Effect of UV-irradiation on DNA replication of the parvovirus minute-virus-of-mice in mouse fibroblasts.

The effect of UV-irradiation on the conversion of the single-stranded DNA of the parvovirus Minute-Virus-of-Mice (MVM) to duplex Replicative Forms (RF) was studied after infection of mouse A9 fibroblasts. UV-irradiation of the virus prior to infection of unirradiated cells resulted in a dose-dependent, single-hit, inhibition of RF formation. Restriction fragment analysis indicated that this inhibition could be ascribed to the introduction of absolute blocks which prevent elongation of the newly synthesized complementary strand. Cell exposure to UV-light prior to infection with UV-irradiated MVM enhanced the fraction of input viral DNA which was converted to RF. This enhancement required de novo protein synthesis during the interval between cell irradiation and virus infection. These results suggest that DNA replication constitutes a target in the viral life cycle that leads to the UV-enhanced Reactivation of virus survival, however, they do not permit us to identify the step of RF formation which is enhanced in UV-pretreated cells.     

Correlation of increased nuclease activity with enhanced virus reactivation

An increase in nuctease activity, which degraded both unirradiated and ultraviolet (UV)-irradiated DNA, was observed in the extract of monkey Vero cells after irradiation with an appropriate amount of UV. In contrast, no increase was observed with mouse L cells. Neither DNA polymerases nor uracil-DNA glycosylase was enhanced but rather suppressed by UV irradiation in both cell lines. Cytological studies showed that, in Vero cells, the reactivation of UV-irradiated herpes simplex virus was markedly enhanced by irradiating cells with UV before infection. However, no enhancement was observed with L cells. These results suggest that an increase in nuclease activity may be one of underlying mechanisms for the enhanced reactivation of DNA viruses.     

Reactivation of herpes simplex virus in a cell line inducible for simian virus 40 synthesis

The reactivation of UV-irradiated herpes simplex virus (HSV) was investigated in irradiated and unirradiated transformed hamster cells in which infectious simian virus 40 (SV40) can be induced. Reactivation was enhanced when the cells were treated with UV light or mitomycin C prior to infection with HSV. The IV dose-response curve of this enhanced reactivation was strikingly similar to that found for induction of SV40 virus synthesis in cells treated under identical condictions. This is the first time that two SOS functions described in bacteria have been demonstrated in a single mammalian cell line.     

Enhanced mutagenesis parallels enhanced reactivation of herpes virus in a human cell line.

U.v. irradiation of human NB-E cells results in enhanced mutagenesis and enhanced reactivation of u.v.-irradiated H-1 virus grown in those cells ( Cornelis et al., 1982). This paper reports a similar study using herpes simplex virus (HSV) in NB-E cells. The mutation frequency of HSV (resistance of virus plaque formation to 40 micrograms/ml iododeoxycytidine ) increased approximately linearly with exposure of the virus to u.v. radiation. HSV grown in unirradiated cells gave a slope of 1.8 X 10(-5)m2/J, with 3.2 X 10(-5)m2/J for HSV grown in cells irradiated (3 J/m2) 24 h before infection. There was no evidence for mutagenesis of unirradiated virus by irradiated cells, as seen with H-1 virus. Enhanced reactivation of irradiated HSV in parallel cultures increased virus survival, manifested as a change in slope of the final component of the two-component survival curve from a D0 of 27 J/m2 in unirradiated cells to 45 J/m2 in irradiated cells. Thus, enhanced mutagenesis and enhanced reactivation occurred for irradiated HSV in NB-E cells. The difference in the enhanced mutagenesis of HSV (dependent on damaged DNA sites) and of H-1 virus (primarily independent of damaged DNA sites) is discussed in terms of differences in DNA polymerases.     

Changes in the properties of the resident L virus in somatic cell hybrid lines

Production of the N-tropic L virus by hybrids of A9 cells (subline of mouse L cells) was found to be influenced by the genotype of the partner cells inasmuch as it was suppressed by B-type cells. Five hybrid lines with the B-type partner were subjected to prolonged in vitro passage during which all lost chromosomes, accompanied in some by phenotypic changes. These five hybrid cell lines resumed virus production detectable either by antigen induction on JLS-V9 cells, and/or by focus formation on mouse embryo fibroblasts. By infection of both N- and B-type embryo cells, the host range of the reappearing viruses was found to be different from the L virus; it was B-tropic in one and NB-tropic in two cases. The results indicate that rearrangements and loss of genetic material in the hybrid cells may influence the infective properties of their resident C-type viruses.     

Rescue of Simian Virus 40 from Cell Lines Transformed at High and at Low Input Multiplicities by Unirradiated or Ultraviolet-irradiated Virus

The relation between simian virus 40 (SV40) input multiplicity during transformation of primary mouse kidney cultures and the subsequent rescue of SV40 from clonal lines of transformed cells has been studied. Primary mouse kidney cultures were transformed with unirradiated SV40 at input multiplicities varying from 0.06 to 200 plaque-forming units (PFU) /cell or with SV40 irradiated with ultraviolet (UV) light to a survival of 0.04 to 0.01. All of the transformed lines contained the intranuclear SV40 T antigen, but cell-free extracts prepared from the transformed cell lines failed to yield infectious virus when assayed on monkey kidney cell (CV-1) monolayers. After fusion with susceptible CV-1 cells induced by UV-inactivated Sendai, all of the lines transformed by unirradiated virus yielded infectious SV40. The frequency of induction and the incidence of successful trials did not depend on the multiplicity of infection. “Good” yielders were obtained from mouse kidney cells transformed at the low input multiplicity of 0.06 PFU /cell. In contrast, only 4 of 12 clonal lines transformed at moderately low input multiplicity, and none of the lines transformed at very low input multiplicity with UV-irradiated virus yielded infectious SV40. The four positive lines have been classified as “poor” or “rare” yielders.     

Identification of a cellular receptor for mouse mammary tumor virus and mapping of its gene to chromosome 16

Pseudotypes of vesicular stomatitis virus (VSV) containing envelope glycoproteins provided by C3H mammary tumor virus (MTV) instead of the normal VSV G-proteins were prepared and used to assay the presence of an MTV receptor on cells. The assay was specific as demonstrated by competition studies with excess MTV particles and neutralization of the pseudotypes with anti-MTV serum or monoclonal antibodies directed against MTV gp52. The MTV receptor was abundantly present on mouse cells but hardly detectable on nonmurine cells, including the Chinese hamster cell line E36. Somatic cell hybrids between E36 cells and GRS/A spontaneous leukemia cells (GRSL cells) and between E36 and GRS/A primary mammary tumor cells were made. The hybrids retained all Chinese hamster chromosomes but segregated mouse chromosomes. From the analysis of the isoenzymes and chromosomes of the hybrid cell lines we conclude that the gene for the receptor (MTVR-1) is located on mouse chromosome 16.     

Embryonal carcinoma cells (and their somatic cell hybrids) are resistant to infection by the murine parvovirus MVM, which does infect other teratocarcinoma-derived cell lines

Minute virus of mice (MVM), a non-defective parvovirus, has been shown to infect cultures of non-pluripotent differentiated teratocarcinoma-derived cells, but pluripotent (and "nullipotent") embryonal carcinoma cells derived from the same teratocarcinoma resist MVN infection. Somatic cell hybrids between an embryonal carcinoma line and Friend erythroblastic leukemia cells are also resistant to MVM, even though Friend cells are susceptible. Among three blastocyst-derived lines tested, only one, a parietal yolk sac cell line, resists MVM infection. These results suggest that teratocarcinoma cultures may provide useful systems in which to study the cellular factors which mediate susceptibility to this teratogenic and oncolytic virus.     

Specific binding sites for a parvovirus, minute virus of mice, on cultured mouse cells.

The early interactions between parvoviruses and host cells have not been extensively described previously. In this study we have characterized some aspects of viral binding to the cell surface and demonstrated the existence of specific cellular receptor sites for minute virus of mice (MVM) on two murine cell lines that are permissive for viral growth. The interaction had a pH optimum of 7.0 to 7.2, and both the rate and extent of the reactions were slightly affected by temperature. Mouse A-9 cells (L-cell derivative) had approximately 5 X 10(5) specific MVM binding sites per cell, and Friend erythroleukemia cells had 1.5 X 10(5) MVM sites per cell. In contrast, the nonpermissive mouse lymphoid cell line L1210 lacked specific viral receptors. Also, cloned lines of A-9 cells resistant to viral infection have been isolated. One of these lines lacked the "specific" virus attachment sites but exhibited low levels of nonsaturable virus binding. Based on these examples, infectivity is correlated with the presence of specific viral receptors on the cell surface.     

High sensitivity but normal DNA-repair activity after UV irradiation in Epstein--Barr virus-transformed lymphoblastoid cell lines from Chediak--Higashi syndrome

We established lymphoblastoid cell lines from 2 children with Chediak--Higashi syndrome (CHS), 2 xeroderma pigmentosum (XP) patients and control donors after transformation of peripheral lymphocytes by Epstein--Barr virus (EBV). We used these lymphoblastoid cell lines to investigate repair activity after ultraviolet irradiation. Cell survival of both CHS lymphoblastoid cell lines after irradiation by UV and treatment by 4-nitroquinoline 1-oxide (4NQO) fell between those of the XP and control cell lines. Unscheduled DNA synthesis of CHS cells after UV irradiation occurred at rates similar to those of control cells.     

Ultraviolet enhanced reactivation of a human virus: effect of delayed infection

The ability of UV-irradiated herpes simplex virus to form plaques was examined in monolayers of CV-1 monkey kidney cells preexposed to UV radiation at different intervals before virus assay. From analysis of UV reactivation (Weigle reactivation) curves it was found that as the interval between cell UV irradiation (0-20 J/m2) and initiation of the virus assay was increased over a period of five days, (1) the capacity of the cells to support unirradiated virus plaque formation, which was decreased immediately following UV exposure to the monolayers, increased and returned to approximately normal levels within five days, and (2) at five days an exponential increase was observed in the relative plaque formation of irradiated virus as a function of UV fluence to the monolayers. For high UV fluence (20 J/m2) to the cells, the relative plaque formation by the UV-irradiated virus at five days was about 10-fold higher than that obtained from assay on unirradiated cells. This enhancement in plaque formation is interpreted as a delayed expression of Weigle reactivation. The amount of enhancement resulting from this delayed reactivation was several fold greater than that produced by the Weigle reactivation which occurred when irradiated herpes virus was assayed immediately following cell irradiation.     

Expression of nervous system antigen-3 by lines of mouse fibroblasts and kidney cells and continued expression in hybrid cells

In a survey of the expression on cultured mouse cells of the cell surface antigen known as nervous system antigen-3 (NS-3), it was found that RAG, a renal adenocarcinoma line, expressed that antigen. It was also observed that 3T3, a fibroblast line of unknown tissue origin, expressed NS-3. Cells of these two lines were hybridized with cells of two mouse L cell lines that did not express NS-3. Four hybrid clones were tested for both the 3T3 × L cell cross and the RAG × L cell cross, and all the hybrids were found to be NS-3 positive. All the hybrids had at least 40% as much activity as the NS-3 positive parent. Of the four parental mouse cell lines used, only 3T3 expressed Thy-1.2 antigen on the cell surface. In contrast to the continued expression of NS-3 on hybrid cells, Thy-1.2 antigen was not detectable on two clones of 3T3 × L cell hybrids that were tested.     

Expression of minute virus of mice structural proteins in murine cell lines transformed by bovine papillomavirus-minute virus of mice plasmid chimera.

Recombinant plasmids containing the genomes of both bovine papillomavirus type I and minute virus of mice (MVM) were constructed and used to transform mouse C127 cells. Transformed lines that express MVM gene products with high efficiency were isolated and characterized. These transformants synthesize large amounts of MVM structural polypeptides and spontaneously assemble them into empty virion particles that are released into the culture medium. These lines were, however, genetically unstable; they slowly generated subpopulations that failed to express MVM-specific proteins, and they possessed episomal DNA in which both MVM and bovine papillomavirus sequences were deleted or rearranged, or both. Clonal isolates of these transformants were also superinfectible by infectious MVM virus. Therefore, in spite of their instability, they should be useful host cell lines for transcomplementing mutations introduced into the MVM genome and for growing defective viruses as virions.     

Bystander effects in unicellular organisms

Radiation-induced bystander effects have been seen in mammalian cells from diverse origins. These effects can be transmitted through the medium to cells not present at the time of irradiation. We have developed an assay for detecting bystander effects in the unicellular eukaryote, the fission yeast Schizosaccharomyces pombe. This assay allows maximal exposure of unirradiated cells to cells that have received electron beam irradiation. S. pombe cells were irradiated with 16-18 MeV electrons from a pulsed electron LINAC. When survival of the irradiated cells decreased to approximately 50%, forward-mutation to 2-deoxy-d-glucose resistance increased in the unirradiated bystander cells. Further increase in dose had no additional effect on this increase. In order to detect this response, it was necessary for the irradiated cell/unirradiated cell ratio to be high. Other cellular stresses, such as heat treatment, UV irradiation, and bleomycin exposure, also caused a detectable response in untreated cells grown with the treated cells. We discuss evolutionary implications of these results.     

Establishment of hybridomas secreting human monoclonal antibodies against tetanus toxin and hepatitis B virus surface antigen

Mouse-human heterohybrids (M X H) were constructed and compared with other cell lines (human or mouse) as parental cells to obtain hybrids secreting human monoclonal antibody (MoAb). One of the M X H lines, HM-5, was far superior to the others and useful for establishing hybrids secreting human MoAb. Using HM-5 as a parental cell line, we have obtained 2 hybrids secreting human anti-tetanus toxoid MoAb with neutralizing activity and a hybrid secreting human anti-hepatitis B virus surface antigen (HBsAg) MoAb which recognizes the a-determinant of HBsAg.     

Construction of a primary RH panel of Italian ryegrass genome via UV-induced protoplast fusion

Symmetric and asymmetric somatic hybrids were produced via protoplast fusion between common wheat ( TRITICUM AESTIVUM L.) cv. "Jinan 177" and Italian ryegrass ( LOLIUM MULTIFLORUM Lam.). The ryegrass without or with UV irradiation was used as a donor, providing a small amount of chromatin. In these somatic hybrids, most ryegrass chromosomes have been confirmed preferential elimination and the somatic hybrid calli and plants showed wheat-like morphology. Some of the hybrid lines were used for the analysis of distribution and heredity of donor DNA in the hybrid genome and the possibility of establishing a radiation hybrid (RH) panel of the ryegrass in the present experiment. These hybrids, subcultured for two and three years, retained the ryegrass DNA examined by RFLP and GISH analysis, respectively. Distribution of the ryegrass DNA in the wheat genomes of 20 single-cell individuals, randomly selected from hybrid cell lines produced, were analyzed by 21 ryegrass genome specific SSR markers. The average frequencies of molecular marker retention in symmetric hybrid lines (UV 0), as well as asymmetric hybrid lines from UV 30 s and 1 min were 10.88, 15.48 and 33.86, respectively. It was suggested that the UV dose increased the introgression of donor DNA into wheat genome. The ryegrass SSR fragments in most asymmetric hybrid cell lines remained stable over a period of 2 approximately 3 years. This revealed that those asymmetric somatic hybrids are suitable for the introgression of ryegrass DNA into wheat, and for RH panel and RH mapping.     

Characteristics of the interferon system of an embryonal carcinomal cell are recessive in intraspecific somatic cell hybrids

We have obtained hybrids of PCC4-aza 1, a mouse embryonal carcinoma stem cell line, and two different thymidine kinase deficient mouse cell lines. We have examined the ability of the parental and hybrid cells to produce interferon after infection with the Newcastle Disease virus and to enter the antiviral state when treated with mouse interferon. The interferon system of PCC4-aza 1 is inactive; this characteristic is recessive in the hybrids obtained.     

Evidence for an indirect effect of radiation on mammalian chromosomes : I. Indirectly-induced chromosome loss from somatic cell hybrids

Mouse cells UV-irradiated with doses of 0–72 J/m² were fused with unirradiated Chinese hamster cells, and the chromosome constitutions of cell hybrids were examined. The number of mouse chromosomes retained by hybrids decreased with UV dose, and, unexpectedly, the number of hamster chromosomes also decreased in a dose-dependent manner. It is suggested that some component contributed by the irradiated mouse parent cell has indirectly induced damage and loss of hamster chromosomes.     

Presence of human chromosome 21 alone is sufficient for hybrid cell sensitivity to human interferon.

Human/mouse somatic cell hybrids with chromosome 21 as the only detectable human genetic material were sensitive to both human leukocyte and fibroblast interferons. The presence of additional human chromosomes decreased the amount of interferon needed to attain a given level of virus resistance. Decreased cytopathic effects, decreased virus yields, and the appearance of a specific phosphorylated protein associated with interferon treatment were all observed in hybrids maintaining only human chromosome 21. The phosphorylated protein found in extracts of these human interferon-treated hybrid cells was of mouse origin.