Levels of bovine papillomavirus RNA and protein expression correlate with variations in the tumorigenic phenotype of hamster cells.

Three independent cell lines were established from primary cultures of LSH hamster embryo cells infected with bovine papillomavirus type 1 (BPV-1). Although these cell lines differed in their in vitro saturation densities, none was capable of colony formation in soft agar. Interestingly, two cell lines (BPV-HE1 and BPV-HE3) were tumorigenic in nude mice, syngeneic hamsters, and allogeneic hamsters, whereas BPV-HE2 was not. All three cell lines contained similar numbers of the BPV-1 genome (approximately 50 to 200 copies per cell). However, the nontumorigenic BPV-HE2 cell line contained very low levels of BPV-specific RNA and only small amounts of the BPV-1 E5 transforming protein. The efficiency and rate of tumor formation by BPV-HE1 and BPV-HE3 correlated directly with the apparent amount of viral E5 protein. This analysis suggests that there is a threshold level of BPV protein synthesis required for tumorigenicity, there is a continuum of tumorigenic phenotypes which may depend upon the level of BPV protein expression, and BPV-transformed hamster cells can withstand allogeneic transplantation.     

鹅圆环病毒感染性克隆的构建及其转染试验

设计带BamHⅠ酶切标记位点的引物,PCR扩增鹅圆环病毒(Goose circovirus,GoCV)全长基因组,将2个基因组顺式连接插入到pGEM-T Easy载体中,获得GoCV全长基因组头尾串联二聚体感染性克隆质粒pGEMT-2GoCV。EcoRⅠ酶切线性化pGEMT-2GoCV,与脂质体混合转染GoCV阴性鹅胚和雏鹅,常规PCR检测发现GoCV在转染鹅体内增殖,鹅胚转染组于孵出第2周和第4周检出血清阳性,且其中一个个体于4周龄扑杀时检出法氏囊阳性,雏鹅转染组于转染后2周检出血清阳性。试验进一步对扩增片段进行了BamHⅠ标记位点的检测,并应用GoCV实时荧光定量PCR方法对转染阳性样品进行了定量,结果显示阳性法氏囊组织中病毒含量为1.57×106拷贝/mg,阳性血清含病毒拷贝数在3.52×104～5.92×105拷贝/μL。综上,本试验构建的GoCV全长基因组头尾串联二聚体感染性克隆DNA可以转染鹅胚和雏鹅并增殖出带标记的GoCV克隆。     

Plasmids containing mouse rDNA do not recombine with cellular ribosomal genes when introduced into cultured mouse cells.

We have examined the fate of plasmids containing a segment of a mouse rDNA repeat after they were introduced by transfection into cultured mouse cells. In addition to the rDNA segment, the plasmids contained the thymidine kinase gene from herpes simplex virus 1 to allow for selection of the plasmid after transfection into thymidine kinase-deficient mouse cells. Thus far, no cases of homologous recombination between transfected plasmid DNAs and host cell sequences have been documented. We reasoned that the high repetition frequency of the rRNA genes in the mouse genome (200 copies per diploid cell) might create a favorable situation for obtaining homologous recombination events between the plasmids containing rDNA and host cell rDNA sequences. The plasmids were introduced into cells in both the presence and the absence of carrier DNA and both as covalently closed circles and linear molecules. The sites of plasmid integration in the genomes of various cell lines were examined by DNA restriction digests and hybridization, molecular cloning, and nuclear fractionation. In the seven cell lines examined, there was no evidence that the plasmids had integrated into the rRNA gene clusters of the cell. Thus, the apparent absence of site-specific integration of cloned DNAs introduced into mammalian cells does not appear to be due simply to the small target presented by most host cell sequences.     

Episomal and integrated copies of Epstein-Barr virus coexist in Burkitt lymphoma cell lines.

The Epstein-Barr virus genome is present in more than 95% of the African cases of Burkitt lymphoma. In this tumor, the viral genome is usually maintained in multiple episomal copies. Viral integration has been described only for Namalwa, a cell line lacking episomes. In this study, we have addressed the question of whether integrated and episomal copies can coexist in Burkitt lymphoma cells. Gel electrophoresis was used to demonstrate the presence of episomal as well as free linear DNA in three Burkitt lymphoma cell lines. The numbers of episomal copies per cell were estimated to be 5 to 10 in BL36 and BL137 cells and below 1 in BL60 cells, indicating that BL60 does not represent a homogeneous cell population. Fluorescence in situ hybridization was combined with chromosomal banding to study the association of the viral DNA with metaphase chromosomes. A symmetrical pattern of signals at both chromatids located at the same chromosomal sites in many if not all metaphases was taken as evidence for viral integration. In each of the three cell lines, one site of integration was identified: at chromosome 11p15 in BL36 cells, at chromosome 1p34 in BL137 cells, and at the site of a reciprocal t(11;19) translocation in BL60 cells. Integrated, episomal and linear copies of Epstein-Barr virus DNA thus coexist in Burkitt lymphoma cells. The biological significance of viral integration in Burkitt lymphoma cells remains to be elucidated.     

Replication of bovine papillomavirus type 1 (BPV-1) DNA in Saccharomyces cerevisiae following infection with BPV-1 virions

Saccharomyces cerevisiae protoplasts exposed to bovine papillomavirus type 1 (BPV-1) virions demonstrated uptake of virions on electron microscopy. S. cerevisiae cells looked larger after exposure to BPV-1 virions, and cell wall regeneration was delayed. Southern blot hybridization of Hirt DNA from cells exposed to BPV-1 virions demonstrated BPV-1 DNA, which could be detected over 80 days of culture and at least 13 rounds of division. Two-dimensional gel analysis of Hirt DNA showed replicative intermediates, confirming that the BPV-1 genome was replicating within S. cerevisiae. Nicked circle, linear, and supercoiled BPV-1 DNA species were observed in Hirt DNA preparations from S. cerevisiae cells infected for over 50 days, and restriction digestion showed fragments hybridizing to BPV-1 in accord with the predicted restriction map for circular BPV-1 episomes. These data suggest that BPV-1 can infect S. cerevisiae and that BPV-1 episomes can replicate in the infected S. cerevisiae cells.     

Production and characteristics of seven rat embryo cell lines transformed by adenovirus type 5 and its DNA

Seven cell lines transformed by adenovirus type 5 and its DNA were obtained. It was shown that different cell lines contain the fragments of viral DNA which differ in length and number of copies per DNA of diploid cells. They contain from the left end 6% of the viral DNA to complete or almost complete viral genome. All studied cell lines were sensitive to reinfection with adenovirus type 5. They produced no virus being cocultivated with cell sensitive to the virus. No cell line was able to induce tumors even in immunosuppressed newborn rats. All cell lines formed colonies in soft agar. The level of virus-specific antigens was higher in cells that contained a large part of the viral genome. The methods used did not allow to correlate the biological properties of the transformed cells with the length and the number of copies of the integrated part of the viral genome.     

Comparison of the ability of lactate dehydrogenase-elevating virus and its virion RNA to infect murine leukemia virus-infected or -uninfected cell lines.

Lactate dehydrogenase-elevating virus (LDV) has a strict species specificity. Cells or cell lines other than a particular subset of mouse primary macrophages which can support LDV replication in vitro have not been identified. LDV induces neurological disorders in old C58 or AKR strains, in which the involvement of multiple copies of the endogenous N-tropic murine leukemia virus (MuLV) genome and the Fv-1 locus of the mouse has been implicated. Our previous studies have demonstrated that LDV could infect and replicate in cell lines of the mouse or other species in vitro when they were infected with MuLV. The significance of and the precise mechanism underlying this phenomenon, however, remain unclear. We demonstrated in this study the efficient infection and replication of the virus in vitro by inoculation of its RNA mixed with liposome. No significant difference either in the efficiency of RNA transfection or in the ability to support its replication was observed among the various species' cell lines examined. In addition, by RNA transfection the virus replicated with equal efficiency in MuLV-infected and -uninfected cells or in macrophages derived from mice irrespective of their age. In contrast, the pattern of the infection by virus particles was quite different; LDV replication was observed only in macrophages (particularly from newborn mice) and MuLV-infected cells. By using various LDV isolates, it was demonstrated that the capability of replication between neurovirulent, LDV type C, and the other avirulent strains was almost the same in mouse cell lines when their RNA was introduced into the cells. Higher infectivity of LDV-C to MuLV-infected cells may be due to its efficient incorporation of the particles into MuLV-infected cells.     

Ploidy in cyanobacteria

A recently developed real-time PCR method for the determination of genome copy numbers was optimized for the application to cyanobacteria. Three species were chosen to represent a fresh water species, a salt water species, and two strains of a widely used laboratory species. Synechococcus PCC 7942 and Synechococcus WH7803 were found to contain 3-4 genome copies per cell and are thus oligoploid, confirming earlier publications. In contrast, Synechocystis PCC 6803 is highly polyploid. The motile wild-type strain contains 218 genome copies in exponential phase and 58 genome copies in linear and in stationary growth phase. The GT wild-type strain contains 142 genome copies in exponential phase and 42 genome copies in linear and stationary growth phase. These are the highest numbers found for any cyanobacterial species. Notably these values are much higher than the value of 12 genome copies published for the 'Kazusa' strain more than 20 years ago. The results reveal that for Synechocystis PCC 6803 strain differences exist and that the ploidy level is highly growth phase-regulated. A compilation of the ploidy levels of all investigated cyanobacterial species gives an overview of the genome copy number distribution and shows that monoploid, oligoploid, and polyploid cyanobacteria exist.     

Presence of herpes simplex virus type 2 DNA in hamster cells oncogenically transformed by ultraviolet-inactivated virus.

Herpes simplex virus type 2 (HSV-2) DNA has been detected by molecular hybridization in hamster fibroblast cells oncogenically transformed by ultraviolet-irradiated virus. At early passages after cloning in soft agar, about 40% of the HSV-2 genome was present in all the transformed cell lines at one to six copies per cell. In cell lines derived from tumors induced by these cells, the same percentage of the HSV-2 genome was also found with more uniform number of copies (between two and three). Thus the presence of viral DNA seems to be necessary for the maintenance of the transformed state in these cell lines.     

Structure of bovine papillomavirus type 1 DNA in a transformed mouse cell line

Linearized bovine papillomavirus type 1 (BPV-1) DNA was introduced into mouse C127 cells, where it recircularized and replicated as an intact monomeric, extrachromosomal circular form in the resulting transformants. These cells contained a mixture of complex high molecular weight forms that were converted to a linear form of approximately BPV-1 size upon digestion with an enzyme that cuts once within the BPV-1 genome. Further analysis of one of these cell lines revealed that these high molecular weight forms consisted of two components. One was detected on agarose gels as a diffuse smear of slow-migrating material representing linear forms that were tightly associated with host chromosomes, probably by integration. The second component was composed of discrete-sized oligomeric open and supercoiled extrachromosomal circular forms of up to approximately 48 X 10(3) base-pairs (6 tandemly linked BPV-1 genomes) in size. No catenated (interlocked) forms could be detected.     

Effect of Helper Virus on the Number of Murine Sarcoma Virus DNA Copies in Infected Mammalian Cells

Cell lines of four mammalian species were each examined for the number of Moloney murine sarcoma virus (M-MSV) DNA copies in total cellular DNA after M-MSV transformation. Sarcoma-positive, leukemia-negative (S+L-) M-MSV-transformed cells were compared to M-MSV-transformed cells infected with a replicating leukemia virus. Both unfractionated M-MSV complementary DNA and complementary DNA representing the MSV-specific and the MSV-murine leukemia virus-common regions of the M-MSV genome were hybridized to total cellular DNA of various species. DNAs of mouse, cat, dog, and human S+L-cells contained from less than one to a few proviral M-MSV DNA copies per haploid genome. In contrast, helper virus-coinfected, M-MSV-producing cells of each species showed a 3- to 10-fold increase in M-MSV proviral DNA over that found in corresponding S+L- cells. MSV-specific and MSV-murine leukemia virus-common nucleotide sequences were each increased to a similar degree. A corresponding examination of cellular DNA of leukemia virus-infected normal or S+L- mammalian cells was performed to establish the resulting number of leukemia proviral DNA copies. The infection of normal or S+L- mammalian cells with several leukemia-type viruses that did not have nucleotide sequences closely related to the cell before infection resulted in the appearance of one to three corresponding leukemia proviral DNA copies.     

Equine connective tissue tumors contain unintegrated bovine papilloma virus DNA

Bovine papilloma virus (BPV) appears to be the etiological agent of common equine connective tissue tumors. We investigated the physical state of the viral DNA within such tumors and found no indication for integration into the host genome. The BPV genomes were present as free circular episomes. Two equine sarcoids were shown to contain multiple copies of free circular BPV type 1 (BPV-1) DNA. When the tumors were digested with several single-cut restriction enzymes, there were only form III BPV-1 DNA sequences could be revealed. One of the sarcoids contained, apart from wild-type BPV-1 DNA, a class of smaller BPV-1 circular DNA molecules bearing a deletion of approximately 9% of the BPV-1 genome. This deletion is located in the physical map between the relative units 0 and 0.32.     

Loss of bovine papillomavirus DNA replication control in growth-arrested transformed cells.

The bovine papillomavirus type 1 (BPV-1) genome replicates as a plasmid within the nuclei of BPV-1-transformed murine C127 cells at a constant multiple copy number, and spontaneous amplification of the viral DNA is rarely observed. We report here that a mutant BPV-1 plasmid within a contact-inhibited C127 cell line replicated as a stable multicopy plasmid in exponentially growing cells but amplified to a high level in confluent cell culture. In situ hybridization analysis revealed that most of the mutant viral DNA amplification occurred in a minor subpopulation of cells within the culture. These consisted of giant nondividing cells with greatly enlarged nuclei, a cell form which was specifically induced in stationary-phase cultures. These observations indicated that expression of a viral DNA replication factor was cell growth stage specific. Consistent with this hypothesis, considerable amplification of wild-type BPV-1 DNA associated with characteristic giant cell formation was observed in typical wild-type virus-transformed C127 cultures following a period of growth arrest achieved by serum deprivation. Further observations indicated that induction of the giant-cell phenotype was dependent on BPV-1 gene expression and implicated a viral E1 replication factor in this process. Moreover, heterogeneity in virus genome copy numbers within the giant-cell population suggested a complex regulation of induction of DNA synthesis in these cells. It appears that this process represents a mechanism employed by the virus to ensure maximal viral DNA synthesis within a growth-arrested cell. Fundamental questions concerning the integration of the virus-cell control circuitry in proliferating and resting cells are discussed.     

Bovine papilloma virus transcription: polyadenylated RNA species and assessment of the direction of transcription

The bovine papilloma virus type 1 (BPV-1)-specific RNA species were identified in virus-induced bovine warts, hamster tumors, and transformed hamster and mouse cells. In each case two major species were present (1.1 and 1.3 kilobases [kb]). Also two species of 1.6 and 1.8 kb appearing in variable amounts were found. Only in the keratinized periphery of the warts, where virus replication takes place, was it possible to reveal an additional 2-kb RNA species. In this tissue, however, the 1.6-kb species was not detected. The basal part of a bovine wart contained an additional minor, 2.9-kb, BPV-1-specific RNA sequence. By hybridization with purified defined BPV-1 DNA fragments it was shown that most of the coding sequences of the 2-kb species were transcribed from a region between 0.02 and 0.19 map units. The majority of the coding sequences of the smaller species in transformed cells were located in the region between 0.31 and 0.61 map units. The putative 5' ends mapped between 0.72 and 0.96 map units. Oligodeoxythymidylic acid-primed [(32)P]cDNA was synthesized from various RNA preparations to generate probes for the detection of 3' termini of the polyadenylated BPV-1 RNAs. By hybridization across the BPV-1 genome only one signal between the map positions 0.30 and 0.40 was obtained when RNA from transformed cells and from a tumor was used as a template. In contrast, RNA from the periphery of a wart led to the detection of an additional signal which was confined to the region between 0.96 and 1.00 map units. From the arrangement of both the 3' termini and the coding areas along the viral genome it appears that several RNA species are transcribed from one DNA strand.     

Oncogenic transformation by by equine herpesviruses. II. Coestablishment of persistent infection and oncogenic transformation of hamster embryo cells by equine herpesvirus type 1 preparations enriched for defective interfering particles.

Infection of permissive hamster embryo cells with virus preparations enriched for defective interfering (DI) particles of equine herpesvirus type 1 (EHV-1) resulted in persistent infection and oncogenic transformation. Six cell lines, designated DI-5 to -10, exhibited biological properties (immortality, increased saturation density, growth in soft agar, etc.) inherent to transformed cells, but 2 to 18% of the total cells in these cell lines were shown to release virus as judged by electron microscope studies and infectious center assays. The released virus was shown to be standard EHV-1 and not to contain DI particles as determined by density measurements of the viral DNA in the analytical ultracentrifuge and by interference assays using the released virus. Tumorigenicity studies revealed that inoculation of these persistently infected cells into newborn LSH inbred hamsters resulted in a lethal, fulminating hepatitis, whereas inoculation into older immunocompetent hamsters (+4 weeks) led to the development of metastatic fibrous sarcomas. Tumor cell lines (DI-5T to -10T) established from these sarcomas were shown to be transplantable and virus nonproducers. Hybridization analyses of cellular DNAs from DI transformed and tumor cell lines using 32P-labeled genomic EHV-1 DNA as probes indicated that the whole virus genome was detectable in multiple copies (23 to 45) in the transformed cells and that DNA sequences representing only 43.5 to 56.6% of the virus genome were present in amounts of 2 to 4 copies per cell in the DI tumor cells. Expression of these viral DNA sequences as demonstrated by the detection of virus-neutralizing antibodies, 50% neutralizing dose titers ranging from 1:50 to 1:1,000, in the sera of animals inoculated with either the virus-producing transformed cells or the virus-nonproducing tumor cells. Further, EHV-1-specific proteins were detected in the membrane and the perinuclear region of bothDI transformed and tumor cells by indirect immunofluorescent assays using antisera against EHV-1 structural antigens, EHV-1 nonstructural antigens, or preparations of EHV-1 DI particles. The roles of DI particles in mediating persistent infection and cellular transformation are discussed.