Kent W. Wilcox

Binding and repression of the latency-associated promoter of herpes simplex virus by the immediate early 175K protein

We demonstrate that the immediate early 175K protein (IE175K) of herpes simplex virus type 1 binds to the cap site of the latency-associated promoter (LAP) in an unusual manner. The complex formed on the LAP cap site was significantly larger than that formed on the IE175K cap site and the requirements for binding were qualitatively distinct with respect to both the primary sequence determinants at the site, and the regions of IE175K protein required for binding compared to those for the IE175K cap site. Although purified IE175K was sufficient for this large complex formed on the LAP cap site, the DNA-binding domain was unable to bind efficiently. This contrasted strikingly with the IE175K cap site where, using precisely analogous probes, the DNA-binding domain exhibited a strong interaction. Surprisingly, from dissociation kinetics we show that binding of the intact protein to the LAP cap site is considerably more stable than the binding of IE175K to its own cap site (half-lives of the complexes 15 min and < 1 min respectively), and this was reflected in more efficient repression of LAP-driven expression than IE175K promoter-driven expression by IE175K. Moreover, primary sequence requirements for IE175K binding to the LAP cap site region differed from previously identified IE175K recognition sequences in that in addition to a partially conserved consensus sequence, neighbouring bases were necessary for binding. Although the LAP cap site exhibits a pseudopalindromic arrangement of core consensus sites, we show that this is not the basis for the higher order, more stable binding to this region. Together these results indicate that IE175K forms an unusual complex at the LAP cap site, broadening the range of previously defined sequences recognized by IE175K.

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.

Herpes-simplex-virus-infected cells produce a protein that binds to the TAR DNA region of the human immunodeficiency virus type 1 long terminal repeat

Activation of human immunodeficiency virus type 1 (HIV-1) provirus by herpes simplex virus type 1 (HSV-1) infection is mediated by both HSV-1 gene products and cellular transactivators. Previously, several key factors such as NF-kappaB-specific proteins p55 and p85, HLP-1 protein that binds to the LBP-1 sequences of the HIV-1 long terminal repeat (LTR) and the viral transactivator ICPO were found to play a role in the transcriptional activation of HIV-1 LTR expression. In this report, we describe binding of herpesvirus-specific protein TDP150 to the TAR DNA region of the HIV-1 LTR. Our data suggest that TDP150 may be related to the herpesvirus transactivator protein ICP4; both proteins are 150-kD DNA-binding proteins produced in HSV-infected cells in the presence of an inhibitor of viral DNA replication. However, the appearance of TDP150-binding activity is delayed by several hours compared to that of ICP4 and the DNA-binding specificity of TDP150 differs from that of purified ICP4. These results suggest that TDP150 is not identical to ICP4; whether it is its analogue remains to be determined. TAR DNA alone can confer responsiveness of heterologous promoter to HSV-1 infection, suggesting that this region can function as an enhancer in HSV-1-infected cells. Deletion of the TAR region from the HIV-1 LTR has not changed significantly the HSV-1-mediated stimulation. Copyright 1994 S. Karger AG, Basel