Polyomavirus EGFP-pseudocapsids: analysis of model particles for introduction of proteins and peptides into mammalian cells.

A vector for preparation of mouse polyomavirus capsid-like particles for transfer of foreign peptides or proteins into cells was constructed. Model pseudocapsids carrying EGFP fused with the C-terminal part of the VP3 minor protein (EGFP-VLPs) have been prepared and analysed for their ability to be internalised and processed by mouse cells and to activate mouse and human dendritic cells (DC) in vitro. EGFP-VLPs entered mouse epithelial cells, fibroblasts and human and mouse DC efficiently and were processed by both, lysosomes and proteasomes. Surprisingly, they did not induce upregulation of DC co-stimulation molecules or maturation markers in vitro; however, they did induce interleukin 12 secretion.

Ori-somes, nucleoprotein complexes descending from origin regions of animal chromosomal DNA replication. A micromorphological study.

Micromorphology of nucleoprotein (NP) complexes designated according to their descent and shape as Ori-somes is presented. These NP complexes of three different types harbor molecules of cytoplasmic "small" polydisperse DNA, which descend from origin regions of chromosomal DNA replication and are equipped, as shown previously, with early DNA-synthesizing activities. By negative staining the Ori-somes are visualized as particles of irregular shape, sometimes of a subunit-like structure. Micromorphological differences in size and structural compactness noted among individual Ori-somes are dependent on their type similarly as earlier shown physico-chemically and biochemically. Such differences were also confirmed by two different spreading techniques. The most unravelled structures with electron diffuse centers belong to Ori-somes of component B associated with most active DNA synthesis. In contrast, the Ori-somes of components A and C, associated with pronounced RNA synthesis, revealed large electron-dense centers. The incidence of replicative structures present in Ori-somes corresponds with the level of their DNA-synthesizing activities.

Caveolae are involved in the trafficking of mouse polyomavirus virions and artificial VP1 pseudocapsids toward cell nuclei.

Electron and confocal microscopy were used to observe the entry and the movement of polyomavirus virions and artificial virus-like particles (VP1 pseudocapsids) in mouse fibroblasts and epithelial cells. No visible differences in adsorption and internalization of virions and VP1 pseudocapsids ("empty" or containing DNA) were observed. Viral particles entered cells internalized in smooth monopinocytic vesicles, often in the proximity of larger, caveola-like invaginations. Both "empty" vesicles derived from caveolae and vesicles containing viral particles were stained with the anti-caveolin-1 antibody, and the two types of vesicles often fused in the cytoplasm. Colocalization of VP1 with caveolin-1 was observed during viral particle movement from the plasma membrane throughout the cytoplasm to the perinuclear area. Empty vesicles and vesicles with viral particles moved predominantly along microfilaments. Particle movement was accompanied by transient disorganization of actin stress fibers. Microfilaments decorated by the VP1 immunofluorescent signal could be seen as concentric curves, apparently along membrane structures that probably represent endoplasmic reticulum. Colocalization of VP1 with tubulin was mostly observed in areas close to the cell nuclei and on mitotic tubulin structures. By 3 h postinfection, a strong signal of the VP1 (but no viral particles) had accumulated in the proximity of nuclei, around the outer nuclear membrane. However, the vast majority of VP1 pseudocapsids did not enter the nuclei.

Ultrastructural analysis of v-myb oncogene product cooperation with components of avian cell nuclear matrix.

The cooperation of the v-Myb oncoprotein with extracted nuclear matrix of avian haematopoietic cells expressing the v-myb oncogene was studied by means of immunoelectron microscopy. The nuclear matrix was extracted by a gentle method of detergent treatment at moderate ionic strength and visualized either in ultrathin LR White sections, in unembedded resin-free sections, and in addition by the aqueous spreading technique. Using anti-Myb polyclonal antibody we have shown interaction of the v-Myb protein product with extracted nuclear matrix. This oncoprotein, however, was easily released from the structure by a detergent as well as by DNAase treatment and ammonium sulphate extraction. Prefixation of structures before detergent treatment prevented this extraction. The v-Myb protein marker was distributed in clusters or associated with fibrillar structures in most cases. Single markers decorating these fibrillar or less dense structures were also detected.

Production of polyomavirus structural protein VP1 in yeast cells and its interaction with cell structures.

The gene for mouse polyomavirus major structural protein VP1 was expressed in Saccharomyces cerevisiae from the inducible GAL7 promoter. VP1 pseudocapsids were purified from cell lysates. Their subpopulation contained fragments of host DNA, which, in contrast to those of VP1 pseudocapsids produced in insect cells, did not assemble with cellular histones into pseudonucleocores. VP1 pseudocapsids accumulated in the yeast cell nuclei. A strong interaction of VP1 with tubulin fibres of the mitotic spindle was observed. The fibres of spindles were larger in diameter, apparently due to tight VP1 binding. Substantial growth inhibition of yeast cells producing VP1 was observed.

Virus-like gene transfer into cells mediated by polyoma virus pseudocapsids.

Mouse polyoma virus-like particles (or pseudocapsids) are composed solely of recombinant viral coat protein. They can interact with DNA and transport it to cells, resulting in gene expression both in tissue culture and in mice. We demonstrate that DNA transfer in vitro depends on partial packaging of DNA within the virus-like capsid. Cell surface sialic acid residues and an intact microtubule network, required for viral infectivity, are also necessary for pseudocapsid-mediated gene expression from heterologous DNA. Thus, gene delivery in this system requires pathways utilised by polyoma virions, rather than proceeding via the 'nonspecific' endosomal route typical of nonviral systems such as liposomes or calcium phosphate precipitates. Despite the fact that all cells appear to internalise pseudocapsid/DNA complexes, only a proportion show productive gene delivery. Bulk internalisation of complexes is dependent on actin fibres, but not cell surface sialic acid or microtubules, indicating that a second transport pathway exists for pseudocapsids which is nonproductive for gene transfer. The model suggested by these data demonstrates the virus-like properties of the pseudocapsid system, and provides a basis for further development to produce a highly effective gene delivery vehicle. Gene Therapy (2000) 7, 2122-2131.

Different morphological changes of two colorectal adenocarcinoma cell lines after sodium butyrate treatment.

The principal aim of this study was to determine whether the sodium butyrate, cell differentiation-inducing compound, induces identical morphological changes in two colorectal adenocarcinoma cell lines which exhibit the different changes in the alkaline phosphatase activity after treatment with this agent. Ultrastructural analysis showed that these two cell lines possessed different sensitivity to the presence of sodium butyrate. Particularly different changes were observed in the chromatin structure of the cell lines tested. Our study demonstrates that sodium butyrate initiates cell differentiation, modifies the cell components, but the characteristics and extent of this modification depends on the cell line used.

Interactions of heterologous DNA with polyomavirus major structural protein, VP1.

'Empty' polyomavirus pseudocapsids, self-assembled from the major structural protein VP1, bind DNA non-specifically and can deliver it into the nuclei of mammalian cells for expression [Forstová et al. (1995) Hum. Gene Ther. 6, 297-3061. Formation of suitable VP1-DNA complexes appears to be the limiting step in this route of gene delivery. Here, the character of VP1-DNA interactions has been studied in detail. Electron microscopy revealed that VP1 pseudocapsids can create in vitro at least two types of interactions with double-stranded DNA: (i) highly stable complexes, requiring free DNA ends, where the DNA is partially encapsidated; and, (ii) weaker interactions of pseudocapsids with internal parts of the DNA chain.

Baculovirus expression system cells expressing v-myb oncogene: the distribution of RNA and DNA in specific nuclear compartments with respect to structures interacting with anti-v-Myb antibody.

The distribution of RNA, total DNA and newly synthesized DNA within nucleoli-like structures in insect cells overexpressing v-myb oncogene was investigated. Three types of these structures which revealed interaction with anti-v-Myb oncoprotein antibody were found at the ultrastructural level. Specific staining by toluidine blue at pH 5.2 showed the presence of RNA in these nucleoli-like structures. To detect total DNA, the in situ terminal deoxynucleotidyl transferase-immunogold technique was used. In addition to an expected labeling of host condensed chromatin, the labeling of the three types of nucleoli-like structures differed from each other. While the compact (type I) and ring-shaped (type II) nucleoli-like structures were labeled only on their periphery and in the proximity of baculovirus particles that interacted with them, the structures with an appearance of nucleolonemas (type III) were labeled strongly. Incorporation of 5-bromo-2-deoxyuridine, in spite of a poor labeling of newly synthesized DNA, confirmed these results. We suggest that the nucleoli-like structures of type I and II are of nucleolar origin. The type III more likely represents virogenic stroma or viral DNA storage site.

Interaction of V-Myb oncoprotein with spread chromatin of avian haematopoietic cells.

Interactions of v-Myb oncoprotein with spread chromatin of avian LSCC-BM2 cells expressing v-myb oncogene were studied by means of immunoelectron microscopy. The application of this technique using anti-Myb polyclonal antibody combined with the Miller type spreading for visualisation of chromatin revealed the presence of Myb protein on stretched chromatin fibres. Intense labelling was apparent on the chromatin dispersed by hypotonic treatment, where the label was present frequently in clusters, although individual marks along the fibrillar molecules were also found. The combination of hypotonic and detergent treatment resulted in better dispersal of chromatin, more frequent detection of active transcription units, but also in removal of some proteins from chromatin fibres. The labelling of chromatin with anti-Myb antibody was substantially reduced in this case and was dependent on detergent concentration used. The marker was found less frequently on chromatin fibres usually present in clusters on remaining protein structures. Our findings confirmed direct interaction of v-Myb protein with chromatin structure. This interaction is apparently affected by detergent treatment.

Enhancement by polylysine of transient, but not stable, expression of genes carried into cells by polyoma VP1 pseudocapsids.

Gene transfer to provide long-term expression of a therapeutic product, without introducing unwelcome genetic information, is a goal being sought for therapy of both hereditary and acquired diseases. Polyoma virus pseudocapsids, generated from a VP1-expressing recombinant baculovirus, lack viral DNA and have been successfully used to introduce small exogenous genes stably into cells in vitro by a process designated 'pseudofection'; although pseudocapsids protect only about 3 kbp of exogenous DNA, low efficiency transfer of a larger fragment (6.2 kbp) has been observed. Here, expression of a 7.2 kbp plasmid (pCMV beta) encoding the beta-galactosidase gene was assessed to monitor not only efficiency, but the ability of pseudocapsids to transfer larger-sized DNA on their own, or in the presence of the polycation, poly-L-lysine, added to protect nonencapsidated DNA. When complexed to pseudocapsids only, the efficiency of expression of the transferred beta-galactosidase gene (in human or rodent cells), although low, appeared to stabilise with time. In the presence of polylysine, unencapsidated DNA was shown to be protected against DNase activity, but electron microscopy (EM) revealed the formation of large mixed aggregates. The addition of pseudocapsids to these aggregates, and measurement of mobilities of the complexes in CsCl equilibrum centrifugation, indicated that they contained negligible amounts of VP1. For subsequent pseudofection experiments, DNA was complexed first with pseudocapsids, then polylysine was added. The latter did not appear to displace pseudocapsids from DNA, and was found to increase the efficiency of short-term expression both in in vitro and in vivo experiments. Gene expression, analysed histochemically or by the polymerase chain reaction, revealed transcriptional activity of the input gene, with expression first diminishing, then stabilising over time. The presence of pseudocapsids, in complexes with DNA with or without polylysine, allowed for stable and persistent gene expression.

Electron-microscopic immunocytochemical study of the intracellular transport of the viral glycoproteins in ts1, a mutant of Moloney murine leukemia virus.

The transport and localization of env-proteins of ts1 virus (a paralytogenic temperature-sensitive mutant of Moloney murine leukemia virus) in infected cells of the TB cell line have been studied at the ultrastructural level. It was found that the envelope precursor polyprotein gPr80-env of ts1 was inefficiently transported out of the endoplasmic reticulum at the restrictive temperature. It was speculated that inefficient transport correlates with inefficient processing of gPr80env into gp70 and Prp15E and leads to paralytic disease.

Micromorphology of cytoplasmic nucleoprotein complexes harboring an extrachromosomal DNA closely related to avian myeloblastosis virus core-bound DNA.

Nucleoprotein (NP) complexes constituting the three basic components (A, B, C) of the postmicrosomal sediment (POMS) of chicken leukemic myeloblasts (CHLMs) which contain extrachromosomal DNA closely related to avian myeloblastosis virus DNA were analyzed electron microscopically. It was shown that these NP complexes resemble micromorphologically, depending on the origin of their POMS components, NP structures involved in three successive stages of early DNA synthesis. Nucleic acids harbored in these NP complexes exhibited micromorphological features typical for replicative structures. It was confirmed electron microscopically that the extrachromosomal DNA of CHLMs replicative in nature and of three length classes is organized into special NP complexes, each of which, as demonstrated, represents a unique reaction machinery of early DNA synthesis.

V-myb oncogene and c-myb proto-oncogene expression in avian cells: morphological changes of the cells and topographic localization of myb proteins.

Morphological changes of avian cells expressing the v-myb oncogene or c-myb proto-oncogene were studied by means of electron microscopy. Expression of both genes lead to distinct morphological changes of these cells. The nucleus of LSCC-BM2 cells espressing v-myb gene was of normal size but usually of irregular shape. It contained large unravelled nucleoli with typical interstices in some cells. Small nucleolar structures were also localized in the periphery of nuclear membrane. Nuclear envelope revealed reduced perinuclear space between two membranes. LSCC-BK3 cells expressing the c-myb gene were characterized by distinctly enlarged nucleus, in most cases of irregular shape. It contained only one nucleolus markedly enlarged, often unravelled, with apparent interstitial area. Nucleoli with nucleolonemas were observed in some cells. Nuclear envelope formed by two obscure membranes showed reduced perinuclear space. Topographic localization of v-Myb and c-Myb protein products was not basically different, both being detected in the nucleus of avian cells. v-Myb and c-Myb markers were distributed mostly in clusters, usually associated with interchromatin granules, but some marker was associated also with the nuclear membrane. Both Myb products were never detected in nucleolar structures of avian cells. Morphological changes of avian cells expressing myb genes and topographic localization of Myb proteins in these cells were different from those found in the insect cells expressing myb genes. The observed differences are discussed.

Morphological changes of the insect cells in the baculovirus system as a function of v-myb and c-myb inserts expression and topographic localization of v-Myb and c-Myb proteins.

Structural changes of insect cells Spodoptera frugiperda in the baculovirus expression system after expression of v-myb oncogene and c-myb protooncogene inserts were studied by means of electron microscopy. Expression of v-myb gene insert was accompanied by extensive changes in cell structure, when compared with those of the noninfected and wild-type virus-infected cells. Enormous increase in nuclear content was apparent within 48 hrs after infection, along with changes in nucleolus appearance. Large ring-shaped nucleoli, compact nucleoli and nucleoli with nucleolonemas were detected together with dense nucleolus of normal appearance and small nucleolar structures localized in the nuclear periphery. The cytoplasm practically disappeared 72 hrs after infection. Morphological changes of insect cells expressing the c-myb gene were significantly less distinct, but more frequent unraveling of nucleoli was observed. Both v-Myb and c-Myb proteins were localized in the nucleus of infected cells as was revealed by fluorescence microscopy and electron microscopy. c-Myb marker decorated distinctly the ring-shaped area of nucleolus with some less intensive labelling of the inner part of nucleolus and proximal area on nuclear membrane. v-Myb protein revealed predominantly more compact and homogeneous distribution inside the nucleolus but a small proportion of it was also detected outside the nucleolus in the nuclear compartment. The data obtained on insect cells suggest that Myb proteins may participate also in the processes in which the nucleolus plays a role.

DNA looping by the HMG-box domains of HMG1 and modulation of DNA binding by the acidic C-terminal domain.

We have compared HMG1 with the product of tryptic removal of its acidic C-terminal domain termed HMG3, which contains two 'HMG-box' DNA-binding domains. (i) HMG3 has a higher affinity for DNA than HMG1. (ii) Both HMG1 and HMG3 supercoil circular DNA in the presence of topoisomerase I. Supercoiling by HMG3 is the same at approximately 50 mM and approximately 150 mM ionic strength, as is its affinity for DNA, whereas supercoiling by HMG1 is less at 150 mM than at 50 mM ionic strength although its affinity for DNA is unchanged, showing that the acidic C-terminal tail represses supercoiling at the higher ionic strength. (iii) Electron microscopy shows that HMG3 at a low protein:DNA input ratio (1:1 w/w; r = 1), and HMG1 at a 6-fold higher ratio, cause looping of relaxed circular DNA at 150 mM ionic strength. Oligomeric protein 'beads' are apparent at the bases of the loops and at cross-overs of DNA duplexes. (iv) HMG3 at high input ratios (r = 6), but not HMG1, causes DNA compaction without distortion of the B-form. The two HMG-box domains of HMG1 are thus capable of manipulating DNA by looping, compaction and changes in topology. The acidic C-tail down-regulates these effects by modulation of the DNA-binding properties.

Avian myeloblastosis virus core-bound 7 S DNA, highly bent minute structures with sequence-directed curvature.

Structural properties and length distribution profile of 7 S avian myeloblastosis virus (AMV) DNA were studied by means of electron microscopy using two different techniques. This DNA represents mostly double strands, the single strands being in minority. We have shown directly that this DNA forms a bent structure typical of the majority of molecules. These bends are sensitive to the distamycin treatment which stretches most of the bent molecules. Some amount (up to 30%) of circular DNA molecules was detected also in DNA preparations, the nature and the size of which are reminiscent of electron microscopic data on microbubbles of replicating DNA. No specific AMV DNA structural features were found using osmium-tetroxide treatment. The basic size of AMV DNA was estimated to be approximately 150 bp, but its multimers were also detected. Their presence and significance is discussed.

Structures of poly(dA-dT, ip5dU) containing various small amounts of the antiherpetic 5-isopropyl-2'-deoxyuridine.

Three different concentrations of the antiherpetic agent 5-isopropyl-2'-deoxyuridine (ip5dU) were introduced into the synthetic DNA poly(dA-dT) to analyze resulting copolymers by electron microscopy, UV absorption and CD spectroscopy. The poly(dA-dT, ip5dU) containing 1.3 and 4.3% ip5dU did not much differ from the parent poly(dA-dT) but poly (dA-dT, ip5dU) with 7.1% ip5dU behaved in an unusual way. Results are explained by the notion that if bulky isopropyls occur sufficiently close to each other then stable hairpins protruding from the double helix are formed, presumably to accommodate the ip5dU-s into the loops.

A stable line of turkey bone marrow cells transformed by the myelocytomatosis virus strain MC31. Ultrastructural characteristics and localization of the DNA replication sites.

Attempts were made to characterize cells of the LSTC-SF2 line by scanning electron microscopy and transmission electron microscopy on the ultrastructural level. The virus-transformed cells are of oval, slightly elongated shape with an undulating surface. The cell nucleus is well outlined, poor in heterochromatin but with a strongly developed nucleolus. The cytoplasm is not rich in organelles except for an abundance of mitochondria with dense granules that are often found in them. With high-resolution autoradiography the DNA synthesis sites were identified mainly in proximity to the nuclear membrane and in the perinuclear spaces. The cells under study can be regarded as immature forms of the blood series and most likely as precursors of cells of the granulocyte or monocyte series.

Microinjection of cloned proviral Rous sarcoma virus DNAs into Xenopus laevis one cell embryos.

Plasmid pATV8 carrying Rous sarcoma virus (RSV) DNA with one long terminal repeat (LTR) or plasmid pAPrC carrying complete RSV proviral DNA was injected into fertilized eggs of Xenopus laevis. The fate of the exogenous DNAs was followed during embryogenesis. In both cases a new form of DNA, comigrating with endogenous DNA, began to appear shortly after the injection. This new form represented a linear dimer of the injected plasmid that had formed by ligation of linear monomers of the plasmid DNAs. All forms of the exogenous pATV8 DNA disappeared gradually during neurulation. No traces of virus-specific RNA were ever found in any of the following stages. The intensity of replication of plasmid pAPrC DNA in fertilized eggs was greater than that of pATV8 DNA and, in contrast to pATV8, a small amount of pAPrC persisted through the neurula till higher stages. In some experiments virus-specific DNA was even observed in 6-day-old embryos. Digestion with Nsi I and Sac I proved integration of the plasmid into the frog genome. At the stage of gastrula and at later stages a weak signal of viral RNA was also detected.