 3-5 months after virus infection might indicate a possibility of establishing latent infection. However, ts mutant showed a significantly lower possibility of latency induction, as compared with highly virulent strains. It was found that the mutant ability to induce latent infection was markedly increased when mice were treated with both ts mutant and Depo-Medrol as immunosuppressive agent. This finding shows both a possibility of increase of frequency of latent infections in the state of immunosuppression, and of activation of the latent infection (recurrence of acute form of infection).     

Temperature sensitive mutant of Herpes simplex virus type 1. I. Pathogenicity and immunogenicity]

The aim of the study was to characterize biological features of the sensitive mutant of HSV-1, derived from McIntyre strain by numerous virus passages at lowered replication temperature (28 degrees C). Pathogenicity of obtained ts mutant for inbred mice lines, CFW/Pzh and BALB/cPzh, was determined. Statistically significant decrease in virulence of the mutant for these mouse lines was demonstrated, as compared with the native virus strain, propagated at 37 degrees C. Immunogenic activity of ts mutant of HSV-1 defined by the possibility of mouse protection against infection with high virulent was determined. Mice, which at the time of immunization with ts mutant received Depo-Medrol--an immunosuppressive agent--were also found to be capable of inducing defense mechanisms to infection with the native strain.     

Herpes simplex virus type 1 infection activates the Epstein-Barr virus replicative cycle via a CREB-dependent mechanism

The reactivation of latent Epstein-Barr virus (EBV) to lytic replication is important in pathogenesis and requires virus-host cellular interactions. However, the mechanism underlying the reactivation of EBV is not yet fully understood. In the present study, herpes simplex virus type 1 (HSV-1) was shown to induce the reactivation of latent EBV by triggering BZLF1 expression. The BZLF1 promoter (Zp) was not activated by HSV-1 essential glycoprotein-induced membrane fusion. Nevertheless, Zp was activated within 6 h post HSV-1 infection in virus entry-dependent and replication-independent manners. Using a panel of Zp deletion mutants, HSV-1 was shown to promote Zp through a cyclic adenosine monophosphate (cAMP) response element (CRE) located in ZII. The phosphorylated cAMP response element-binding (phos-CREB) protein, the cellular transactivator that binds to CRE, also increased after HSV-1 infection. By transient transfection, cAMP-dependent protein kinase A and HSV-1 US3 protein were found to be capable of activating Zp in CREB- and CRE-dependent manners. The relationship between EBV activation and HSV-1 infection revealed a possible common mechanism that stimulated latent EBV into lytic cycles in vivo.     

Herpes simplex virus type 1 pathogenicity in footpad and ear skin of mice depends on Langerhans cell density, mouse genetics, and virus strain.

Skin Langerhans cells have been shown to be very efficient in presenting antigens to T-helper cells and stimulating the immune response. The present study demonstrates their essential role in the control of primary herpetic infections in the skin. Two unrelated stimuli (abrasion and steroids) were shown to cause depletion of the Langerhans cells in the murine epidermis, and both caused enhancement of the virulence of herpes simplex type 1 (HSV-1) in the skin. The Langerhans cell density was found to be lower in the skin of the ear than in the footpad. HSV-1 was consistently more virulent when injected into the ear epidermis than in the footpad. Thus, HSV-1 pathogenicity in mouse skin depends on the mouse age and strain, the virus strain, and the state of the epidermal Langerhans cells. These findings are discussed in relation to the antigen-presenting cell function of the Langerhans cells.     

Latent infection of SCID mice with herpes simplex virus 1 and lethal cutaneous lesions in pregnancy.

Some SCID mice survived primary infection with herpes simplex virus 1 without the development of peripheral lesions but established coculture-positive ganglionic latency when a low dose of a wild-type strain was inoculated intracutaneously. The latency was also evidenced by the development of the fatal zosteriform skin lesions and the isolation of the virus during pregnancy. We consider that the viral entry into neurons without successive replication, rather than the arrest of the lytic infection within the cells, is an important mechanism in the establishment of latency.     

Herpes simplex virus type 1 corneal infection results in periocular disease by zosteriform spread

In humans and animal models of herpes simplex virus infection, zosteriform skin lesions have been described which result from anterograde spread of the virus following invasion of the nervous system. Such routes of viral spread have not been fully examined following corneal infection, and the possible pathologic consequences of such spread are unknown. To investigate this, recombinant viruses expressing reporter genes were generated to quantify and correlate gene expression with replication in eyes, trigeminal ganglia, and periocular tissue. Reporter activity peaked in eyes 24 h postinfection and rapidly fell to background levels by 48 h despite the continued presence of viral titers. Reporter activity rose in the trigeminal ganglia at 60 h and peaked at 72 h, concomitant with the appearance and persistence of infectious virus. Virus was present in the periocular skin from 24 h despite the lack of significant reporter activity until 84 h postinfection. This detection of reporter activity was followed by the onset of periocular disease on day 4. Corneal infection with a thymidine kinase-deleted reporter virus displayed a similar profile of reporter activity and viral titer in the eyes, but little or no detectable activity was observed in trigeminal ganglia or periocular tissue. In addition, no periocular disease symptoms were observed. These findings demonstrate that viral infection of periocular tissue and subsequent disease development occurs by zosteriform spread from the cornea to the periocular tissue via the trigeminal ganglion rather than by direct spread from cornea to the periocular skin. Furthermore, clinical evidence is discussed suggesting that a similar mode of spreading and disease occurs in humans following primary ocular infection.     

Herpes simplex virus infection and propagation in a mouse L cell mutant lacking heparan sulfate proteoglycans.

We have isolated a variant line of mouse L cells, termed gro2C, which is partially resistant to infection by herpes simplex virus type 1 (HSV-1). Characterization of the genetic defect in gro2C cells revealed that this cell line harbors a specific defect in the heparan sulfate synthesis pathway. Specifically, anion-exchange high-performance liquid chromatography of metabolically radiolabeled glycosaminoglycans indicated that chondroitin sulfate moieties were synthesized normally in the mutant cells, whereas heparin-like chains were absent. Because of these properties, we have used these cells to investigate the role of heparan sulfate proteoglycans in the HSV-1 life cycle. In this report, we demonstrate that the partial block to HSV-1 infection in gro2C cells occurs in the virus entry pathway. Virus adsorption assays using radiolabeled HSV-1 (KOS) revealed that the gro2C cell surface is a relatively poor target for HSV-1 in that virus attachment was 85% lower in the mutant cells than in the parental L cell controls. A portion of the 15% residual virus adsorption was functional, however, insofar as gro2C cells were susceptible to HSV-1 infection in plaque assays and in single-step growth experiments. Moreover, although the number of HSV-1 plaques that formed in gro2C monolayers was reduced by 85%, the plaque morphology was normal, and the virus released from the mutant cells was infectious. Taken together, these results provide strong genetic evidence that heparan sulfate proteoglycans enhance the efficiency of HSV attachment to the cell surface but are otherwise not essential at any stage of the lytic cycle in culture. Moreover, in the absence of heparan sulfate, other cell surface molecules appear to confer susceptibility to HSV, leading to a productive viral infection.     

Herpes simplex virus type 1 DNA polymerase. Mechanism-based affinity chromatography

The potent inhibition of herpes simplex type 1 (HSV-1) DNA polymerase by acyclovir triphosphate has previously been shown to be due to the formation of a dead-end complex upon binding of the next 2'-deoxynucleoside 5'-triphosphate encoded by the template after incorporation of acyclovir monophosphate into the 3'-end of the primer (Reardon, J. E., and Spector, T. (1989) J. Biol. Chem. 264, 7405-7411). This mechanism of inhibition of HSV-1 DNA polymerase has been used here to design an affinity column for the enzyme. A DNA hook template-primer containing an acyclovir monophosphate residue on the 3'-primer terminus has been synthesized and attached to a resin support. In the absence of added nucleotides, the column behaves as a simple DNA-agarose column, and HSV-1 DNA polymerase can be chromatographed using a salt gradient. The presence of the next required nucleotide encoded by the template (dGTP) increases the affinity of HSV-1 DNA polymerase for the acyclovir monophosphate terminal primer-template attached to the resin, and the enzyme is retained even in the presence of 1 M salt. The enzyme can be eluted from the column with a salt gradient after removal of the nucleotide from the buffer. Traditionally, the affinity purification of an enzyme relies on elution by a salt gradient, pH gradient, or more selectively by addition of a competing ligand (substrate/inhibitor) to the elution buffer. In the present example, elution of HSV-1 polymerase is facilitated by removal of the substrate from the buffer. This represents an example of mechanism-based affinity chromatography.     

Herpes simplex virus type 1 ICP8: helix-destabilizing properties.

The major single-stranded DNA-binding protein, ICP8, of herpes simplex virus type 1 (HSV-1) is one of seven virus-encoded polypeptides required for HSV-1 DNA replication. To investigate the role of ICP8 in viral DNA replication, we have examined the interaction of ICP8 with partial DNA duplexes and found that it can displace oligonucleotides annealed to single-stranded M13 DNA. In addition, ICP8 can melt small fragments of fully duplex DNA. Unlike a DNA helicase, ICP8-promoted strand displacement is ATP and Mg2+ independent and exhibits no directionality. It requires saturating amounts of ICP8 and is both efficient and highly cooperative. These properties make ICP8 suitable for a role in DNA replication in which ICP8 destabilizes duplex DNA during origin unwinding and replication fork movement.     

Cytotoxicity of a replication-defective mutant of herpes simplex virus type 1.

Replication-defective mutants of herpes simplex virus type 1 (HSV-1) may prove useful as vectors for gene transfer, particularly to nondividing cells. Cgal delta 3 is an immediate-early gene 3 (IE 3) deletion mutant of HSV-1 that expresses the lacZ gene of Escherichia coli from the human cytomegalovirus immediate-early control region but does not express viral early or late genes. This vector was able to efficiently infect and express lacZ in cells refractory to traditional methods of gene transfer. However, 1 to 3 days postinfection, Cgal delta 3 induced cytopathic effects (CPE) in many cell types, including neurons. In human primary fibroblasts Cgal delta 3 induced chromosomal aberrations and host cell DNA fragmentation. Other HSV-1 strains that caused CPE, tested under conditions of viral replication-inhibition, included mutants of the early gene UL42, the virion host shutoff function, single mutants of IE 1, IE 2, and IE 3, and double mutants of IE 3 and 4 and IE 3 and 5. Inhibition of viral gene expression by UV irradiation of virus stocks or by preexposure of cells to interferon markedly reduced the CPE. We conclude from these studies that HSV-1 IE gene expression is sufficient for the induction of CPE, although none of the five IE gene products appear to be solely responsible. After infection of human fibroblasts with Cgal delta 3 at a low multiplicity of infection, we were able to recover up to 6% of the input virus 2 weeks later by a superinfection-rescue procedure, even though the virally transduced human cytomegalovirus-lacZ transgene was not expressed at this time. It is therefore likely that inhibition or inactivation of viral IE gene expression, either for establishing latency or for the long-term transduction of foreign genes by HSV-1 vectors, is essential to avoid the death of infected cells.     

Herpes simplex virus type 1 oriL is not required for virus replication or for the establishment and reactivation of latent infection in mice.

During the course of experiments designed to isolate deletion mutants of herpes simplex virus type 1 in the gene encoding the major DNA-binding protein, ICP8, a mutant, d61, that grew efficiently in ICP8-expressing Vero cells but not in normal Vero cells was isolated (P. K. Orberg and P. A. Schaffer, J. Virol. 61:1136-1146, 1987). d61 was derived by cotransfection of ICP8-expressing Vero cells with infectious wild-type viral DNA and a plasmid, pDX, that contains an engineered 780-base-pair (bp) deletion in the ICP8 gene, as well as a spontaneous approximately 55-bp deletion in oriL. Gel electrophoresis and Southern blot analysis indicated that d61 DNA carried both deletions present in pDX. The ability of d61 to replicate despite the deletion in oriL suggested that a functional oriL is not essential for virus replication in vitro. Because d61 harbored two mutations, a second mutant, ts+7, with a deletion in oriL-associated sequences and an intact ICP8 gene was constructed. Both d61 and ts+7 replicated efficiently in their respective permissive host cells, although their yields were slightly lower than those of control viruses with intact oriL sequences. An in vitro test of origin function of isolated oriL sequences from wild-type virus and ts+7 showed that wild-type oriL, but not ts+7 oriL, was functional upon infection with helper virus. In an effort to determine the requirement for oriL in latency, ts+7 was compared with wild-type virus for its ability to establish, maintain, and be reactivated from latent infection in a murine eye model. The mutant was reactivated as efficiently as was wild-type virus from trigeminal ganglia after cocultivation with permissive cells, and each of the seven reactivated isolates was shown to carry the approximately 150-bp deletion characteristic of ts+7. These observations demonstrate that oriL is not required for virus replication in vitro or for the establishment and reactivation of latent infection in vivo.     

Ultrastructural localization of glycoproteins in rabbit fibroblasts altered by Herpes simplex virus type 1 infection

The periodic acid-thiocarbohydrazide (SO2)--OsO4 method was used to examine the distribution of glycoproteins in rabbit fibroblast cells infected with Herpes simplex virus type 1. In non-infected cells, a low level of staining was seen over the plasma membrane and the membranes of the Golgi apparatus. At 17 hr post-infection, the intensity of reaction was increased to include not only a relatively heavy staining of the plasma membrane, including the numerous microvilli characteristic of infected cells, and of the newly proliferated Golgi membranes, but also the envelopes of intracytoplasmic and extracellular virions. A very faint but only occasional staining also was associated with the virus-induced reduplications of the inner nuclear membrane and the envelopes of associated enveloping nucleocapsids. We suggest that such differences in the intensity of staining may be related either to the amount of glycoproteins or to the sequential maturation of the viral glycoproteins. We also observed that the structurally modified portions of the Golgi membranes at the position where intracytoplasmic naked nucleocapsids bud into the Golgi cisternae usually exhibit a more intense reaction for glycoproteins than do the adjacent portions of the Golgi membranes. This supports the evidence for an envelopment of nucleocapsids in the cytoplasm, but it does not indicate whether this event obligatorily follows or only occasionally takes the place of the envelopment of nucleocapsids at the inner nuclear membrane. In either event, the envelopes of all mature virions exhibit a prominent reaction to glycoproteins.     

Herpes simplex virus glycoprotein D mediates interference with herpes simplex virus infection.

We showed that the expression of a single protein, glycoprotein D (gD-1), specified by herpes simplex virus type 1 (HSV-1) renders cells resistant to infection by HSV but not to infection by other viruses. Mouse (LMtk-) and human (HEp-2) cell lines containing the gene for gD-1 under control of the human metallothionein promoter II expressed various levels of gD-1 constitutively and could be induced to express higher levels with heavy metal ions. Radiolabeled viruses bound equally well to gD-1-expressing and control cell lines. Adsorbed viruses were unable to penetrate cells expressing sufficient levels of gD-1, based on lack of any cytopathic effects of the challenge virus and on failure to detect either the induction of viral protein synthesis or the shutoff of host protein synthesis normally mediated by a virion-associated factor. The resistance to HSV infection conferred by gD-1 expression was not absolute and depended on several variables, including the amount of gD-1 expressed, the dosage of the challenge virus, the serotype of the challenge virus, and the properties of the cells themselves. The interference activity of gD-1 is discussed in relation to the role of gD-1 in virion infectivity and its possible role in permitting escape of progeny HSV from infected cells.     

Attenuated, replication-competent herpes simplex virus type 1 mutant G207: safety evaluation in mice

Herpes simplex virus type 1 (HSV-1) mutants that are attenuated for neurovirulence are being used for the treatment of cancer. We have examined the safety of G207, a multimutated replication-competent HSV-1 vector, in mice. BALB/c mice inoculated intracerebrally or intracerebroventricularly with 10(7) PFU of G207 survived for over 20 weeks with no apparent symptoms of disease. In contrast, over 80% of animals inoculated intracerebrally with 1.5 x 10(3) PFU of HSV-1 wild-type strain KOS and 50% of animals inoculated intracerebroventricularly with 10(4) PFU of wild-type strain F died within 10 days. Similarly, after intrahepatic inoculation of G207 (3 x 10(7) PFU) all animals survived for over 10 weeks, whereas no animals survived for even 1 week after inoculation with 10(6) PFU of KOS. After intracerebroventricular inoculation, LacZ expression was initially observed in the cells lining the ventricles and subarachnoid space; expression decreased until almost absent within 5 days postinfection, with no apparent loss of ependymal cells. G207 DNA could be detected by PCR in the brains of mice 8 weeks after intracerebral inoculation; however, no infectious virus could be detected after 2 days. As a model for latent HSV in the brain, we used survivors of an intracerebral inoculation of HSV-1 KOS at the 50% lethal dose. Inoculation of a high dose of G207 at the same stereotactic coordinates did not result in reactivation of detectable infectious virus or symptoms of disease. We conclude that G207 is safe at or above doses that were efficacious in mouse tumor studies.