Uncontrolled nuclear division in murine cells abortively transformed by simian virus 40.

Cytochalasin B (CB) prevents cytoplasmic cleavage without directly affecting nuclear division. Secondary cultures of mouse embryo fibroblasts or 3T3 cells show controlled nuclear division when treated with CB: only binucleated cells are formed. Many CB-treated transformed cells show uncontrolled nuclear division and become highly multinucleated. When CB-treated normal cells are concurrently infected with high inputs of SV40, many of these cells become highly multinucleated. It is suggested that these highly multinucleated cells represent abortively transformed cells since the actual number of transforming units (focus-forming units) of simian virus 40 (SV40) is too low to account for the appearance of these cells. Also, if the CB treatment is begun 6 days after SV40 inoculation, the large increase in highly multinucleated cells is not observed. Most cells stably transformed by SV40 or adenovirus show uncontrolled nuclear division when treated with CB. However, 3T3 cells transformed by SV40 or adenovirus and analyzed shortly after transformation are an exception and show controlled nuclear division. This property of 3T3 cells is apparently overcome by high inputs of SV40 since abortively transformed cells become highly multinucleated.     

Suppression of in vivo tumor formation induced by simian virus 40- transformed cells in mice receiving antiidiotypic antibodies

This study characterizes four private idiotypes (Id) associated with monoclonal antibodies (mAb) to simian virus 40 (SV40) tumor antigen (T-Ag), and to a cellular protein, p53. Anti-Id recognized Id determinants associated with the antibody-combining site. BALB/c mice receiving a pool of anti-Id directed against mAb recognizing distinct amino and carboxyl terminal epitopes of T-Ag before receiving a tumorigenic dose of SV40-transformed cells showed suppression of tumor formation. Serum obtained from these mice before tumor challenge contained anti-anti-Id that failed to bind T-Ag. These data support the potential role of regulatory idiotopes in tumor immunity.     

Demonstration of infectious DNA in transformed cells. III. Correlation of detection of infectious DNA-protein complexes with persistence of virus in simian adenovirus SA7-induced tumor cells.

Primary tumors induced in newborn hamsters by simian adenovirus SA7 were investigated in transfection experiments. Infectious DNA-protein complexes were readily detected in both the supernatant and pellet fractions obtained by a modified Hirt extraction procedure; DEAE-dextran was required for infectivity to become manifest. Infectivity could be abolished by exposure to DNase, but it was unaffected by SA7-specific antiserum or RNase, and only partially inactivated by trypsin treatment. SA7 tumor cells serially passaged either in tissue culture or in hamsters yielded infectious DNA complexes much less frequently and appeared to evolve into nonyielder cell lines. When large numbers of cells were lysed, intact virus could be recovered from all the primary tumors and from some of the subcultured cell lines. There was a correlation between the persistence of complete virus and the presence of infectious DNA-protein complexes. When the tumor cells carried very small amounts of intact virus, infectious DNA complexes could still be detected; when virus could no longer be detected in the tumor cells, infectious DNA complexes could no longer be found. The results suggest that a portion of the infectious DNA moieties exists as viral DNA-protein complexes in the tumor cells.     

Papovavirus structural polypeptides: comparison of human and rabbit papilloma viruses with simian virus 40

A comparative study of the structural polypeptides of purified human papilloma virus, rabbit papilloma virus, and simian virus 40 (SV40) was performed by electrophoretic analysis on 10% polyacrylamide gels containing sodium dodecyl sulfate either by Coomassie blue staining or by electrophoresing purified virus preparations labeled with radioactive iodine. Analysis of human papilloma virus on stained gels revealed 6 bands with a major polypeptide of molecular weight 63,000 daltons comprising 60% of the virion protein. Analysis of rabbit papilloma virus on stained gels revealed 5 bands with a major polypeptide of molecular weight 60,000 daltons comprising 48% of the virion protein. Under conditions which yielded 6 polypeptides for SV40, in agreement with published data, 4 and 7 polypeptides were regularly detected in iodinated preparations of human and rabbit papilloma viruses, respectively. Isoelectrofocusing of iodinated virions revealed isoelectric points of pH 3.7 for SV40 and pH 4.0 for both rabbit and human papilloma viruses.     

Transformation of human cells by temperature-sensitive mutants of simian virus-40.

Conditions necessary for the establishment and maintenance of transformation of human cells by wild type and temperature-sensitive mutants of SV40 were examined. For both early and late mutants, the frequency of transformation was found to be up to 5-fold higher, and virus yield 100-fold lower, at 39 degrees than at 33 degrees. No such effect was observed with the wild type virus under the same conditions. This observation is apparently at variance with previously published work, but may be explained by the semipermissive nature of the cells that we used. Increasing the temperature to 40.5 degrees caused cells transformed by the early mutant, tsA30, to lose T-antigen as detectable by staining, and also to lose the ability to grow to high density, while it produced no effect on cells transformed by wild type virus.     

Surface antigens on hamster cells transformed by SV40, BK virus and JC virus

We studied the surface antigens on hamster cells transformed by SV40, BK and JC viruses using a complement-dependent cytotoxicity test. Antiserum was obtained from guinea pigs immunized with an SV40-transformed hamster cell line, and used after appropriate absorption with a range of cell types. The cell lines transformed by these viruses were all found to possess the following surface antigens: 1) a unique set of organ antigens, 2) fetal antigen(s), and 3) an antigen tentatively named primate polyoma virus-transformed hamster cell antigen (PPH antigen-1). These antigens were not detected on other hamster cell lines, including those transformed by mouse polyoma-, adeno-, herpes-, or retro-viruses Neither could they be detected on mouse, rat or human cells transformed by SV40 or BK viruses. They were, thus, probably host-derived antigens specifically depressed or unmasked by the primate polyoma viruses. In addition, all hamster cell lines tested, including BHK21, carried an antigen(s) named hamster cell line antigen, which could not be detected on surfaces of normal cells, in primary or secondary culture.     

Lysosome stability during lytic infection by simian virus 40.

By 48 h postinfection, 40--80% of SV40-infected CV-1 cells have undergone irreversible injury as indicated by trypan blue staining. Nevertheless, at this time the lysosomes of these cells appear as discrete structures after vital staining with either acridine orange or neutral red. Lysosomes, vitally stained with neutral red at 24 h postinfection, were still intact in cells stained with trypan blue at 48 h. Acid phosphatase activity is localized in discrete cytoplasmic particles at 48 h, as indicated by histochemical staining of both fixed and unfixed cells.     

Leukocyte migration inhibition detects cross-reacting antigens between cells transformed by Epstein-Barr virus (EBV) and EBV-like simian viruses

The leukocyte migration inhibition (LMI) technique was used to measure the T cell-mediated immune response of Epstein-Barr-virus (EBV)-seropositive human donors to antigens associated with B cell lines of simian origin, transformed by simian EBV-like viruses, Herpesvirus papio (HVP), H. pan, H. gorilla and H. pongo. Extracts of cell lines carrying three of the four simian viruses (from gorilla, chimpanzee and orangutan) induced a positive LMI response, whereas lines carrying baboon-derived HVP were ineffective. None of the simian virus-transformed lines elicited an LMI reaction in human EBV-seronegative individuals. Leukocytes from patients with acute infectious mononucleosis (IM) failed to respond to any of the lines transformed by EBV or the simian EBV-like viruses. Such lines express the virally encoded nuclear antigen, but have only a low level of viral cycle-associated antigens. Extracts of the EBV-carrying human cell line P3HR-1 induced with 12-O-tetradecanoylphorbol-13-acetate to express high levels of early and virus capsid antigens (EA, VCA, respectively) however, elicited a strong response with leukocytes from patients with acute IM. During convalescence, IM patients became responsive to EBV, H. gorilla-, H. pan- and H. pongo-transformed lines, indicating that the LMI reaction induced by these simian virus-transformed lines was directed against the antigens expressed in immortalized cells rather than against antigens of the lytic cycle. It is highly probable that this reaction reflects a cross-recognition of the nuclear antigens associated with these four transforming viruses (excluding H. papio) at the level of human T cells.     

Inhibition of simian virus 40 large tumor antigen expression in human fetal glial cells by an antisense oligodeoxynucleotide delivered by the JC virus-like particle

Human JC virus (JCV) is a neurotropic virus, and the etiological agent of progressive multifocal leukoencephalopathy (PML), a fatal neurological disease. Because of its natural infection tropism, it is possible to use the JCV capsid as a gene-transducing vector for therapeutic purposes in neurological disorders. In the current study, a recombinant JCV virus-like particle (VLP) was generated and purified from yeast. VLP was able to accommodate and protect DNA molecules of up to approximately 2000 bp in length. VLP was able to package and deliver an antisense oligodeoxynucleotide (AS-ODN) against simian virus 40 (SV40) large tumor antigen (LT) into SV40-transformed human fetal glial (SVG) cells in order to inhibit expression of the oncoprotein. Subsequently, apoptosis of VLP-AS-ODN-treated cells was demonstrated after the blocking of LT expression. In addition, JCV VLP was able to deliver ODN into human astrocytoma, neuroblastoma, and glioblastoma cells with high efficiency. In vivo delivery of ODN into a human neuroblastoma tumor nodule by VLP was also demonstrated. These findings suggest that JCV VLP is a gene delivery vector with potential therapeutic use for human neurological disorders.     

Establishment and characterization of a simian virus 40-immortalized rat pancreatic stellate cell line

Activated pancreatic stellate cells (PSCs) have recently been implicated in the pathogenesis of pancreatic fibrosis and inflammation. Primary PSCs can be subcultured only several times because of their limited growth potential. A continuous cell line would be valuable in studying molecular mechanisms of these pancreatic disorders. The aim of this study was to establish an immortalized cell line of rat PSCs. PSCs were isolated from the pancreas of male Wistar rats, and the simian virus 40 T antigen was introduced to PSCs by retrovirus-mediated gene transfer. This procedure yielded an actively growing cell line, designated as SAM-K. This cell line has been passaged repeatedly for almost 2 years, and is thus likely immortalized. SAM-K cells retained morphological characteristics of primary PSCs, and expressed alpha-smooth muscle actin, glial fibrillary acidic protein, type I collagen, fibronectin, and prolyl hydroxylases. The level of p53 expression was very high in SAM-K cells. Proliferation of SAM-K cells was stimulated by serum and platelet-derived growth factor-BB. Interleukin-1beta (IL-1beta) activated nuclear factor-kappaB, activator protein-1, and three classes of mitogen-activated protein (MAP) kinases: extracellular signal-regulated kinase1/2, c-Jun N-terminal kinase, and p38 MAP kinase. IL-1beta induced expression of intercellular adhesion molecule-1 and monocyte chemoattractant protein-1, both of which were abolished in the presence of pyrrolidine dithiocarbamate, a specific inhibitor of nuclear factor-kappaB activation. IL-1beta-induced monocyte chemoattractant protein-1 was partially inhibited by specific inhibitors of MAP kinase kinase (U0126) and of p38 MAP kinase (SB203580) whereas intercellular adhesion molecule-1 expression was not altered by the inhibitors. Thus, SAM-K would be useful for in vitro studies of cell biology and signal transduction of PSCs.     

Genome structure of adenovirus 12 host range mutants adapted to growth in simian Vero cells

Mutants of adenovirus type 12 adapted to growth in Vero cells were studied by restriction enzyme and sequence analysis. Plaque-purified mutants were isolated either after long-term passage of the virus in Vero cells (CS-1, CL-1) or from Vero cells transfected with viral DNA (11-g, 12-k). The plaque-forming activity of these mutants was equal or even slightly higher in Vero cells than in HeLa cells, whereas the activity of wild-type virus in HeLa cells exceeded that in Vero cells by about one to two orders of magnitude. All mutants tested had an insertion of variable length consisting of adenoviral sequences at the right end of the viral genome. Sizes of the insertions were determined to be 294 bp (CS-1, 12-k), 560 (11-g), or 180 bp (CL-1). Additional insertions of multiple size and less than equimolar amount were detected and seem to exist in some of the viral particles. Furthermore, all four mutants tested showed an identical 69-bp deletion in the first exon of the E1a gene.