Change of DNA near the origin of replication enhances the transforming capacity of human papovavirus BK.

A turbid-plaque-forming mutant (pm522) of human papovavirus BK, which has a small deletion at about 0.7 map unit and grows somewhat more slowly in human cells than does wild-type BK virus, transformed hamster and rat cells in culture much more efficiently than did wild-type virus. Another plaque morphology mutant, pm525, forming turbid plaques larger than those of pm522 also had a high transforming capacity. The similar difference in transforming capability between wild-type and plaque morphology viruses was observed with DNAs extracted from virions. Recombinant viruses were constructed from the wild-type DNA fragment lacking HindIII-C (0.62 to 0.73 map unit) and pm522 HindIII-C (including the origin of replication) by the molecular cloning method. Characterization of the recombinants showed that the change near the origin of DNA replication was responsible both for the altered plaque morphology and for the enhanced transforming capacity of the BK virus mutant.     

Viable deletion mutant of human papovavirus BK that induces insulinomas in hamsters.

A plaque morphology mutant (pm-522) of human papovavirus BK, which was rescued from a human papovavirus BK-induced hamster pineocytoma, was characterized and compared with a cloned wild-type virus (wt-501). Mutant pm-522 formed turbid plaques and grew more slowly than wt-501 in human embryonic kidney (HEK) cells. The immunofluorescence assay revealed that more HEK cells underwent abortive infection with pm-522 than with wt-501. Whereas wt-501 induced brain tumors and osteosarcomas, but no insulinomas, in hamsters, pm-522 induced brain tumors and insulinomas. The DNA of pm-522 was found by electrophoresis and electron microscopy to have a deletion (85 +/- 15 base pairs) and an insertion (40 +/- 10 base pairs) between map coordinates 0.708 and 0.725 from the endonuclease EcoRI cleavage site. These results demonstrate the presence of a viable deletion human papovarivus BK mutant capable of inducing insulinomas in hamsters.     

p53-independent endoplasmic reticulum stress-mediated cytotoxicity of a Newcastle disease virus strain in tumor cell lines

While Newcastle disease virus (NDV) causes serious infections in birds, it is apparently nonpathogenic in mammalian species, including humans. Previous observations and small-scale clinical trials indicated that NDV exerts oncolytic effects. Isolates of NDV were found to have selective affinity to transformed cells. We previously showed that the attenuated NDV strain MTH-68/H causes apoptotic cell death in cultures of PC12 rat pheochromocytoma cells. The aim of the present study was to extend MTH-68/H cytotoxicity testing with human tumor cell lines and to analyze certain biochemical aspects of its oncolytic effect. MTH-68/H was found to be able to kill a wide range of transformed cells by apoptosis. While caspase-8 and caspase-9 are not involved in MTH-68/H-induced apoptosis, activation of caspase-3 and caspase-12 was detected in virus-infected PC12 cells. A human glioblastoma cell line with repressible expression of the p53 protein did not show any difference in MTH-68/H sensitivity in its p53-expressing and p53-depleted states, indicating that the apoptotic process induced by MTH-68/H does not depend on p53. Apoptosis was accompanied by virus replication in two tumor cell lines tested (PC12 cells and HeLa human cervical cells), and signs of endoplasmic reticulum stress (phosphorylation of protein kinase R-like endoplasmic reticulum kinase and eIF2alpha) were also detected in transformed cells. In contrast, proliferation of nontransformed mouse and rat fibroblast cell lines and human primary fibroblasts was not affected by MTH-68/H treatment. MTH-68/H thus selectively kills tumor cell cultures by inducing endoplasmic reticulum stress leading to p53-independent apoptotic cell death.     

Transformation of Human Embryonic Kidney Cells by a Viable Deletion Mutant of BK Virus

Infection by a viable deletion mutant of BK virus (pm-522) of human embryonic kidney (HEK) cultures originating from different fetuses generated cell lines, designated pmHEK, resistant to superinfection by the virus. However, infection of HEK cultures by a cloned wild-type BK virus (wt-501) did not generate a cell line. In medium with 10% calf serum all pmHEK cells reached saturation densities significantly higher than those of HEK cells and could grow in medium containing 1% serum. They did not form colonies in soft-agar medium, and had limited life-spans greatly extended beyond that of HEK cells. These results suggest that pmHEK cells are partially transformed. T antigen was uniformly expressed by all pmHEK cells, while V antigen was present in only a small minority of the cells. Thirty to 5,000 copies of the viral DNA per cell were detected, primarily in a nonintegrated form, in all pmHEK cell lines and the clones isolated from one of them. Since at least in two pmHEK lines, the actual quantities of free viral DNA per cell were sig-nificantly greater than those estimated on the basis of the assumption that free viral DNA was produced only by the V-antigen-positive cells, it can be concluded that free viral DNA is also present in the V-antigen-negative cells predominant in the cell populations. Although the existence of a few copies of integrated viral sequences in pmHEK cells was not ruled out, the large amount of free viral DNA present in each cell probably plays a prominent role in the production of T antigen required for maintenance of transformation.     

Persistent BK papovavirus infection of transformed human fetal brain cells. I. Episomal viral DNA in cloned lines deficient in T-antigen expression.

After infection of permissive human fetal brain cells by BK human papovavirus (BKV), the vast majority of the cells were killed by the virus, but rare survivors were recovered after frequent medium changes. These surviving cells grew and formed visible colonies after 5 to 6 weeks and were thereafter established as permanent cell lines. These cells, designated as BK-HFB cells, were persistently infected and shed BKV. Morphologically, they were small polygonal cells and had transformed growth properties. Their plating efficiency on solid substrates or in semisolid medium was high, and they were tumorigenic in athymic nude mice. Cloning experiments in medium containing BKV antiserum revealed that BKV did not persist in the cultures in a simple carrier state. All cloned cell lines were initially T-antigen negative and virus-free. However, every clone began to release BKV and again became persistently infected within 3 weeks after removal of BKV antiserum. After rigorous antibody treatment, four of seven clones still released virus spontaneously upon removal of antiserum; three clones have remained virus-free and are apparently cured. Although these cloned cell lines are T- and V-antigen negative when grown in antiserum-containing medium, they retain "free" or episomal BKV genomes; integrated viral DNA was not detected in any of the clones. These free genomes are indistinguishable from prototype BKV DNA and are found in much larger amounts in virus-shedding cell lines.     

Malignant transformation of hamster kidney cells by BK virus.

Primary hamster kidney cells were transformed by BK virus, a new human papovavirus. Transformed (HKBK) cell produced BK virus T antigen and induced tumors in hamsters that developed antibodies to BK virus T antigen. BK virus was rescued from HKBK cells by Sendai virus-assisted fusion with permissive cells. One out of six cell lines derived from HKBK cell-induced tumors showed the same characteristics as HKBK cells.     

In vitro tumoricidal activity of macrophages against virus-transformed lines with temperature-dependent transformed phenotypic characteristics.

The susceptibility of targets to destruction by tumoricidal rat and mouse macrophages was studied with virus-transformed cell lines in which various elements of the transformed phenotype are only expressed at specific temperatures. BHK cells transformed by the ts3 mutant of polyoma virus, rat embryo 3Y1 cells transformed by a temperature-sensitive A cistron mutant of simian virus 40 (SV40) and the ts-H6-15 temperature-sensitive line of SV40-transformed mouse 3T3 cells were killed in vitro by macrophages at both the permissive (33 °C) or nonpermissive (39 °C) temperatures for expression of the transformed phenotype. 3T3, 3Y1 and BHK cells transformed by wild-type SV40 or polyoma virus were also destroyed by tumoricidal macrophages at both 33 and 39 °C, but untransformed 3T3, 3Y1, and BHK cells were not. Thus, transformed cells are killed by macrophages regardless of whether or not they express cell surface LETS protein or Forssman antigen, display surface changes which permit agglutination by low doses of plant lectins, express SV40 T antigen, have a low saturation density, or exhibit density-dependent inhibition of DNA synthesis.     

Expression of neuronal markers in chick and quail embryo neuroretina cultures infected with Rous sarcoma virus

Cultures of neuroretina (NR) cells from 7-day chick and quail embryos were infected with ts NY-68, a thermosensitive mutant of Rous sarcoma virus (RSV) which transformed NR cells at 36 degrees C. The following differentiation markers for neurones were studied: tetanus toxin-binding sites at the cell surfaces, presence of synapses, and the specific activity of the enzymes choline acetyltransferase (CAT) and glutamic acid decarboxylase (GAD). Appearance of synapses and expression of CAT were similar in control and transformed cultures. Tetanus toxin-binding cells were observed in transformed primary cultures and also in quail NR subcultures. GAD-specific activity was markedly stimulated in chick and quail primary cultures transformed by ts NY-68 and further increased in subcultures of ts NY-68-transformed quail NR cells. Stimulation of GAD activity is controlled by the transforming (src) gene of RSV since it was not observed in cultures infected with RAV-1, a leukosis virus which lacks the src gene. These data show that infection of chick and quail NR cultures with RSV results in the transformation of cells with neuronal markers.     

Unintegrated viral sequences in rat cells transformed by simian virus 40 and a temperature sensitive A gene mutant

The status of viral sequences in rat cells transformed by simian virus 40 (SV40) and its temperature sensitive A gene mutant was investigated. Agarose gel electrophoresis of cell DNA prepared from clones picked from soft-agar and blot hybridization showed that sequences specific to SV40 genome were present both as integrated and unintegrated structures in rat clones. Digestion of rat cell DNA with various endonucleases with or without recognition sites in SV40 DNA and analysis indicated that the unintegrated viral genomes existed as full-length, covalently closed circular molecules. No differences in the free viral genomes were apparent between the clones transformed by the wild type and the mutant virus. The importance of the existence of free viral genomes in nonpermissive cells is discussed.     

Isolation and characterization of herpes simplex virus mutants containing engineered mutations at the DNA polymerase locus.

We have derived Vero cell lines containing the herpes simplex virus DNA polymerase (pol) gene that complement temperature-sensitive pol mutants. These cell lines were used to recover viruses containing new mutations at the pol locus. Two spontaneously arising host-range mutants, 6C4 and 7E4, were isolated. These mutants did not grow efficiently on Vero cells or synthesize late polypeptides but formed plaques on a cell line containing the pol gene (DP6 cells). Whereas mutant 6C4 specified a wild-type-size Pol protein, we detected no full-length Pol protein in 7E4-infected cell extracts. Complementation studies demonstrated that 6C4 and 7E4 contain different mutations and indicated that 6C4 is in a complementation group different from that of pol temperature-sensitive mutant tsC7 or tsD9. A mutant in which 2.2 kilobases of pol sequences were replaced with the Escherichia coli lacZ gene under the control of the herpes simplex virus thymidine kinase promoter was constructed. This mutant formed blue plaques on DP6 cells in the presence of 5-bromo-4-chloro-3-indolyl-beta-D-galactoside. Using this virus in marker rescue experiments, we engineered three mutants containing deletions in the pol coding region which grew efficiently on DP6 cells but not on Vero cells and which differed in their synthesis of Pol polypeptides. The lacZ insertion virus was also used to introduce a deletion in the region upstream of the pol long open reading frame, which removes a short open reading frame that could encode a 10-amino-acid peptide. This mutant grew to similar titers on Vero and DP6 cells, indicating that these sequences are not essential for growth of the virus in tissue culture.     

State of the viral DNA in rat cells transformed by polyma virus. II. Identification of the cells containing nonintegrated viral DNA and the effect of viral mutations.

F2408 rat cells transformed by polyoma virus contained integrated and nonintegrated viral DNA. The presence of nonintegrated viral DNA is under control of the A early viral function. Polyoma ts-a-transformed rat cells lose the free viral DNA when growth at the nonpermissive temperature (40 degrees C), but they reexpress it 1 to 3 days after they are shifted back to the permissive temperature. In contrast, rat cells transformed by a late viral mutant, ts-8, contain free viral DNA at both permissive and nonpermissive temperatures. Treatment of the transformed rat cells with mitomycin C produces a large increase in the quantity of free viral DNA and some production of infectious virus. Experiments of in situ hybridization, with 3H-labeled polyoma complementary RNA as a probe, show that only a minority (approximately 0.1%) of the transformed cells contain nonintegrated viral DNA at any given time. These results suggest that the presence of free viral DNA in polyoma-transformed rat cells is caused by a spontaneous induction of viral DNA replication, occurring with low but constant probability in the transformed cell population, and that the free viral DNA molecules originate from the integrated ones, probably through a phenomenon of excision and limited replication.     

Demonstration of T antigens on the surface of cells transformed with primate polyoma viruses

Primate polyoma virus-transformed hamster, mouse, and rat cell lines were examined by indirect immunofluorescence staining for cell surface-associated T antigens, by using a rabbit antiserum prepared against sodium dodecyl sulfate-denatured large T antigen of simian virus 40 (anti-SV40-SDS-T serum). Positive surface staining was shown not only on SV40-transformed cells, but also on BK and JC virus-transformed cells. In contrast, normal cells and cells transformed with mouse polyoma-, human adeno-, and murine sarcoma viruses were negative. The data on SV40-transformed cells confirmed the reports of others demonstrating the cell surface location of SV40 large T antigen, and the data on BK and JC virus-transformed cells proved that these cells have cell-surface T antigens that cross-react with anti-SV40-SDS-T serum.     

Cell-Dependent Differences in the Production of Infectious Herpes Simplex Virus at a Supraoptimal Temperature

The replication of herpes simplex virus (HSV) was compared in rabbit and hamster cells at optimal and supraoptimal temperatures. Replication occurred in cells of either species at 33 C, but the total infectious virus yield was routinely about 10-fold greater in rabbit cells than in hamster cells. At 39 C, this difference was exaggerated to greater than 100,000-fold. Whereas infectious virus was produced and plaques formed in rabbit kidney cell monolayers at the higher temperature, neither developed in those derived from hamster embryos. Elevating the temperature from 33 C to 39 C at various time intervals after exposure of the cultures to virus revealed that production of infectious virus in hamster cells was completely heat-sensitive up to 6 hr after infection. Specific viral antigens and viral deoxyribonucleic acid (DNA) were synthesized in both rabbit and hamster cell cultures. In addition, cellular DNA synthesis was depressed and cytopathic effects occurred in both cell systems. These cytopathic effects were not observed in cell cultures treated with HSV previously inactivated with ultraviolet light. Compared with parallel cultures at 33 C, the amount of viral DNA synthesized at 39 C was greatly reduced in both systems. In hamster cells, the reduction was twofold greater than in rabbit cells. This cell-dependent thermal inhibition of HSV replication in hamster cells did not occur with vaccinia virus.     

Arrangement of Integrated Avian Sarcoma Virus DNA Sequences Within the Cellular Genomes of Transformed and Revertant Mammalian Cells

We have examined the arrangement of integrated avian sarcoma virus (ASV) DNA sequences in several different avian sarcoma virus transformed mammalian cell lines, in independently isolated clones of avian sarcoma virus transformed rat liver cells, and in morphologically normal revertants of avian sarcoma virus transformed rat embryo cells. By using restriction endonuclease digestion, agarose gel electrophoresis, Southern blotting, and hybridization with labeled avian sarcoma virus complementary DNA probes, we have compared the restriction enzyme cleavage maps of integrated viral DNA and adjacent cellular DNA sequences in four different mouse and rat cell lines transformed with either Bratislava 77 or Schmidt-Ruppin strains of avian sarcoma virus. The results of these experiments indicated that the integrated viral DNA resided at a different site within the host cell genome in each transformed cell line. A similar analysis of several independently derived clones of Schmidt-Ruppin transformed rat liver cells also revealed that each clone contained a unique cellular site for the integration of proviral DNA. Examination of several morphologically normal revertants and spontaneous retransformants of Schmidt-Ruppin transformed rat embryo cells revealed that the internal arrangement and cellular integration site of viral DNA sequences was identical with that of the transformed parent cell line. The loss of the transformed phenotype in these revertant cell lines, therefore, does not appear to be the result of rearrangement or deletions either within the viral genome or in adjacent cellular DNA sequences. The data presented support a model for ASV proviral DNA integration in which recombination can occur at multiple sites within the mammalian cell genome. The integration and maintenance of at least one complete copy of the viral genome appear to be required for continuous expression of the transformed phenotype in mammalian cells.     

Human embryonic kidney cells: stable transformation with an origin-defective simian virus 40 DNA and use as hosts for human papovavirus replication.

An origin-defective mutant DNA of simian virus 40 immortalized human embryonic kidney cells, maintaining a T protein which could function for human papovavirus BK DNA replication but not for human papovavirus JC DNA replication. Neither BK virions nor capsid proteins were produced in these cells. This may indicate that the simian virus 40 T protein in human embryonic kidney cells is competent for maintaining transformation and initiating and completing DNA replication for BK but is not competent for switching to late gene functions. Furthermore, it appears that the JC DNA replication origin cannot efficiently use the simian virus 40 T protein for its DNA synthesis, as suggested by its DNA sequence data (R. Frisque, J. Virol. 46:170-176, 1983; T. Miyamura, H. Jikoya, E. Soeda, and K. Yoshiike, J. Virol. 45:73-79, 1983).