A set of proteins interacting with transcription factor Sp1 identified in a two-hybrid screening.

The two-hybrid system was used to isolate cDNA clones encoding polypeptides that interact with the N-terminal region (activation domains A, B and C) of the Sp1 transcription factor. Among the 65 collected clones, 43 contained cDNA fragments with open reading frames. They corresponded to 13 genes encoding proteins of known function and to 15 genes, the proteins of which have no known function. Six overlapping cDNA clones corresponded to the Hsc70 protein. Host cell factor (HCF-1) and the KIAA0461 gene (encoding a putative Zn-finger protein of unknown function) were both identified through the isolation of three overlapping cDNA clones. Two cDNA fragments encoding the same region of the SREBP-2 transcription factor were independently selected and two overlapping cDNA clones corresponded to the splicing factor SF3A120. Two different cDNA clones encoded the N- and C-terminal region of the Oct-1 transcription factor. Transcription factors Elf-1 and TIEG, as well as HSph2, the putative human homologue of a murine polyhomeotic gene, were each represented by a single clone. Noticeably, for the four identified transcription factors, the DNA-binding domain was excluded from the selected polypeptides. In vitro binding of the selected polypeptides to the Sp1 protein was demonstrated for the four transcription factors and for the SF3A120, Hsc70, HCF-1, HSph2 and pKIAA0461(245) proteins. Four other cDNA clones encoding polypeptides of unknown function were tested in the in vitro binding assay. All four polypeptides were found to interact with Sp1 in this assay.

Transcriptional mechanisms responsible for the overexpression of the keratin 18 gene in cells of a human colon carcinoma cell line.

The keratin 18 (K18) gene is overexpressed in cells of tumorigenic clones isolated from the SW613-S human colon carcinoma cell line, compared to cells of nontumorigenic clones. The isolated minimal promoter (TATA box and initiation site) of the K18 gene has by itself a differential activity in tumorigenic and nontumorigenic cells. An Sp1 binding site located upstream of the TATA box contributes to the high level of expression of the gene in tumorigenic cells. We report here that the Sp1 gene is not differentially expressed between the two cell types and that this is also the case for genes coding for factors of the preinitiation complex known to directly interact with the Sp1 protein. Further, DNase I footprinting experiments and mutagenesis analysis indicated that the mechanism responsible for the differential activity of the minimal K18 promoter apparently does not involve the binding of a factor to a specific sequence. During the course of these experiments, it was found that the initiation site of the K18 promoter is actually located 11 bp upstream of the +1 position previously reported and that the TATA box is the only essential element of the minimal promoter. Treatment of the cells with histone deacetylase inhibitors was more efficient at stimulating the activity of the K18 promoter in nontumorigenic cells than in tumorigenic cells. We propose that overexpression of the K18 gene in tumorigenic cells could result from of a high level of acetylation of histones and/or of factors controlling the activity of the transcription complex.

An Sp1 binding site and the minimal promoter contribute to overexpression of the cytokeratin 18 gene in tumorigenic clones relative to that in nontumorigenic clones of a human carcinoma cell line.

Clones of cells tumorigenic or nontumorigenic in nude mice have been previously isolated from the SW613-S human colon carcinoma cell line. We have already reported that tumorigenic cells overexpress the cytokeratin 18 (K18) gene in comparison with nontumorigenic cells and that this difference is mainly due to a transcriptional regulation. We now report that a 2,532-bp cloned human K18 gene promoter drives the differential expression of a reporter gene in a transient assay. A 62-bp minimal K18 promoter (TATA box and initiation site) has a low but differential activity. Analysis of deletion and substitution mutants as well as hybrid SV40-K18 promoters and reconstructed K18 promoters indicated that an important element for the activity of the K18 promoter is a high-affinity binding site for transcription factor Sp1 located just upstream of the TATA box. This Sp1 binding element, as well as the intron 1 enhancer element, stimulates the basal activity of the minimal promoter through mechanisms that maintain the differential activity. Gel shift assays and the use of an anti-Sp1 antibody have shown that both tumorigenic and nontumorigenic SW613-S cells contain three factors able to bind to the Sp1 binding element site and that one of them is Sp1. A hybrid GAL4-Sp1 protein transactivated to comparable extents in tumorigenic and nontumorigenic cells a reconstructed K18 promoter containing GAL4 binding sites and therefore without altering its differential behavior. These results indicate that the Sp1 transcription factor is involved in the overexpression of the K18 gene in tumorigenic SW613-S cells through its interaction with a component of the basal transcription machinery.

Partial characterization of two serologically related DNA-binding proteins specified by the cottontail rabbit herpesvirus CTHV.

We have previously described two serologically related DNA-binding phosphoproteins of different apparent molecular mass (75 kDa and 35 kDa) produced in cottontail rabbit herpesvirus (CTHV)-infected cells. The 75 kDa protein appeared before the 35 kDa protein in the infectious cycle. Here, we extend the characterization of these proteins. Protease V8 fingerprints of methionine-labelled 35 kDa protein showed four major peptide products, three of which co-migrated with major peptides from digests of the 75 kDa protein. The fourth peptide, with an estimated mass of 10 kDa, reacted with an antiserum recognizing both proteins. In vitro translation of total or poly A-containing RNA isolated from infected cells at 24 h to 72 h post-infection produced only the 75 kDa protein as measured by immunoprecipitation with anti-75/35 kDa serum, suggesting that the 35 kDa protein is derived from the 75 kDa protein by proteolytic cleavage. Virus-specific RNA obtained by prehybridization to CTHV DNA also produced the 75 kDA protein, confirming its viral origin. The putative gene for the 75 kDa protein was localized to a region on the CTHV DNA restricted by PvuII.

Cytochemical study of the localization and organization of parental herpes simplex virus type I DNA during initial infection of the cell.

Changes in the location and structural organization of parental herpes simplex virus type 1 (HSV-1) DNA during its migration from the extracellular space to the interior of the nucleus of the target cell were examined by in situ hybridization using an HSV-1 DNA probe, specific DNA staining, and autoradiography after infection of cells with tritium-labeled viruses. In situ hybridization was carried out on denatured DNA to reveal as much as possible of the HSV-1 sequence present at the surface of the sections, and also on non-denatured DNA which revealed the presence of single-stranded portions of parental DNA, both prior to and during its intracellular migration. The results from in situ hybridization and autoradiography demonstrated that a short interval of about 15 min separated the initial contact of the viruses with the cells from the entry of parental viral DNA into the nucleus. In transit, morphologically intact nucleoids were released into the cytoplasm, and swollen nucleoids which contained partially decondensed viral DNA became detectable in the juxtanuclear cytoplasm and the periphery of the nucleus among the cell chromatin fibers. Completely decondensed parental viral DNA fibers could not be distinguished structurally from cellular DNA, but their position could be revealed by the in situ hybridization label. The infective DNA became randomly distributed within all compartments of the nucleus except the matrix-associated clusters of interchromatin granules.

Genome structure of cottontail rabbit herpesvirus.

The genome structure of a herpesvirus isolated from primary cultures of kidney cells from the cottontail rabbit Sylvilagus floridanus was elucidated by using electron microscopy and restriction enzyme analysis. The genome, which was about 150 kilobase pairs long and which had an average G + C composition of 45%, consisted of two regions with unique base sequences (54 and 47 kilobase pairs) enclosed by reiterations of a 925-base-pair sequence with a variable copy number. The internal repeats were in opposite polarity with respect to the terminal repeats, and both unique regions underwent inversion. The nucleotide sequence of the repeat unit was determined, and virion DNA termini were precisely localized within this sequence. Elements showing homology with the cleavage-packaging signals common to other herpesviruses were detected. The data indicate that this virus is different from the previously described herpesvirus sylvilagus.

DNA-binding proteins induced by the cottontail rabbit herpesvirus CTHV.

Several new polypeptides were detected in cells infected with CTHV, a cottontail rabbit herpesvirus. All of them (Mr 150K, 110K, 93K, 83K, 75K and 35K) accumulated in the nucleus during the infectious cycle, and all except the 150K species bound to DNA-cellulose affinity columns in low-salt buffers. Polyclonal antisera prepared against the 35K DNA-binding protein also recognized the 75K species. Although the 75K protein could be detected earlier in infection than the 35K protein, late in the infectious cycle the latter increased to an abundance approaching that of cellular histones. Treatment of partially purified virions with a non-ionic detergent indicated that the 35K protein, but not the 75K protein, is a component of capsid/tegument structures. The anti-35K/75K serum did not cross-react with herpesvirus sylvilagus virion proteins, which, in an electrophoretic comparison, exhibited both similarities to and differences from the virion proteins of CTHV. Labelling of CTHV-infected cells with [32P]orthophosphate revealed the presence of phosphoproteins electrophoretically comigrating with the 93K, 83K, 75K and 35K proteins.

Effect of dehydrating agents on DNA organization in herpes viruses.

With routine procedures of Epon- or GMA-embedding and a stain specific for DNA, the nucleoid of mature herpes simplex virus-type 1 (HSV-1) assumes the well-known form of a short, compact, hollow cylinder or torus. A new, more complex organization of DNA filaments in encapsidated HSV-1 was found in infected cells after aldehyde fixation, methanol dehydration, and Lowicryl embedment. We have determined that it is the use of methanol as dehydrating agent that permits visualization of this internal structure. The same new spatial organization of DNA can be seen in Epon and GMA sections when methanol dehydration is used. This organization is lost in a methanol-ethanol sequence of dehydration but can be restored in an ethanol-methanol sequence. Dimethylsulfoxide (DMSO) is the only other agent among several reviewed here which resembles methanol in its effect on HSV-1 DNA. Methanol had the same effect on five subfamilies of the herpes group (HSV-1, HSV-2, CCV, CMV, CTHV) but did not alter the nucleoid ultrastructure in simian virus 40 (SV40) and adenovirus type 5 (Ad 5). Therefore, it may sometimes, but not always, provide additional information about the organization of biological structures.

Localization of highly phosphorylated proteins in cells altered by Herpes simplex virus infection.

Highly phosphorylated proteins detectable by their ability to bind bismuth ions were localized in rabbit fibroblasts before and during infection with Herpes simplex viruses type 1 and type 2. The bismuth tartrate procedure of Locke and Huie applied to glutaraldehyde-fixed cells revealed a low level of bismuth binding in a restricted portion of the normal nucleolus in non-infected cells. From 2.5-17 hr post-infection during virus development and maturation, the phosphorylated proteins were more widespread and the intensity of reaction was augmented. Bismuth deposits were then associated with virus-modified pre-existing structures including all of the nucleolar fibrils, the more abundant interchromatin granules, reduplications of some areas of the inner nuclear membrane and the Golgi apparatus. Virus-induced structures which were stained included nuclear dense bodies, the teguments of enveloped virions and the contents of extranuclear enveloped structures devoid of capsids. Following detergent-induced destruction of membranes, staining was lost from the nuclear envelope and cytoplasmic virions, which demonstrated that the highly phosphorylated proteins were tightly bound to nuclear and viral membranes. Bismuth staining of nitrocellulose sheets containing proteins extracted from whole cells revealed no reaction in normal cells but three positive bands were found in infected cells.

Ultrastructural localization of the herpes simplex virus major DNA-binding protein in the nucleus of infected cells.

Using immunocytochemical procedures in conjunction with electron microscopy we have examined the distribution of the major DNA-binding protein (DBP) of herpes simplex virus (HSV) in infected nuclei. In embedded specimens, DBP was preferentially associated with fibrillar material of electron-translucent viral inclusions, and to a lesser extent with peripheral (marginated) host chromatin. The latter association was sensitive to a non-ionic detergent ('Photo flo'). In chromatin spread by the Miller technique, DBP was found to be a component of the 10 nm 'thick filaments' previously described in HSV infection.

Organization of the nucleolar RNP components in cultured rabbit fibroblasts following mild loosening of the nuclear content.

The organization of the ribonucleoprotein (RNP) components of the nucleolus was studied in ultrathin sections by a recent method of mild loosening of the nuclear content. This investigation was carried out in cultured rabbit fibroblasts in which the nucleoli contain clearly separated fibrillar and granular RNP components. In addition, the demarcation between these two particulate components was accentuated by infection with herpes simplex type 1 virus (HSV1), by recovery at 37 degrees C after a heat shock, and by actinomycin D treatment. Except in actinomycin D-treated cells, it was possible to identify two distinct components which are not visible in routinely prepared sections: (1) highly contrasted fibrillar clusters resulting from the loosening of the fibrillar component, (2) a granular network composed of nucleolar granules interconnected by a thin filament of about 5 nm in thickness. In actinomycin D-treated cells, only the granular zone was observed. High resolution autoradiography carried out both on standard fixed and on loosened cells following a 5 min labeling with tritiated uridine revealed that the fibrillar clusters correspond to sections of nucleolar transcription complexes. When the same labeling was followed by 3 h chase only the granular network was labeled indicating that it corresponds to the site of pre-ribosomal ribonucleic acid (pre-rRNA) maturation. Infection by HSV1 did not change this result. Preferential RNP staining and deoxyribonucleic acid (DNA) specific staining failed to reveal the thin filaments interconnecting the nucleolar granules. We suggest that the latter filaments serve as support for pre-rRNA processing and for storage of large pre-ribosomal subunits.

Infectious DNA from herpes simplex virus: infectivity of double-stranded and single-stranded molecules.

The infectious units in native and alkalidenatured preparations of DNA of herpes simplex virus were characterized with respect to their sensitivity to Neurospora crassa endonuclease, their sedimentation properties in high-salt, neutral sucrose gradients, and their sensitivity to hydrodynamic shearing forces. Infectious molecules in native preparations were resistant to N. crassa endonuclease, sedimented at 56 S, and were highly sensitive to shearing forces. After alkaline denaturation, infectious molecules became sensitive to the N. crassa enzyme, sedimented at 200 S, and were relatively resistant to shear. We conclude that both intact duplex molecules ([unk]100 x 10(6) daltons) and intact single strands ([unk]50 x 10(6) daltons) are capable of initiating productive infection.