Tatsuo Suzutani

The role of the UL41 gene of herpes simplex virus type 1 in evasion of non-specific host defence mechanisms during primary infection

The UL41 gene product (vhs) of herpes simplex virus (HSV) is packaged in the virion, and mediates host protein synthesis shutoff at the early stage of the virus replication cycle. In order to clarify the role of vhs in virus replication and virulence, we isolated a completely UL41-deficient mutant (the VRDelta41 strain) and its revertant (the VRDelta41R strain). In the mouse encephalitis model, the replication of strain VRDelta41 was inhibited after 2 days post-infection, resulting in low virulence, by gamma-ray-sensitive cells such as lymphocytes and/or neutrophils. The result suggested that some cytokines, produced in VRDelta41-inoculated brains, activate and induce the migration of gamma-ray-sensitive cells to the infection site. Therefore, cytokines produced by HSV-1-infected human cells were screened, and potent inductions of interleukin (IL)-1beta, IL-8 and macrophage inflammatory protein-1alpha by VRDelta41 infection were observed. Moreover, the VRDelta41 strain showed 20- and 5-fold higher sensitivity to interferon-alpha and -beta compared to the wild-type strain, respectively. These results indicate that one important role of vhs in vivo is evasion from non-specific host defence mechanisms during primary infection through suppression of cytokine production in HSV-infected cells and reduction of the anti-HSV activity of interferon-alpha and -beta.

Enhancement of Interferon-γ Production in Glycyrrhizin-Treated Human Peripheral Lymphocytes in Response to Concanavalin A and to Surface Antigen of Hepatitis B Virus

Abstract The effects of glycyrrhizin, a component of licorice (Glycyrrhiza glabra) roots, on the production of interferon-γ in human peripheral lymphocyte-macrophage cultures by concanavalin A (Con A) was examined. Interferon-γ production in normal lymphocyte-macrophage cultures treated with 10 to 100 μg/ml of glycyrrhizin at 37°C for 12 hr or longer, and then treated with 10 μg/ml of Con A, was enhanced four to eight times compared to control cell cultures. Lymphocyte-macrophage cultures treated with 10 to 100 μg/ml of glycyrrhizin alone did not produce interferon. No significant difference in the adsorption of [3H]Con A to glycyrrhizin-treated and control lymphocyte-macrophage cultures was found, but RNA and protein synthesis of the treated lymphocytes was increased compared to control cells; DNA synthesis, however, was reduced. Collaboration between enriched T-lymphocytes and macrophages, both treated with glycyrrhizin, was needed for the enhancement of interferon-γ production. A smaller increase in interferon production was also observed in the glycyrrhizin-treated peripheral lymphocyte-macrophage cultures derived from an asymptomatic carrier of hepatitis B virus, in response to Con A and surface antigen of hepatitis B virus.

Nucleotide sequence of thymidine kinase gene of sequential acyclovir-resistant herpes simplex virus type 1 isolates recovered from a child with Wiskott-Aldrich syndrome : Evidence for reactivation of acyclovir-resistant herpes simplex virus

Recurrent acyclovir (ACV)‐resistant (ACV‐r) herpes simplex virus type 1 (HSV‐1) infections occurred in a patient with Wiskott–Aldrich syndrome, an X‐linked recessive immunodeficiency syndrome composed of three clinical characteristics of immunodeficiency, thrombocytopenia, and an eczematous dermatitis. The patient had severe and recurrent ACV‐r herpes simplex and was treated with vidarabine in a satisfactory manner from 1993 to 1997. During the 4‐year observation period, two ACV‐sensitive (ACV‐s) HSV‐1 isolates and five ACV‐r HSV‐1 isolates were recovered. The nucleotide sequence of the thymidine kinase (TK) gene from these sequential ACV‐r isolates was compared with the ACV‐s isolates. A single nucleotide deletion of cytosine (C) from homopolymer stretch of four C residues between nucleotide 1061 and 1064 of the open reading frame was found in all ACV‐r isolates. No other differences were observed in the TK nucleotide sequence between ACV‐s and ACV‐r isolates. The TK nucleotide sequences of the two ACV‐s isolates were identical to each other and those of the five ACV‐r isolates were identical to one another. These results suggest that the ACV‐r HSV‐1 might have derived from the ACV‐s strain in the patient body and that TK‐associated ACV‐r HSV‐1 can reactivate from latency. J. Med. Virol. 58:387–393, 1999.

Efficacies of antiherpesvirus nucleosides against two strains of herpes simplex virus type 1 in vero and human embryo lung fibroblast cells

Antiviral activities of five nucleoside analogs against the VR-3 and WT-34 strains of herpes simplex virus type 1 (HSV-1) were investigated in Vero and human embryo lung fibroblast (HEL) cells. In HEL cells, the compounds showed antiviral activities against both strains of HSV-1, but in Vero cells, the antiviral activities of the compounds were reduced in proportion to their antiviral indexes (the 50% inhibitory dose [ID50] for cell growth divided by the 50% plaque reduction dose for virus). The ratio of the ID50 in Vero cells to the ID50 in HEL cells was larger in VR-3-infected cells than in WT-34-infected cells. The following results were obtained. (i) Thymidine kinase (TK; EC 2.7.1.21) activity in the VR-3- or WT-34-infected Vero cells was about half that in VR-3- or WT-34-infected HEL cells. Induction of viral TK was especially low in the VR-3-infected Vero cells. (ii) The ID50 of the plaque reduction assay in hypoxanthine, aminopterin, and thymidine medium revealed that the activity of cellular thymidylate synthetase (EC 2.1.1.45) was important in viral replication in VR-3-infected Vero cells. (iii) The VR-3-infected cells required larger thymidine and thymidine phosphate pools for viral replication than the WT-34-infected cells did, although uptake of 1-beta-D-arabinofuranosyl-E-5-(2-bromovinyl) uracil into infected cells was equal for both strains. (iv) In the VR-3-infected Vero cells, the quantity of 1-beta-D-arabinofuranosyl-E-5-(2-bromovinyl)uracil triphosphate was smaller than that in VR-3-infected HEL cells and WT-34-infected Vero and HEL cells.

Effects of various nucleosides on antiviral activity and metabolism of 1-beta-D-arabinofuranosyl-E-5-(2-bromovinyl)uracil against herpes simplex virus types 1 and 2.

Uptake of 1-beta-D-arabinofuranosyl-E-5-(2-bromovinyl)uracil (BV-araU) into herpes simplex virus type 1 (HSV-1)- and 2 (HSV-2)-infected cells was elevated about 190 to 40 times, compared with that into mock-infected human embryo lung fibroblast cells. Uptake was not enhanced by infection with thymidine kinase-negative HSV-1 and HSV-2 mutants, however. In HSV-1-infected cells, 9.7% of BV-araU was phosphorylated to BV-araU triphosphate, but only 1.1% was phosphorylated in HSV-2-infected cells. The antiviral effect, uptake, and turnover of BV-araU were inhibited significantly by thymidine (dThd), moderately by deoxyuridine, and not at all by deoxycytidine. On the other hand, the antiviral activity of acyclovir (ACV) was inhibited only by dThd. The effect of BV-araU was influenced by dThd and dThd phosphates (mono-, di-, and triphosphates), and the effect of ACV was influenced only by dThd, which competitively inhibited the phosphorylation of ACV to ACV monophosphate. The combination of 5-fluorodeoxyuridine (FUdR)-BV-araU or FUdR-ACV had a synergistic effect on HSV-1 and HSV-2 replication. The effect of FUdR on the turnover of BV-araU in HSV-1- and HSV-2-infected cells was analyzed by high-performance liquid chromatography. In HSV-1-infected cells, 86% of the BV-araU was phosphorylated to BV-araU triphosphate, but no effect was observed in HSV-2-infected cells, in which 98% of the BV-araU remained as BV-araU monophosphate.

Role of gamma delta TCR+ lymphocytes in the augmented resistance of trehalose 6,6'-dimycolate-treated mice to influenza virus infection.

Trehalose 6,6-dimycolate (TDM), an immunomodulator, potentiates non-specific resistance in mice to influenza virus infection. When mice were injected intravenously with TDM, the striking proliferation of a minority of T-lymphocytes bearing gamma/delta T-cell receptors (gamma delta T-cells) that accumulated in granulomatous lungs was thought to be associated with the maintenance of acquired resistance to lethal influenza virus infection. To clarify the cellular basis of the defence against influenza virus, mice were depleted of gamma delta T-cells, alpha/beta (alpha beta) T-cells, or natural killer (NK) cells by in vivo administration of corresponding antibodies prior to influenza virus infection. The depletion of gamma delta T-cells significantly abrogated the augmented resistance of TDM-treated mice to infection, as did depletion of either alpha beta T-cells or NK cells. To gain insight into the functional ability of gamma delta T-cells, we evaluated the cytotoxic activity of this T-cell subset against a panel of target cell lines that were stably transfected with the influenza virus haemagglutinin (HA) gene from A/PR/8/34(H1N1) and A/Aichi/2/68(H3N2) strains. The gamma delta T-cells from TDM-treated mice showed profound cytotoxicity against the target cells expressing HA of either the H1 or H3 subtype, in a non-major histocompatibility complex-restricted manner. Taken together, these results indicate that gamma delta T-cells play a non-specific role, in conjunction with alpha beta T-cells and NK cells, in protecting mice against influenza virus infection, and that the recognition and destruction of HA-expressing target cells by the activated gamma delta T-cells is one of the steps involved in this anti-influenza virus immunosurveillance.

Analysis of the Relationship between Cellular Thymidine Kinase Activity and Virulence of Thymidine Kinase-Negative Herpes Simplex Virus Types 1 and 2

The virulence of thymidine kinase‐negative herpes simplex virus type 1 (HSV‐1; VRTK− strain) and type 2 (HSV‐2; UWTK− strain) was studied in comparison with that of their parental strains (VR‐3 and UW‐268, respectively) in an encephalitis model of adult (4‐week‐old) and newborn (3‐day‐old) mice. Viral thymidine kinase (TK) activity was essential for the maximum expression of virulence of HSV‐1, because the 50% lethal dose (LD50) of VRTK− was 60 times higher than that of VR‐3 in the brains of newborn mice expressing high levels of cellular TK activity. However, the UWTK− strain showed the same virulence as the parental strain in newborn mice, despite the lack virulence in adults, suggesting that replication of the UWTK− strain was completely supported by cellular TK activity. This difference in the role of viral and cellular TKs for virus growth between HSV‐1 and HSV‐2 was confirmed with the one‐step growth of virus strains in L‐M and L‐M(TK−) cells.

Role of interferon in the augmented resistance of trehalose-6,6'-dimycolate-treated mice to influenza virus infection.

Mice inoculated intravenously with 10 to 100 micrograms trehalose-6,6-dimycolate in an oil-in-water emulsion (TDM emulsion) acquired high resistance to intranasal infection by influenza virus at 7 to 14 days, but not at 1 day, after treatment. Mice inoculated with an oil-in-water emulsion without TDM (control emulsion) did not resist infection. The activity of the reticuloendothelial system of mice inoculated with TDM emulsion or control emulsion was greatly stimulated 1 day and 14 days after treatment. Interferon production in response to influenza virus was augmented in lung and serum of TDM emulsion-treated mice. The augmented interferon production was greatly diminished in the TDM emulsion-treated mice by treatment with anti-Thy-1.2 monoclonal antibody. Production of haemagglutination-inhibiting antibody in the TDM emulsion-treated or control emulsion-treated mice was higher than that in untreated mice, although no difference was observed between the TDM emulsion-treated and control emulsion-treated mice. On the other hand, TDM emulsion treatment of mice did not influence the appearance of antibody-producing cells, nor the activity of natural killer cells in the mice. The enhanced resistance of mice was diminished by inoculating anti-interferon-alpha/beta serum before influenza virus infection. No detectable interferon activity was observed in lung and blood of mice inoculated with anti-interferon-alpha/beta serum prior to influenza virus infection. These results suggest that the augmented early interferon production in T-lymphocytes of TDM emulsion-treated mice in response to influenza virus may play an important role in the enhanced resistance.

Direct and mononuclear cell mediated effects on interleukin 6 production by glioma cells in infection with herpes simplex virus type 1

To clarify the mechanism of interleukin (IL)‐6 elevation in the cerebrospinal fluid of viral meningitis and/or encephalitis patients, we investigated how herpes simplex virus type 1 (HSV1)‐infection enhances IL‐6 production in human glioma cells (the U373MG and T98G cells). Although human glioma cells did not show enhanced IL‐6 production by direct HSV1‐infection, the cell‐free supernatant from HSV1‐stimulated mononuclear cells (MNC) culture and lipopolysaccharide, as a positive control, markedly elevated IL‐6 production at both mRNA and polypeptide levels. Ultra violet‐irradiated HSV1 induced the secretion of the IL‐6 inducing factor(s) from MNC, whereas heat‐inactivated HSV1 did not show this activity. This finding indicated that the adsorption of virus on the surface of MNC may be sufficient for induction of secretion. The supernatant from the culture of HSV1‐stimulated MNC contained detectable amounts of IL‐1β, tumor necrosis factor (TNF) α, interferon (IFN) γ and IL‐6, and its IL‐6‐inducing activity was inhibited only by anti‐IL‐1β antibodies. Moreover, recombinant IL‐1β markedly enhanced IL‐6 production in glioma cells with a concomitant elevation of its mRNA level. Taken together, the results suggest that in HSV1‐infection of the CNS, enhancement of IL‐6 production in glial cells is mediated not by direct infection to glial cells but rather by IL‐1β released from HSV1‐stimulated MNC. These findings may help elucidate the mechanisms underlying cerebro‐parenchymal inflammatory progression and repair in herpes simplex encephalitis. J. Med. Virol. 63:252–258, 2001.

Structure-activity relationship of the affinity of 5-substituted uracil nucleoside analogues for varicella-zoster virus thymidine kinase and their activity against varicella-zoster virus

We investigated structure-activity relationships of 5-substituted uracil nucleoside analogues for their selective antiviral activity against varicella-zoster virus (VZV) and affinity for VZV thymidine kinase (TK). Anti-proliferative activity of the compounds was measured using human lymphoblastoid cells. Most 2-deoxyribofuranosyluracil, arabinofuranosyluracil (araU) and 2-deoxy-2-fluoro-arabinofuranosyluracil derivatives showed selective anti-VZV activity as well as activity against herpes simplex virus types 1 and 2. 2-Deoxyuridine derivatives showed higher affinity than the corresponding araU analogues. A correlation was seen between the 50% effective doses for VZV and the Ki values for VZV TK, except for 5-ethyl-2-deoxyuridine and 5-ethyl araU that showed relatively high affinity for VZV TK without showing any activity against VZV. 5-Halogenovinyluracil nucleosides showed the highest affinity and the most potent and selective anti-VZV activity. 2-Deoxy-2-fluoro-arabinofuranosyluracil derivatives exhibited high anti-VZV potency though they showed relatively low affinity for VZV TK. Some 3-deoxythymidine analogues having anti-human immunodeficiency virus activity were inactive against herpesviruses.