Amounts of Epstein-Barr Virus DNA in Somatic Cell Hybrids Between Burkitt Lymphoma-Derived Cell Lines

The amounts of Epstein-Barr virus DNA in somatic cell hybrids between human lymphoid cell lines were found to be higher than in the parental cell lines, although the hybrids were not induced to spontaneous virus production.     

The Herpes Simplex Virus Type 1 Infected Cell Protein 22

As one of the immediate-early(IE)proteins of herpes simplex virus type 1(HSV-1),ICP22 is a multifunctional viral regulator that localizes in the nucleus of infected cells.It is required in experimental animal systems and some nonhuman cell lines,but not in Vero or HEp-2 cells.ICP22 is extensively phosphorylated by viral and cellular kinases and nucleotidylylated by casein kinase Ⅱ.It has been shown to be required for efficient expression of early(E)genes and a subset of late(L)genes.ICP22,in conjunction wit...     

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.     

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.     

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).     

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.     

Expression of Herpes Simplex Virus 1 MicroRNAs in Cell Culture Models of Quiescent and Latent Infection

To facilitate studies of herpes simplex virus 1 latency, cell culture models of quiescent or latent infection have been developed. Using deep sequencing, we analyzed the expression of viral microRNAs (miRNAs) in two models employing human fibroblasts and one using rat neurons. In all cases, the expression patterns differed from that in productively infected cells, with the rat neuron pattern most closely resembling that found in latently infected human or mouse ganglia in vivo.     

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.     

Clinical Reactivations of Herpes Simplex Virus Type 2 Infection and Human Immunodeficiency Virus Disease Progression Markers

Background

The natural history of HSV-2 infection and role of HSV-2 reactivations in HIV disease progression are unclear.

Methods

Clinical symptoms of active HSV-2 infection were used to classify 1,938 HIV/HSV-2 co-infected participants of the Women''s Interagency HIV Study (WIHS) into groups of varying degree of HSV-2 clinical activity. Differences in plasma HIV RNA and CD4+ T cell counts between groups were explored longitudinally across three study visits and cross-sectionally at the last study visit.

Results

A dose dependent association between markers of HIV disease progression and degree of HSV-2 clinical activity was observed. In multivariate analyses after adjusting for baseline CD4+ T cell levels, active HSV-2 infection with frequent symptomatic reactivations was associated with 21% to 32% increase in the probability of detectable plasma HIV RNA (trend p = 0.004), an average of 0.27 to 0.29 log10 copies/ml higher plasma HIV RNA on a continuous scale (trend p<0.001) and 51 to 101 reduced CD4+ T cells/mm³ over time compared to asymptomatic HSV-2 infection (trend p<0.001).

Conclusions

HIV induced CD4+ T cell loss was associated with frequent symptomatic HSV-2 reactivations. However, effect of HSV-2 reactivations on HIV disease progression markers in this population was modest and appears to be dependent on the frequency and severity of reactivations. Further studies will be necessary to determine whether HSV-2 reactivations contribute to acceleration of HIV disease progression.     

Effect of Heparin on Hemagglutinin of Herpes Simplex Virus Type 1

Heparin inhibited the hemagglutinin activity of herpes simplex virus (HSV) type 1. The minimal inhibitory concentration of heparin required to inhibit 8 hemagglutination (HA) U of HSV ranged from 0.005 to 0.01 U/ml. Mouse erythrocytes failed to combine with the HA inhibitory factor of heparin. On the other hand, mouse erythrocytes treated with heparinase had greatly reduced agglutinability by HSV. Virus-heparin complex formation was observed by sedimenting heparin with the virus particles.     

Suppression of Herpes Simplex Virus Infection by Phosphonoacetic Acid

Disodium phosphonoacetate when administered orally or topically to mice experimentally infected with herpes simplex virus was able to significantly reduce the mortality associated with the agent. In addition, this compound was able to reduce herpesvirus lesions on the corneas of infected rabbits.     

Differentiation Between Herpes Simplex Virus Type 1 and Type 2 Strains by Immunoelectroosmophoresis

A method has been elaborated to differentiate between herpes simplex type 1 and type 2 viruses by immunoelectroosmophoresis. With rabbit immune sera cross-absorbed with heterologous virus antigen, a distinct difference was shown between the two virus types. Herpes simplex type 1 virus tested against cross-absorbed type 1 antiserum gave two precipitin lines. Herpes simplex type 2 virus gave one precipitin line when tested against cross-absorbed homologous serum. When the viral antigens were tested against cross-absorbed heterologous immune sera, no or only very weak precipitin reactions were observed. The test is easy and rapid, requires relatively small quantities of antigen and antibody, and is suitable for typing of herpes simplex virus in diagnostic routine work.