Jerry L. Taylor

Interferon Coordinately Inhibits the Disruption of PML-Positive ND10 and Immediate-Early Gene Expression by Herpes Simplex Virus

Interferons (IFNs) are important components of the innate immune response, limiting herpes simplex virus (HSV) infection. In recombinant HSV-infected cells, IFN inhibited expression of beta-galactosidase from the immediate-early gene, ICP4, promoter. The extent of inhibition was dependent on IFN dose, IFN type, cell type, and multiplicity of infection (moi). IFN inhibited gene transcription, leading to a complete block in ICP4 promoter-driven gene expression in 90% of cells. The same IFN treatments resulted in an increase in the size and number of nuclear domain 10 (ND10) structures that stained positive by immunofluorescence for the promyelocytic leukemia (PML) protein. In cultures infected at low moi with a recombinant HSV producing ICP4 as a fusion protein with green fluorescence protein, the appearance of green fluorescence in the nucleus coincided with loss of PML-positive ND10 in the same nucleus, even in the rare ICP4-expressing IFN-treated cells. IFN-dependent inhibition was nearly complete when the immediate-early promoter was in the viral genome but was minimal when the promoter was stably integrated into the cellular genome. These data reveal that IFN can completely block viral gene expression in infected cells and that enhancement of the ND10 structure, which is the site of initiation of HSV replication, correlates with the block in viral gene expression.

SP100B, a repressor of gene expression preferentially binds to DNA with unmethylated CpGs

SP100A and SP100B are mammalian nuclear proteins encoded by alternatively‐spliced transcripts from the SP100 gene. The N‐terminal portion of SP100B (aa 1–476) is identical to SP100A and contains an HP1 interaction domain. The C‐terminal portion of SP100B (aa 477–688) contains an HMG2 interaction domain and a SAND domain. The SAND domain is a DNA‐binding domain identified in several nuclear proteins involved in transcriptional regulation. We have previously reported that SP100B represses expression of genes present on transfected DNA in a SAND domain‐dependent manner. The goal of the present study was to characterize the DNA binding properties of full‐length SP100B expressed in mammalian cells. SP100B associated with DNA whereas SP100A did not. The SP100B SAND domain was essential for DNA binding. Deletion of the HP1‐ or HMG2‐binding domain had no effect on DNA binding. SP100B preferentially associated with sequences containing CpG dinucleotides. Our results did not reveal any preference of SP100B for bases flanking CpG dinucleotides. The number of CpGs in a DNA sequence and spacing between CpGs influenced SP100B binding, suggesting that multimers of SP100B bind DNA in a cooperative manner. Binding of SP100B was abrogated by methylation of the cytosine residue within the context of the CpG dinucleotide. We propose that the preference of SP100B for non‐methylated CpGs provides a mechanism to target SP100B to foreign DNA, including plasmid DNA or viral DNA genomes, most of which are hypomethylated. J. Cell. Biochem. 98: 1106–1122, 2006.

SP100B is a repressor of gene expression.

Mammalian cell nuclei exhibit discrete sites where specific proteins characteristically localize. PML nuclear bodies (PML NBs) (nuclear domain 10s (ND10s)) are the primary localization site for the promyelocytic leukemia (PML) protein and the SP100 autoantigen. The observations that some PML and SP100 isoforms can function as transcriptional regulators, that both the size and number of PML bodies increase in response to interferon treatment, and that many mammalian viruses encode proteins that mediate disruption of PML bodies suggest that these sites suppress viral infection, perhaps by repressing viral gene expression. We hypothesized that a component of PML NBs functions as a repressor of gene expression. To test this hypothesis, we characterized the effect of PML or SP100 isoforms on expression of transfected reporter genes. PML‐I, PML‐VI, and SP100A did not repress reporter gene expression. In contrast, SP100B repressed reporter gene expression, especially under conditions in which the reporter gene expression was elevated by a viral transactivator or addition of trichostatin A to the culture medium. The SP100B DNA binding domain was required for repression. SP100B had no detectable effect on the amount, methylation pattern, or topological form of plasmid DNA in the nuclei of transfected cells. The demonstrated repressive activity of SP100B supports the hypothesis that SP100B is a component of an innate immune response that represses expression of ectopic DNA.

Induction in rainbow trout of an acute phase (C-reactive) protein by chemicals of environmental concern

1. Rainbow trout, Salmo gairdneri, produce elevated amounts of a serum acute phase (C-reactive) protein (CRP) when administered a variety of chemicals of environmental importance. 2. Compounds administered in doses which induce the cytochrome(s) P450 catalytic enzymes in trout hepatic microsomes also induce serum CRP. 3. However, an interferon-inducing virus does not induce CRP. Interferon induction by the virus is not significantly inhibited by chemicals which induce trout cytochrome(s) P450. 4. Simultaneous administration of chemicals and virus or virus alone results in depression of P450 protein production and only minor induction of CRP. 5. Thus, as with mammals, a reciprocating relationship appears to exist between the hemeprotein monooxygenase and immune systems of this freshwater teleost, and C-reactive protein appears to fit the reciprocating scheme closer to the cytochromes P450 response.

Combined anti-herpes virus activity of nucleoside analogs and interferon

Addition of interferon (IFN) to nucleoside analog therapy for herpetic keratitis has been shown to significantly increase the efficacy of therapy compared to nucleoside alone. We have analysed several nucleoside analogs and recombinant IFN-alpha 2 to determine which combinations have increased anti-herpes simplex virus type 1 (HSV) activity. Synergistic anti-HSV activity between IFN-alpha 2 and the acyclic guanosine analogs, acyclovir (ACV) and ganciclovir (DHPG), was demonstrated in cytopathic effect reduction assay in human corneal cell cultures as well as in Vero cells. In this assay system IFN-alpha 2 alone had little detectable antiviral activity at titers of greater than or equal to 2,000 IU/ml, however, treatment of cells with about 100 IU/ml of IFN-alpha 2 for 24 hrs prior to infection decreased the ED50 of ACV approximately 2- to 3-fold and of DHPG approximately 5- to 6-fold in Vero cells. Combinations of IFN-alpha 2 with bromovinyldeoxyuridine (BVdU) in Vero cells or human corneal stromal cells did not increase the antiviral activity of BVdU. Combinations of IFN-alpha 2 with trifluorothymidine (TFT) also did not increase the effective antiviral activity of this nucleoside and resulted in decreased uptake of TFT from the medium. These studies document that combinations of acyclic nucleoside analogs, ACV and DHPG, with IFN-alpha 2 resulted in synergistic anti-HSV activities in both Vero and human corneal stromal cells, while the pyrimidine analogs, TFT and BVdU, were not synergistic with IFN-alpha 2. IFN-alpha 2 treatment of cells induced modifications of nucleoside (e.g., thymidine and TFT), but not nucleobase (e.g., ACV) uptake. These studies suggest that selective inhibition of nucleoside versus nucleobase uptake may contribute to the mechanism of IFN/nucleobase synergy in the inhibition of HSV replication.

Combined effects of interferon-α and acyclovir on herpes simplex virus type 1 DNA polymerase and alkaline DNase

Treatment of cells with combinations of human interferon-alpha (IFN-alpha) and the nucleoside analog, acyclovir (ACV), leads to the synergistic inhibition of herpes simplex virus type 1 (HSV-1) replication. We have examined the effect of these agents on the replication of HSV-1 DNA and the synthesis of early viral enzymes to understand the mechanism(s) responsible for this synergistic activity. Combination treatment with 100 IU/ml IFN-alpha and 5 microM ACV led to HSV-1 DNA levels more than 8-fold lower than in cells treated with ACV alone, while IFN-alpha treatment alone had no detectable effect on viral DNA synthesis. Steady state levels of DNA polymerase were reduced approximately 50% by IFN-alpha and 25% by ACV, but combination treatment did not decrease enzyme levels to an extent greater than the sum of these effects. In contrast, the activity of another early viral enzyme, alkaline DNase, was reduced less than 20% by IFN-alpha alone or combination treatment and was unaffected by ACV treatment. No decrease in the level of mRNA encoding either enzyme was detected in IFN-alpha-treated cells although ACV treatment reduced polymerase mRNA levels. These studies suggest that the synergistic anti-HSV activities of IFN-alpha with ACV could be mediated, in part, through some post-transcriptional mechanism induced by IFN-alpha treatment, leading to the reduction in production of viral early enzymes, especially DNA polymerase.

The determination of effective antiviral doses using a computer program for sigmoid dose-response curves.

A computer program was designed to construct best fit sigmoid dose-response curves for determination of the dose required to reduce the yield of virus by 50%, effective antiviral dose (ED50). A single antiviral agent, 9-beta-D-arabinofuranosyladenine, was examined for effectiveness against four strains of herpes simplex virus type 1. The resulting ED50 values were compared with those obtained by probit analysis. The statistical parameters obtained from sigmoid curve fit program were utilized to evaluate statistical differences between ED50 values for resistant and sensitive virus strains and to evaluate the goodness-of-fit of the regression line to the data. In addition, using this analytical method, it was shown that a change in one experimental variable, i.e., multiplicity of infection, in the yield reduction assay significantly affected the apparent ED50 value. The computer program was easily utilized for analysis of data obtained from both plaque reduction and yield reduction assays and generated the parameters necessary for statistical comparison of relative antiviral activity of any antiviral agent.

Interferon production in inbred mice during herpetic eye disease

Low levels (less than 5 units/eye) of interferon (IFN) were detected in the eyes of BALB/c and C57BL/6 mice one to five days after instillation of 10(7) pfu/eye of herpes simplex virus type 1 (HSV) onto scarified corneas. This dose of virus produced herpetic keratitis characterized by dendritic epithelial lesions one day post infection in both strains of mice. The disease progressed to severe necrotizing stromal keratitis in the eyes of all BALB/c mice, but only three of 10 eyes of C57BL/6 mice by 21 days after infection. Footpad immunization 30 days prior to ocular infection protected both strains from stromal disease, but did not enhance IFN production in the eye. At lower inoculating doses of virus (less than or equal to 10(5) pfu/eye), C57BL/6 mice showed greater resistance to stromal disease and produced less virus over a shorter period of time than BALB/c mice. No IFN was detected at any time after infection with doses of virus less than 10(7) pfu/eye, nor was IFN detected in plasma of any infected mice. The failure to detect high levels of IFN in homogenates of eyes did not reflect an inability of ocular tissues to produce IFN since IFN-beta was detected as early as two hours after topical treatment with the potent IFN inducer, carboxymethylacridanone (CMA). The two mouse strains produced similar levels of IFN in the eye in response to CMA. These data indicated that the relative resistance of mice to HSV eye infection was not related to the rapid local production of IFN, nor was resistance related to systemic IFN production in plasma or spleen.

[39] Induction of interferon in mice by 10-carboxymethyl-9-acridanone

Publisher Summary 10-Carboxymethyl-9-acridanone (CMA) is a small-molecular-weight compound structurally related to the acridines. It has been shown to induce very high titers of interferon in mice when given intraperitoneally (IP), subcutaneously (SC), intramuscularly (IM), intravenously (IV), or orally and to be effective in protecting mice against lethal infections with several RNA and DNA viruses but without effect in cell culture. The compound is unique among small molecular weight, nonviral interferon inducers because of the high titers of interferon induced and because, unlike tilorone, CMA, under the conditions studied thus far, does not induce a hyporeactive state to interferon induction. The interferon induced by CMA is similar to virus-induced interferon. The interferon is resistant to pH 2, nonsedimentable, and sensitive to trypsin and heat treatment; it is not expressed in the presence of actinomycin D. The molecular weight of CMA-induced mouse plasma interferon is determined to be 25,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These parameters are the same as for bluetongue virus-induced mouse plasma interferon analyzed simultaneously.

The effects of interferon-α and acyclovir on herpes simplex virus type-1 ribonucleotide reductase

Herpes simplex virus-type 1 (HSV-1) encodes both the small (UL40) and large (UL39) subunits of the enzyme, ribonucleotide reductase. Treatment of HSV-1-infected cells with interferon-alpha (IFN-alpha) reduced the levels of both enzyme subunits. Reduced steady state levels of the large subunit were demonstrated by immunoblot using polyclonal antibody specific for the viral enzyme. Reduction in the amount of small subunit was shown by a reduction in the electron spin resonance signal derived from the iron-containing tyrosyl free-radical present in this subunit. Treatment of cells with 100 IU/ml of IFN-alpha decreased levels of both subunits resulting in a reduction in enzyme activity as measured by conversion of CDP to dCDP. The decrease in the amount of the large subunit was not due to a reduction in the level of its mRNA. The combination of IFN-alpha and ACV treatment of human cornea stromal cells did not result in a further reduction in amounts of ribonucleotide reductase relative to that detected with IFN-alpha alone. The IFN-alpha-induced reduction in ribonucleotide reductase activity is the likely cause of decreased levels of dGTP which we have previously demonstrated in IFN-alpha-treated, infected cells.