Origin of replication in episomal bovine papilloma virus type 1 DNA isolated from transformed cells.

The origin of replication of bovine papilloma virus type 1 (BPV-1) has been determined by isolating replicative intermediates (RI) of BPV-transformed hamster embryo fibroblasts (HEF-BPV). These RI were treated with single cut restriction enzymes to determine the start-position (origin) of the extending replication eyes using electron microscopic techniques. 'Cairns'-type RI molecules were shown to contain one replication eye in monomeric as well as in dimeric molecules. The position of this eye was localized at 6940 +/- 5% bp in the physical map. In a second set of experiments BPV-1 DNA fragments cloned in pBR322 were tested for transient episomal replication. Transfected cells were harvested after increasing periods of time and screened for replication with isoschizomeric restriction enzymes to differentiate between input and replicated DNA. The part of the BPV genome harboring the replication origin spans the BPV ClaI-C restriction fragment corresponding to the non-coding region of the BPV genome and coincides with the DNase I-hypersensitive control region in the chromatin, isolated from transformed cells.     

A polyoma-derived plasmid vector maintained episomally in both E. coli and mouse hepatoma cells

We describe a recombinant plasmid, pBBPY1, containing polyoma virus sequences which persists episomally in mouse hepatoma (MH) cells and can be shuttled between these cells and bacteria. This plasmid is composed of a subgenomic fragment of a polyoma virus mutant that includes two origins of replication; sequences of plasmid pML2; the xanthine-guanine phosphoribosyltransferase gene of Escherichia coli (Ecogpt) under the control of SV40 early-region promoter and RNA processing signals, providing a dominant selectable marker for mammalian transfection. MH cells from colonies growing in HAT medium (hypoxanthine, aminopterin and thymidine) were found to contain vector DNA molecules in an episomal state, the majority of them unrearranged. When HAT-selective pressure was applied for only 3 days, the resulting cells contained up to 50-100 copies of intact plasmid, i.e. 20-fold more than cells grown under standard selection conditions with continuous HAT-selective pressure. Contrary to standard conditions, transient selection does not alter the epithelial morphology nor ability of transfected hepatoma cells to produce albumin.     

Transformation of rodent cells by a cloned DNA fragment of herpes simplex virus type 2.

Transformation of rodent cells with isolated restriction endonuclease fragments of herpes simplex virus type 2 DNA identified a region of the genome located between map positions 0.58 and 0.62. These sequences were cloned into pBR322, and the recombinant plasmid was used to transform primary rat embryo cells and NIH 3T3 cells. The transformants were selected for their ability to form dense foci on a monolayer or to form colonies in semisolid medium. In contrast to the parental rat or mouse cells, cell lines transformed with the cloned herpes simplex virus type 2 fragment grow to high saturation densities, replicate in medium containing 1% serum, form colonies in dilute methylcellulose, show reduced levels of fibronectin, and are tumorigenic in nude mice and in their syngeneic hosts. Southern blot hybridizations have detected sequences homologous to the viral fragment in high-molecular-weight DNA from the transformed cell lines that are not present in DNA from normal rodents. In all cases, the plasmid DNA was present in less than one copy per cell, and the patterns of viral sequences changed with passage of the cell line in vivo.     

The SU and TM envelope protein subunits of bovine leukemia virus are linked by disulfide bonds, both in cells and in virions

After the polyprotein precursor of retroviral envelope proteins is proteolytically cleaved, the surface (SU) and transmembrane (TM) subunits remain associated with each other by noncovalent interactions or by disulfide bonds. Disulfide linkages confer a relatively stable association between the SU and TM envelope protein subunits of Rous sarcoma virus and murine leukemia virus. In contrast, the noncovalent association between SU and TM of human immunodeficiency virus leads to significant shedding of SU from the surface of infected cells. The SU and TM proteins of bovine leukemia virus (BLV) initially were reported to be disulfide linked but later were concluded not to be, since TM is often lost during purification of SU protein. Here, we show that SU and TM of BLV do, indeed, associate through disulfide bonds, whether the envelope proteins are overexpressed in transfected cells, are produced in virus-infected cells, or are present in newly produced virions.     

Transformation of human epidermal cells by transfection with plasmid containing simian virus 40 DNA linked to a neomycin gene in a defined medium

A human epidermal cell culture was transformed by transfection with a recombinant plasmid containing simian virus 40 DNA with a deletion at the origin and an antibiotic (neomycin or G418) marker. A calcium phosphate-mediated DNA transfection method was optimized for introducing exogenous DNA into cells maintained in a fully defined medium. The transformed cells were propagated for more than 200 population doublings and did not appear to go through a "crisis" period. The growth characteristics of the transformed cells were similar to those of untransformed cells. Major keratins synthesized in the transformed cells were similar to those found in normal epidermal cells. Transformed cells initially transfected with the recombinant plasmid could be propagated for more than 30 passages. Actively growing cells could then be repeatedly selected from cell populations based upon their neomycin (G418)-resistant phenotype for at least another 30 passages. Simian virus 40 T-antigen and extrachromosomal DNA containing plasmid- and SV40-specific DNA sequences were detected in the transformed cells. Because of their nononcogenic phenotype and defined growth requirements, the transformed cells provide a model for examining structural changes during cell proliferation and differentiation, and for exploring the multistage carcinogenesis of human epithelial cells.     

Human immunodeficiency virus type 1 DNA nuclear import and integration are mitosis independent in cycling cells

An essential step in human immunodeficiency virus type 1 (HIV-1) replication is the movement of the viral preintegration complex from the cytoplasm into the nucleus. The pathway(s) and timing for HIV-1 DNA nuclear entry in cycling cells have not been established. Here, we show that if cycling cells are infected before S phase, viral DNA can be integrated prior to passage of the host DNA replication fork through the integration site, as indicated by stable inheritance in both daughter cells. We conclude that efficient nuclear entry can occur independently of mitotic nuclear disassembly in cycling cells.     

An improved strategy for rapid direct sequencing of both strands of long DNA molecules cloned in a plasmid.

A strategy for kilo-base sequencing of a target DNA cloned in plasmid pWR34 is described. A long target DNA is progressively shortened from one end, by digestion with BAL31 nuclease or exonuclease III and nuclease S1, followed by cleaving off the shortened vector DNA. The family of the shortened target DNA molecule is next cloned in between the StuI site on one end, and a cohesive-ended restriction site on the other end, within the polylinker region of pWR34. DNA fragments cloned into this plasmid are sequenced directly by using a synthetic oligonucleotide primer, which binds to one side of the polylinker region using the dideoxynucleotide chain-termination method. The plasmid DNA, easily obtained by adoption of a rapid mini-preparation, is usually pure enough for direct DNA sequencing. Thus, both strands of any DNA several thousand base pairs in length can be completely sequenced (using two different primers) with ease within a short time, without the need for constructing a physical map.     

Nuclear import of plasmid DNA in digitonin-permeabilized cells requires both cytoplasmic factors and specific DNA sequences.

Although much is known about the mechanisms of signal-mediated protein and RNA nuclear import and export, little is understood concerning the nuclear import of plasmid DNA. Plasmids between 4.2 and 14.4 kilobases were specifically labeled using a fluorescein-conjugated peptide nucleic acid clamp. The resulting substrates were capable of gene expression and nuclear localization in microinjected cells in the absence of cell division. To elucidate the requirements for plasmid nuclear import, a digitonin-permeabilized cell system was adapted to follow the nuclear localization of plasmids. Nuclear import of labeled plasmid was time- and energy-dependent, was inhibited by the lectin wheat germ agglutinin, and showed an absolute requirement for cytoplasmic extract. Addition of nuclear extract alone did not support plasmid nuclear import but in combination with cytoplasm stimulated plasmid nuclear localization. Whereas addition of purified importin alpha, importin beta, and RAN was sufficient to support protein nuclear import, plasmid nuclear import also required the addition of nuclear extract. Finally, nuclear import of plasmid DNA was sequence-specific, requiring a region of the SV40 early promoter and enhancer. Taken together, these results confirm and extend our findings in microinjected cells and support a protein-mediated mechanism for plasmid nuclear import.     

Molecular cloning, refined physical map and heterogeneity of methylation sites of papilloma virus type 1a DNA

The entire genome of human papilloma virus type 1a was cloned in Escherichia coli using the plasmid pBR322 as vector. The integrity and the homogeneity of the viral DNA thus obtained was confirmed by restriction endonucleases analysis. Viral DNA isolated from a single wart was partially methylated at only one out of the four HpaII sites, d(C-C-G-G). Recognition sites for Bg/I, Bg/II, PstI and PvuII restriction endonucleases were located on the cloned genome.     

A region immediately adjacent to the origin of replication of bovine papilloma virus type 1 interacts in vitro with the nuclear matrix.

We have investigated the interaction of the 69% transforming fragment of the Bovine Papilloma Virus type 1 (BPV1) with the nuclear matrix from 1361.5 cells (NIH-3T3 cells transformed by a BPV chimeric construct). In vitro studies performed with end-labelled DNA fragments and nuclear matrices prepared using a high-salt extraction procedure demonstrate the binding of a 672 bp fragment adjacent to the viral origin of replication and containing the plasmid maintenance sequence (PMS-1). This fragment can be cleaved into two pieces (393 and 279 bp), both interacting equally well with the nuclear matrix. This indicates that a least two regions of the 672 bp DNA fragment are involved in the interaction.     

Type 1 human T-cell leukemia virus small envelope protein expressed in mouse cells by using a bovine papilloma virus-derived shuttle vector.

In an attempt to express the small (transmembrane) envelope protein p21e of type 1 human T-cell leukemia (lymphotrophic) virus (HTLV-1) exclusive of other viral gene products, we have constructed a recombinant plasmid clone (pMBE-1) in a bovine papillomavirus-derived mammalian expression vector. Mouse C127 cells transfected with the pMBE-1 plasmid expressed the introduced HTLV-1 viral gene(s) as demonstrated by Northern blot and indirect immunofluorescence with natural human antisera. The transfected mouse cells were injected into BALB/c mice, and a monoclonal antibody was recovered which specifically recognizes a 21-kilodalton protein present in HTLV-1 virions, indicating that the pMBE-1 plasmid encodes the small envelope protein.     

A bovine papilloma virus vector with a dominant resistance marker replicates extrachromosomally in mouse and E. coli cells

We describe the construction of a bovine papilloma virus-based vector (pCGBPV9) which contains a dominant selectable marker and replicates autonomously in both mouse and Escherichia coli cells. This vector contains the complete bovine papilloma virus genome, a ColE1 replication origin and a dominant selectable marker conferring resistance to kanamycin in bacteria and G418 in eukaryotic cells. A high number of G418R colonies are obtained after transfer of pCGBPV9 into mouse C127 cells. These G418R colonies contain vector DNA which replicates autonomously at approximately 10-30 copies per cell. The molecules are in most cases unrearranged and can be rescued into E. coli cells by bacterial transformation.     

Combined prime-boost vaccination against tick-borne encephalitis (TBE) using a recombinant vaccinia virus and a bacterial plasmid both expressing TBE virus non-structural NS1 protein

Background  

Heterologous prime-boost immunization protocols using different gene expression systems have proven to be successful tools in protecting against various diseases in experimental animal models. The main reason for using this approach is to exploit the ability of expression cassettes to prime or boost the immune system in different ways during vaccination procedures. The purpose of the project was to study the ability of recombinant vaccinia virus (VV) and bacterial plasmid, both carrying the NS1 gene from tick-borne encephalitis (TBE) virus under the control of different promoters, to protect mice against lethal challenge using a heterologous prime-boost vaccination protocol.     

Structure of defective DNA molecules in Epstein-Barr virus preparations from P3HR-1 cells.

Epstein-Barr virus (EBV), isolated from P3HR-1 cells, induces early antigen and viral capsid antigen upon infection of human B-lymphoblasts. The strong early antigen- and viral capsid antigen-inducing activity is only observed in P3HR-1 virus preparations harboring particles with defective genomes, suggesting that this biological activity is directly associated with the defective DNA population. After infection of EBV genome-carrying Raji or EBV genome-negative BJAB cells, defective genomes of P3HR-1 EBV DNA are replicated in excess, depending on the multiplicity of infecting EBV particles. Hybridization of the DNA from such infected cells with 32P-labeled EBV DNA after HindIII cleavage reveals six hypermolar fragments. Mapping of these fragments shows that they form one defective genome unit containing four nonadjacent regions (alpha, beta, gamma, and delta) of the nondefective P3HR-1 EBV DNA. Two of the segments (alpha and beta) contain ca. 17 and 13 megadaltons, respectively, from the terminal regions of the P3HR-1 genome, whereas the two smaller segments (gamma and delta) contain ca. 3.7 and 3.0 megadaltons, respectively, originating from the central portion of the genome. In the defective molecule, the regions gamma and delta are present in the opposite orientation compared with nondefective P3HR-1 EBV DNA. Tandem concatemers are formed by fusion of the alpha and beta regions. Our model suggests that tandem concatemers of three defective genome units can be packaged into virions in P3HR-1 cells.     

Human chromosome 12 encodes a species-specific factor which increases human immunodeficiency virus type 1 tat-mediated trans activation in rodent cells.

The human immunodeficiency virus type 1 (HIV-1) tat protein functions at a much lower level in rodent cells than in human cells. This species-specific difference in trans activation appears to be due to the lack of a functional homolog of a human cofactor for tat in rodent cells. Using HIV-1 long terminal repeat-driven human growth hormone as a reporter plasmid, we found that the tat-mediated trans activation functions at a level 5- to 20-fold lower in rodent cells than in human cells. Stable rodent-human hybrid cells containing only human chromosome 12 support a dramatically higher degree of trans activation. Thus, human chromosome 12 encodes a species-specific HIV-1 tat cofactor which, at least partially, restores high levels of tat-mediated trans activation. Chromosome 6 also appears to provide an additional factor which enhances HIV-1 tat-mediated trans activation in murine cells.     

Bluetongue virus type 17 can exist in a latent state in MDBK cells.

An infection with bluetongue virus type 17 can be regulated by the temperature of incubation to be either persistent, producing low levels of virus; lytic, producing a high titer of released virus; or latent, producing no detectable virus. The persistent and latent states are reversible.