Ex Vivo Infection of Murine Epidermis with Herpes Simplex Virus Type 1

To enter its human host, herpes simplex virus type 1 (HSV-1) must overcome the barrier of mucosal surfaces, skin, or cornea. HSV-1 targets keratinocytes during initial entry and establishes a primary infection in the epithelium, which is followed by latent infection of neurons. After reactivation, viruses can become evident at mucocutaneous sites that appear as skin vesicles or mucosal ulcers. How HSV-1 invades skin or mucosa and reaches its receptors is poorly understood. To investigate the invasion route of HSV-1 into epidermal tissue at the cellular level, we established an ex vivo infection model of murine epidermis, which represents the site of primary and recurrent infection in skin. The assay includes the preparation of murine skin. The epidermis is separated from the dermis by dispase II treatment. After floating the epidermal sheets on virus-containing medium, the tissue is fixed and infection can be visualized at various times postinfection by staining infected cells with an antibody against the HSV-1 immediate early protein ICP0. ICP0-expressing cells can be observed in the basal keratinocyte layer already at 1.5 hr postinfection. With longer infection times, infected cells are detected in suprabasal layers, indicating that infection is not restricted to the basal keratinocytes, but the virus spreads to other layers in the tissue. Using epidermal sheets of various mouse models, the infection protocol allows determining the involvement of cellular components that contribute to HSV-1 invasion into tissue. In addition, the assay is suitable to test inhibitors in tissue that interfere with the initial entry steps, cell-to-cell spread and virus production. Here, we describe the ex vivo infection protocol in detail and present our results using nectin-1- or HVEM-deficient mice.     

Interleukin-18 Protects Mice against Acute Herpes Simplex Virus Type 1 Infection

We examined the effects of interleukin-18 (IL-18) in a mouse model of acute intraperitoneal infection with herpes simplex virus type 1 (HSV-1). Four days of treatment with IL-18 (from 2 days before infection to 1 day after infection) improved the survival rate of BALB/c, BALB/c nude, and BALB/c SCID mice, suggesting innate immunity. One day after infection, HSV-1 titers were higher in the peritoneal washing fluid of control BALB/c mice than in that of IL-18-treated mice. A genetic deficiency of gamma interferon (IFN-γ), however, diminished the survival rate and the inhibition of HSV-1 growth at the injection site in the mice. Anti-asialo GM1 treatment had no influence on the protective effect of IL-18 in infected mice. IL-18 augmented IFN-γ release in vitro by peritoneal cells from uninfected mice, while no appreciable IFN-γ production was found in uninfected mice administered IL-18. Although IFN-γ has the ability to induce nitric oxide (NO) production by various types of cells, administration of the NO synthase inhibitor N^G-monomethyl-l-arginine resulted in superficial loss of the improved survival, but there was no influence on the inhibition of HSV-1 replication at the injection site in IL-18-treated mice. Based on these results, we propose that IFN-γ produced before HSV-1 infection plays a key role as one of the IL-18-promoted protection mechanisms and that neither NK cells nor NO plays this role.Interleukin-18 (IL-18) is a newly cloned murine and human cytokine (28, 36) previously called gamma interferon (IFN-γ)-inducing factor. It is synthesized by activated macrophages and has a structural relationship to the IL-1 family (5). Precursor IL-18 is processed by IL-1β-converting enzyme and is cleaved into mature IL-18 (11). IL-18 induces IFN-γ production by murine helper T cells and NK cells and stimulates T-cell proliferation and NK activation (18, 28). Moreover, IL-18 augments the Fas ligand-mediated cytotoxic activity of the Th1 clone and the NK cell clone (8, 35). Thus, IL-18 shares some biological activities with IL-12, although no significant homology between the two cytokines has been detected at the protein level (34). Furthermore, treatment with IL-12 and IL-18 has a synergistic effect on IFN-γ production (2, 14, 38, 40).According to a review by Nash (27), not only nonspecific or innate immunity, such as that from IFN, NK cells, or macrophages, but also specific or adaptive immunity is important in protection against herpesvirus infection. Herpes simplex virus is known to be an IFN inducer (13). IFN is produced at an early stage of virus infection. In addition to the direct inhibition of viral replication, it enhances the efficiency of the adaptive (specific) immune response by stimulating increased expression of major histocompatibility complex class I and II or by activating macrophages and NK cells. In protection from infection by herpesviruses, especially cytomegalovirus, NK cells have been major effector cells because of the correlation of increased susceptibility to cytomegalovirus infection with the absence or reduction of NK cell activity, as seen in Chediak-Higashi syndrome patients and beige mice (27). Upon target cell disruption, NK and cytotoxic T cells share not only the perforin but also the Fas ligand as an effector molecule (4, 20, 37). Recently, nitric oxide (NO) was reported to be involved in host defense against bacteria, fungi, parasites, and viruses (10, 16, 19, 39). NO produced by herpes simplex virus type 1 (HSV-1)-infected macrophages is reported to inhibit viral replication (7). CD4⁺ T cells, macrophages, IFN-γ, and tumor necrosis factor (TNF) are important in adaptive immunity against HSV-1 infection. The Th2 response exacerbates HSV-1-induced disease (25).Recently a protective role of IL-18 was reported in microbial infections (6, 17). Here, we demonstrate that IL-18 treatment protects mice from acute viral infection via both IFN-γ-dependent and -independent pathways. Although IFN-γ has the ability to induce NO production by a variety of cells, including macrophages (9), it is not likely to be important, at least in the inhibition of HSV-1 proliferation at the injection site of IL-18-treated mice. Furthermore, the protective effect of IL-18 on HSV-1 infection also does not seem to require complete NK cell activity in our experimental system, whereas our colleagues have already reported that deletion of NK cells by administration of anti-asialo GM1 antibody resulted in lowering of the improved survival rate of tumor-bearing mice treated with IL-18 (23).     

一种1型单纯庖疹病毒载体系统的建立

1型单纯疱疹病毒（HSV-1）作为溶瘤病毒和病毒载体的研究已有很长的历史. 本研究利用细菌人工染色体技术建立了一种HSV-1载体系统. 首先,将HSV-1内部反向重复序列（internal inverted repeat sequences, IR）两侧的片段克隆入pKO5获得穿梭质粒pKO5/BN,其电转含pHSV-BAC的大肠杆菌后筛选获得删除IR区重组DNA的 pHSVΔIR-BAC. pHSVΔIR-BAC转染Vero细胞获得删除IR区的重组病毒HSVΔIR（MH1001）.上述pKO5/BN和含pHSVΔIR BAC的大肠杆菌构成了HSV-1载体系统. 利用该系统获得了表达绿色荧光蛋白EGFP的重组病毒HSVΔIR/EGFP（MH1002）.MH1001和MH1002在感染的Vero细胞中增殖水平略低于野生型HSV-1,但无显著差异|Western印迹检测表明,重组病毒早期蛋白质ICP0、ICP4、ICP8、ICP22、ICP27在感染细胞中的表达水平下降|免疫荧光及激光共聚焦检测表明,重组病毒与野生型病毒均存在于细胞质中.以上结果表明,删除IR区的重组HSV-1保留了复制能力,能够携载并表达外源基因,建立的HSV-1载体系统可用于构建携载外源基因的复制型重组HSV-1.     

Modified VP22 Localizes to the Cell Nucleus during Synchronized Herpes Simplex Virus Type 1 Infection

The UL49 gene product (VP22) of herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) is a virion phosphoprotein which accumulates inside infected cells at late stages of infection. We previously (J. A. Blaho, C. Mitchell, and B. Roizman, J. Biol. Chem. 269:17401-17410, 1994) discovered that the form of VP22 packaged into infectious virions differed from VP22 extracted from infected-cell nuclei in that the virion-associated form had a higher electrophoretic mobility in denaturing gels. Based on these results, we proposed that VP22 in virions was "undermodified" in some way. The goal of this study is to document the biological and biochemical properties of VP22 throughout the entire course of a productive HSV-1 infection. We now report the following. (i) VP22 found in infected cells is distributed in at least three distinct subcellular localizations, which we define as cytoplasmic, diffuse, and nuclear, as measured by indirect immunofluorescence. (ii) Using a synchronized infection system, we determined that VP22 exists predominantly in the cytoplasm early in infection and accumulates in the nucleus late in infection. (iii) While cytoplasmic VP22 colocalizes with the HSV-1 glycoprotein D early in infection, the nuclear form of VP22 is not restricted to replication compartments which accumulate ICP4. (iv) VP22 migrates as at least three unique electrophoretic species in denaturing sodium dodecyl sulfate-DATD-polyacrylamide gels. VP22a, VP22b, and VP22c have high, intermediate, and low mobility, respectively. (v) The relative distribution of the various forms of VP22 derived from infected whole-cell extracts varies during the course of infection such that low-mobility species predominate at early times and high-mobility forms accumulate later. (vi) The highest-mobility forms of VP22 partition with the cytoplasmic fraction of infected cells, while the lowest-mobility forms are associated with the nuclear fraction. (vii) Finally, full-length VP22 which partitions in the nucleus incorporates radiolabel from [32P]orthophosphate whereas cytoplasmic VP22 does not. Based on these results, we conclude that modification of VP22 coincides with its appearance in the nucleus during the course of productive HSV-1 infection.     

PKR-Dependent Xenophagic Degradation of Herpes Simplex Virus Type 1

The lysosomal pathway of autophagy is the major catabolic mechanism for degrading long-lived cellular proteins and cytoplasmic organelles. Recent studies have also shown that autophagy (xenophagy) may be used to degrade bacterial pathogens that invade intracellularly. However, it is not yet known whether xenophagy is a mechanism for degrading viruses. Previously, we showed that autophagy induction requires the antiviral eIF2alpha kinase signaling pathway (including PKR and eIF2alpha) and that this function ofeIF2alpha kinase signaling is antagonized by the herpes simplex virus (HSV-1) neurovirulence gene product, ICP34.5. Here, we show quantitative morphologic evidence of PKR-dependent xenophagic degradation of herpes simplex virions and biochemical evidence of PKR and eIF2alpha-dependent degradation of HSV-1 proteins, both of which are blocked by ICP34.5. Together, these findings indicate that xenophagy degrades HSV-1 and that this cellular function is antagonized by the HSV-1 neurovirulence gene product, ICP34.5. Thus, autophagy-related pathways are involved in degrading not only cellular constituents and intracellular bacteria, but also viruses.     

Herpes Simplex Virus Type 1 Infection of Isogenic Epstein-Barr Virus Genome-Negative and -Positive Burkitt''s Lymphoma-Derived Cell Lines

The Epstein-Barr virus (EBV) genome-negative Burkitt's lymphoma-derived cell lines BJAB and Ramos and their in vitro EBV-converted sublines BJAB-B1, BJAB-A5, BJAB-B95-8, and AW-Ramos were infected with high multiplicities of herpes simplex virus type 1 (HSV-1; 10 to 70 PFU/cell). Cultures were monitored for cell growth and HSV-1 DNA synthesis. EBV-converted BJAB cultures were more permissive for HSV-1 infection than BJAB cultures. Significant cell killing and HSV-1 DNA synthesis were observed during the first 48 h of infection in the EBV-converted BJAB cultures but not in the BJAB cultures. The EBV-converted BJAB-B1 cell line contains an appreciable fraction of EBV-negative cells. Therefore, it was cloned. EBV-positive and -negative cells were identified by using EBV-determined nuclear antigen anti-complement immunofluorescence. Two types of subclones were identified: (i) those which contained both EBV-determined nuclear antigen-positive and -negative cells and (ii) those which contained only EBV-determined nuclear antigen-negative cells. When levels of HSV-1 DNA synthesis were measured in these subclones, it was found that the former were more permissive for HSV-1 infection than the latter. Thus, the presence of the EBV genome in BJAB cells correlates with increased permissiveness of these cells for HSV-1 during the first 48 h of infection. Nonetheless, persistent HSV-1 infections were established in both BJAB and EBV-converted BJAB-B1 cultures. No differences in extent of permissiveness for HSV-1 infection were found for Ramos and EBV-converted AW-Ramos cells.     

Ribonucleotides Linked to DNA of Herpes Simplex Virus Type 1

Cells of a continuous cell line derived from rabbit embryo fibroblasts were infected with herpes simplex type 1 virus (HSV-1) and maintained in the presence of either [5-(3)H]uridine or [methyl-(3)H]thymidine or (32)PO(4) (3-). Nucleocapsids were isolated from the cytoplasmic fraction, partially purified, and treated with DNase and RNase. From the pelleted nucleocapsids, DNA was extracted and purified by centrifugation in sucrose and cesium sulfate gradients. The acid-precipitable radioactivity of [5-(3)H]uridine-labeled DNA was partially susceptible to pancreatic RNase and alkaline treatment; the susceptibility to the enzyme decreased with increasing salt concentration. No drop of activity of DNA labeled with [(3)H]thymidine was observed either after RNase or alkali treatment. Base composition analysis of [5-(3)H]uridine-labeled DNA showed that the radioactivity was recovered as uracil and cytosine. In the cesium sulfate gradient, the purified [5-(3)H]uridine-labeled DNA banded at the same position as the (32)P-labeled DNA. The present data tend to suggest that ribonucleotide sequences are present in HSV DNA, that they are covalently attached to the viral DNA, and that they can form double-stranded structures.     

Role of Interleukin-2 and Herpes Simplex Virus 1 in Central Nervous System Demyelination in Mice

We have reported previously that ocular infection of different strains of mice with recombinant herpes simplex virus 1 (HSV-1) constitutively expressing interleukin-2 (IL-2) provokes central nervous system (CNS) demyelination and optic neuropathy, as determined by changes in visual evoked cortical potentials and pathological changes in the optic nerve and CNS, whereas recombinant viruses expressing IL-4, gamma interferon, IL-12p35, IL-12p40, or IL-12p70 do not induce this neuropathy. The goal of this study was to dissect the mechanism underlying the interplay between the immune system (elevation of IL-2) and an environmental factor (infection with HSV-1) that elicits this pathology. Similar results were obtained upon delivery of IL-2 into the mouse brain using osmotic minipumps or injection of mice with recombinant IL-2 protein, IL-2 DNA, or IL-2 synthetic peptides prior to infection with wild-type (wt) HSV-1 strains McKrae and KOS. The critical role of IL-2 is further supported by our data, indicating that a single mutation at position T27A in IL-2 completely blocks the HSV-1-induced pathology. This study shows a novel model of autoimmunity in which viral infection and enhanced IL-2 cause CNS demyelination.     

Sequestration of PML and Sp100 Proteins in an Intranuclear Viral Structure during Herpes Simplex Virus Type 1 Infection

We investigated the intranuclear distribution of PML and Sp100 in HeLa cells at the ultrastructural level and examined their relocalization in response to herpes simplex virus type 1 (HSV-1) infection. In the absence of infection, we observed that both are components, not only of nuclear bodies, but also of interchromatin granule-associated zones, which suggests a potential role for PML and Sp100 in splicing events. Prolonged HSV-1 infection induced dramatic changes in nuclear organization which consisted of the morphological disappearance of some nuclear structures (nuclear bodies, interchromatin granule-associated zones, coiled bodies) and of the development of a centrally located electron-translucent viral region which pushed the cellular clusters of interchromatin granules to the nuclear border. Concomitantly, dense bodies, concentric arrays of reduplicated inner nuclear membrane, and translucent patches containing a few viral capsids occurred at the nuclear border. PML and Sp100 were exclusively detected over the finely granular material of the vital translucent patches which also contains small amounts of p80-coilin and U1 and U2 snRNAs. An antiserum raised against capsid proteins intensely labeled the viral translucent patches at the level of their finely granular material and enclosed viral capsids. Our data, therefore, suggest that these viral structures, in addition to being the site of accumulation of vital capsid proteins and, possibly, a capsid-works, are also a site of sequestration of cell factors including PML and Sp100. Viral capsid proteins could interfere with and inactivate PML and Sp100 and be implicated in the shutoff of host cell metabolism induced by HSV-1 infection.