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 latency

Herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) are ubiquitous human pathogens. They share with other herpesviruses the ability to establish lifelong latent infection of the host. Periodic reactivation from latency is responsible for most of the clinical disease burden of HSV infection. This review focuses on what we have learned from molecular studies in model systems of HSV latency, and the implications these findings have for treating recurrent HSV disease.     

Herpes simplex virus type 1 immediate-early protein Vmw110 reactivates latent herpes simplex virus type 2 in an in vitro latency system.

Reactivation of latent herpes simplex virus type 2 (HSV-2) by the immediate-early protein Vmw110 was studied by using an in vitro latency system. Adenovirus recombinants that express Vmw110 reactivated latent HSV-2. An HSV-1 mutant possessing a deletion in a carboxy-terminal region of Vmw110 reactivated latent HSV-2, whereas mutant FXE, which has a deletion in the second exon, did not. Therefore, Vmw110 alone is required to reactivate latent HSV-2 in vitro, and the region of Vmw110 defined by the deletion in FXE is important for this process.     

Herpes simplex virus latency and the immune response

Following infection, herpes simplex virus establishes latency in the nervous system and recurrences of lytic replication occur periodically. Molecular events which may determine how virus enters latency, how it is maintained and what occurs during reactivation have been investigated. The role of the immune response in limiting infection of the nervous system, influencing the latent state and removing virus from peripheral sites following reactivation has also been studied.     

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 type 1 dUTPase mutants are attenuated for neurovirulence, neuroinvasiveness, and reactivation from latency.

Herpes simplex virus type 1 (HSV-1) encodes a dUTPase which has been shown to be dispensable for normal viral replication in cultured cells (S. J. Caradonna and Y. Cheng, J. Biol. Chem. 256:9834-9837, 1981; F. B. Fisher and V. G. Preston, Virology 148:190-197, 1986). However, the importance of this enzyme in vivo has not been determined. In this report, HSV-1 strain 17 syn+ and two isogenic engineered dUTPase-negative mutants were characterized in the mouse model. Both mutants replicated with wild-type kinetics and achieved wild-type titers in cultured cells. The mutants were 10-fold less neurovirulent than 17 syn+ following intracranial inoculation and more than 1,000-fold less virulent following footpad inoculation. The dUTPase- mutants replicated with wild-type kinetics in the footpad and entered and replicated efficiently in the peripheral nervous system of the mouse. However, their replication in the central nervous system was significantly reduced. The dUTPase- strains established latent infections but displayed a greatly reduced reactivation frequency in vivo. Neurovirulence, neuroinvasiveness, and reactivation frequency were all restored by recombination with wild-type dUTPase sequences. These results have important implications with regard to anti-herpesvirus therapeutic strategies.     

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.     

Herpes simplex virus type 1-specific cytotoxic T lymphocytes recognize virus nonstructural proteins.

The specificity of herpes simplex virus type 1-specific cytotoxic T cells was examined with target cells expressing either input viral structural antigens or antigens resulting from permissive infection or cells from an interrupted infection in which they expressed predominantly nonstructural immediate-early proteins. These studies indicated that only an insignificant minority of cytotoxic T cells recognized the input viral antigens, whereas a significant proportion (20 to 35%) recognized target cells that expressed the immediate-early proteins despite the absence of serologically detectable viral antigens upon the infected cell surface. The finding that a significant proportion of cytotoxic T-cell populations obtained from the draining lymph nodes of mice acutely infected with herpes simplex virus type 1 also recognized immediately-early gene-expressing target cells indicates the importance of nonstructural herpes simplex virus proteins to antiviral immunity in vivo.     

Herpes simplex type 1 activation by Epstein-Barr virus nuclear antigen 1

We have investigated the effect of Epstein-Barr virus nuclear antigen 1 (EBNA-1), a nuclear protein encoded by EBV, on herpes simplex virus type 1 (HSV-1) infection either in cells constitutively expressing EBNA-1 or in transient expression assays. Rat-1 cells and rat embryo fibroblasts (REF) immortalized by c-myc or E1A were transfected with a specific EBV DNA fragment coding for EBNA-1. Cloned cell lines which constitutively expressed this antigen were infected with HSV-1. Our results indicate that in EBNA-1-expressing cells, virus growth was higher than in control cells for different virus strains or rodent cell lines. This increase was maximal when cells were infected at low multiplicity, as determined by virus growth, and correlated with the stimulation of viral DNA synthesis. REF + c-myc and Vero cells were contransfected by an EBNA-1 expression vector driven by Moloney murine leukaemia virus LTR and HSV-1 immediate-early (α0) or early thymidine kinase upstream promoter regulatory regions linked to chloramphenicol acetyltransferase (CAT) coding sequences as effectors. In both cell lines, stimulation of CAT expression by EBNA-1 was observed only with the immediate-early promoter. These results suggest that EBNA-1 can transactivate immediate-early HSV-1 expression.     

Herpes simplex virus type 1 glycoprotein D inhibits T-cell proliferation

Herpes simplex virus type 1 (HSV-1) glycoprotein D (gD) binds to its cellular receptor, herpesvirus entry mediator (HVEM), to enter into activated T cells. Since gD is expressed on the cell surface of activated T cells after infection and can interact with HVEM, a co-stimulatory molecule for T cells, we hypothesized that the membrane-bound gD can exert an immunomodulatory effect on activated T cells. In this report, we demonstrated the following: (1) The gD expression was detected on the cell surface of activated T cells after HSV-1 infection. (2) Recombinant soluble gD protein or gD-expressing mouse fibroblasts inhibited T-cell proliferation that was induced by OKT3 [anti-CD3 monoclonal antibody (mAb)]. (3) The co-expression of gD and HVEM resulted in the inhibition of the nuclear factor (NF)-kappaB activation that was induced by the HVEM overexpression. Taken together, our results suggest that the inhibitory effect of gD may be due to its ability to actively inhibit the signaling pathway that is mediated by HVEM on the cell surface level, which may be a novel immune evasion mechanism that is utilized by HSV-1.     

Herpes simplex virus type 1 targets the MHC class II processing pathway for immune evasion

HSV type 1 (HSV-1) has evolved numerous strategies for modifying immune responses that protect against infection. Important targets of HSV-1 infection are the MHC-encoded peptide receptors. Previous studies have shown that a helper T cell response and Ab production play important roles in controlling HSV-1 infection. The reduced capacity of infected B cells to stimulate CD4(+) T cells is beneficial for HSV-1 to evade immune defenses. We investigated the impact of HSV-1 infection on the MHCII processing pathway, which is critical to generate CD4(+) T cell help. HSV-1 infection targets the molecular coplayers of MHC class II processing, HLA-DR (DR), HLA-DM (DM), and invariant chain (Ii). HSV-1 infection strongly reduces expression of Ii, which impairs formation of SDS-resistant DR-peptide complexes. Residual activity of the MHC class II processing pathway is diminished by viral envelope glycoprotein B (gB). Binding of gB to DR competes with binding to Ii. In addition, we found gB associated with DM molecules. Both, gB-associated DR and DM heterodimers are exported from the endoplasmic reticulum, as indicated by carbohydrate maturation. Evaluation of DR, DM, and gB subcellular localization revealed abundant changes in intracellular distribution. DR-gB complexes are localized in subcellular vesicles and restrained from cell surface expression.     

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.