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.     

Effect of gamma- and ultraviolet-irradiation on survival and totipotency of haploid tobacco cells in culture

Summary Haploid tobacco (Nicotiana tabacum L.) cell suspensions subjected to varying doses of gamma and UV rays showed LD₅₀ of about 3.7 Krad and 2,700 ergs/mm² respectively. On exposure of UV-irradiated cells to visible light, an increase in suvival was observed. Regeneration of shoot buds from gamma-irradiated cells was completely inhibited at a dose of 2 Krad, while the same phenomenon with UV-irradiated cells was observed at 8,000 ergs/mm². Regeneration pattern was similar for both photoreactivated and non-photoreactivated cells.     

Supersensitive double mutants in yeast

Summary Double mutants containing the radiosensitive alleles uxsl (UVR^-EXR^-) and either uvsl-2 or uvsl 9-2 (both UVR^-EXR⁺) have been constructed. We find that these double mutants are extremely sensitive to UV: their LD₃₇ doses are 0.3–0.5 ergs/mm² as compared to 16–40 ergs/mm² for the radiosensitive parental strains carrying only a single mutant gene and 456 ergs/mm² for wild-type.     

Analysis of Minimal Functions of Simian Virus 40 III. Evidence for “Host Cell Repair” of Oncogenicity and Infectivity of UV-Irradiated Simian Virus 40

The in vitro transforming capacity of simian virus 40 (SV40) for Syrian hamster cells is highly resistant to inactivation by UV light in comparison to infectivity. In the same cell system, we demonstrated a "host cell repair mechanism" sensitive to caffeine which is, to a large extent, responsible for the high resistance to UV inactivation of the transforming capacity of SV40. The survival of infectivity of UV-irradiated SV40 in CV-1 cells was also sensitive to caffeine, again indicating host cell repair. On the other hand, depression of normal cell DNA synthesis by hydroxyurea during the first 24 h postinfection only modestly reduced, and to a similar extent, the transforming capacity of UV-irradiated and nonirradiated SV40.     

Inactivation of T Antigen-Forming Capacities of Simian Virus 40 and Adenovirus 12 by Ultraviolet Irradiation

Methods to measure T antigen-forming capacities of simian virus 40 (SV40) and adenovirus 12 (Ad12) were investigated, and a method to measure the capacity in terms of T antigen-forming units was employed by the use of cytosine arabinoside. Plaque-forming units and T antigen-forming units of SV40, SV40 deoxyribonucleic acid, or Ad12 were inactivated by ultraviolet (UV) irradiation at the same rate, roughly following a single-hit curve. T-antigen formation by UV-irradiated SV40 and Ad12 was enhanced in cells multiply infected and in cells in a growing state. These observations showed that it was difficult or impossible to estimate the size of the gene for T antigen by UV inactivation.     

Effect of UV Light on RNA-Directed DNA Polymerase Activity of Murine Oncornaviruses

The UV inactivation of RNA-directed DNA polymerase activity of Rauscher leukemia virus was shown to be due to damage to the protein. The UV dose resulting in 37% survival of viral polymerase activity at 254 nm was 2.4 × 10⁴ to 3.1 × 10⁴ ergs/mm². The inactivation rate of p30, a major internal viral protein, was much slower.     

A study of beating frequency of a single myocardial cell : II. Ultraviolet micro-irradiation of the nucleus and of the cytoplasm

Single rat myocardial cells were irradiated with the UV micro-irradiation technique over a nuclear or cytoplasmic area of 5 μm of diameter. The contractile response was studied immediately after the irradiation. After 10³ ergs mm⁻² of UV light (254 nm), 4% and 21% of the cells irradiated in the nucleus and the cytoplasm, respectively, showed a temporary increase of the beating rhythm. Moreover, cytoplasmic regions rich in mitochondria were more excitable than other cytoplasmic regions. The ultrastructure and the survival of these cells 24 h after the irradiation did not differ from the control cells. The change of the contractile response according to the localization of the irradiation indicates that the main target organelles are mitochondria; the role of the membrane is not excluded when higher doses of irradiation are considered.     

Properties of strains of Escherichia coli K12 carrying mutant lex-1 and uvrA6 alleles

Summary Strains with both uvrA6 and the lex-1 mutations are more sensitive to ultraviolet light (UV) than isogenic strains with only one of the mutations. The lex ^- uvrA^-double mutant has the same sensitivity to methyl-methane-sulfonate as the lex ^- uvrA⁺single mutant. UV-irradiated cultures of lex ^- uvrA⁺and lex ^- uvrA^-strains do not produce more streptomycinresistant mutants per survivor than unirradiated cultures. UV-irradiated cultures of a lex ⁺ uvrA^-strain produce large yields of mutants at both low (4 ergs/mm²) and high (25 ergs/mm²) doses of UV compared with the lex ⁺ uvrA⁺ strain which produce an intermediate yield of mutants at 25 ergs/mm², and a small yield at 4 ergs/mm², not significantly greater than unirradiated cultures. A dose of UV which does not induce mutations in strains with the lex-1 mutation produces only a small decrease in DNA synthesis in the lex ^- uvrA⁺strain. The results are interpreted to mean that the lex-1 mutation probably does not affect the same pathway of DNA repair as the uvrA ⁺product (i.e. excision of thymine dimers), and that the absence of UV-induced mutations in irradiated cultures of lex ^-strains is probably not due to a cessation of DNA replication.     

Inactivation of murine norovirus, feline calicivirus and echovirus 12 as surrogates for human norovirus (NoV) and coliphage (F+) MS2 by ultraviolet light (254 nm) and the effect of cell association on UV inactivation

Aims: To determine inactivation profiles of three human norovirus (NoV) surrogate viruses and coliphage MS2 by ultraviolet (UV) irradiation and the protective effect of cell association on UV inactivation. Methods and Results: The inactivation rate for cell‐free virus or intracellular echovirus 12 was determined by exposure to 254‐nm UV light at fluence up to 100 mJ cm^?2. The infectivity of murine norovirus (MNV), feline calicivirus (FCV) and echovirus 12 was determined by cell culture infectivity in susceptible host cell lines, and MS2 infectivity was plaque assayed on Escherichia coli host cells. The UV fluencies to achieve 4‐log₁₀ inactivation were 25, 29, 30 and 70 (mJ cm^?2) for cell‐free FCV, MNV, echovirus 12 and MS2, respectively. However, a UV fluence of 85 mJ cm^?2 was needed to inactivate intracellular echovirus 12 by 4 log₁₀. Conclusions: Murine norovirus and echoviruses 12 are more conservative surrogates than FCV to predict the UV inactivation response of human NoV. Intracellular echovirus 12 was 2·8‐fold more resistant to UV irradiation than cell‐free one. Significance and Impact of the Study: Variation in UV susceptibilities among NoV surrogate viruses and a likely protective effect of cell association on virus susceptibility to UV irradiation should be considered for effective control of human NoV in water.     

The role of pyrimidine dimers in postreplication repair in Neurospora

Summary Using the Micrococcus luteus dimer specific endonuclease assay of Wilkins (1973), and photoreactivation we have examined the induction and fate of ultraviolet induced pyrimidine dimers in the excision defective strain, uvs-2, of Neurospora crassa.Dimer induction was fluence dependent from 0 to 800 ergs/mm² UV. An interdimer distance of 19.6x10⁶ DNA molecular weight was found after a fluence of 220 ergs/mm². We confirm the earlier report that this mutant is completely excision defective (Worthy and Epler 1972). Photoreactivation (PR), which greatly enhanced survival (by 10 fold after 440 ergs/mm² UV), reduced significantly (40–44%) the number of UV-endonuclease sensitive sites found in irradiated DNA. This treatment also alleviated immediately some of the temporary blocks to high molecular weight DNA synthesis (elongation or ligation) seen in irradiated cells.We have also attempted to elucidate the mechanism of cellular postreplication repair used to overcome the UV inhibition to DNA synthesis. It was determined that during postreplication repair, Neurospora does not use recombination to bypass dimers and that single stranded DNA gaps opposite dimers do not appear to be present during the time when DNA being synthesized is made only in short pieces.     

Transcription of Simian Virus 40 II. Hybridization of RNA Extracted from Different Lines of Transformed Cells to the Separated Strands of Simian Virus 40 DNA

The amount of simian virus 40 (SV40) DNA present in various SV40-transformed mouse cell lines and “revertants” isolated from them was determined. The number of viral DNA copies in the different cell lines ranged from 1.35 to 8.75 copies per diploid quantity of mouse cell DNA and from 2.2 to 14 copies per cell. The revertants had the same number of viral DNA copies per diploid quantity of mouse cell DNA as their parental cell lines. (However, they showed an increased number of viral DNA copies per cell due to their increased amount of DNA.) By using separated strands of SV40 DNA, the extent of each DNA strand transcribed into stable RNA species was determined for the transformed and “revertant” cell lines. From 30 to 80% of the “early” strand and from 0 to 20% of the “late” strand was present as stable RNA species in the cell lines tested. There was no alteration in the pattern of the stable viral RNA species present in three concanavalin A-selected revertants, whereas in a fluorodeoxyuridine-selected revertant there appeared to be less viral-specific RNA present in the cells.     

Assembly of T-Antigen Double Hexamers on the Simian Virus 40 Core Origin Requires Only a Subset of the Available Binding Sites

Initiation of simian virus 40 (SV40) DNA replication is dependent upon the assembly of two T-antigen (T-ag) hexamers on the SV40 core origin. To further define the oligomerization mechanism, the pentanucleotide requirements for T-ag assembly were investigated. Here, we demonstrate that individual pentanucleotides support hexamer formation, while particular pairs of pentanucleotides suffice for the assembly of T-ag double hexamers. Related studies demonstrate that T-ag double hexamers formed on “active pairs” of pentanucleotides catalyze a set of previously described structural distortions within the core origin. For the four-pentanucleotide-containing wild-type SV40 core origin, footprinting experiments indicate that T-ag double hexamers prefer to bind to pentanucleotides 1 and 3. Collectively, these experiments demonstrate that only two of the four pentanucleotides in the core origin are necessary for T-ag assembly and the induction of structural changes in the core origin. Since all four pentanucleotides in the wild-type origin are necessary for extensive DNA unwinding, we concluded that the second pair of pentanucleotides is required at a step subsequent to the initial assembly process.     

Reinitiation of DNA Synthesis and Cell Division in Senescent Human Fibroblasts by Microinjection of Anti-p53 Antibodies

In human fibroblasts, growth arrest at the end of the normal proliferative life span (induction of senescence) is dependent on the activity of the tumor suppressor protein p53. In contrast, once senescence has been established, it is generally accepted that reinitiation of DNA synthesis requires loss of multiple suppressor pathways, for example, by expression of Simian virus 40 (SV40) large T antigen, and that even this will not induce complete cell cycle traverse. Here we have used microinjection of monoclonal antibodies to the N terminus of p53, PAb1801 and DO-1, to reinvestigate the effect of blocking p53 function in senescent human fibroblasts. Unexpectedly, we found that both antibodies induce senescent cells to reenter S phase almost as efficiently as SV40, accompanied by a reversion to the “young” morphology. Furthermore, this is followed by completion of the cell division cycle, as shown by the appearance of mitoses, and by a four- to fivefold increase in cell number 9 days after injection. Immunofluorescence analysis showed that expression of the p53-inducible cyclin/kinase inhibitor p21^sdi1/WAF1 was greatly diminished by targeting p53 with either PAb1801 or DO-1 but remained high and, moreover, still p53 dependent in cells expressing SV40 T antigen. As previously observed for induction, the maintenance of fibroblast senescence therefore appears to be critically dependent on functional p53. We suggest that the previous failure to observe this by using SV40 T-antigen mutants to target p53 was most probably due to incomplete abrogation of p53 function.     

Evaluation of water treatment plant UV reactor efficiency against Cryptosporidium parvum oocyst infectivity in immunocompetent suckling mice

Aim: To assess the efficiency of a medium‐pressure UV reactor under full‐scale water treatment plant (WTP) conditions on the infectivity of Cryptosporidium parvum oocysts in an Naval Medical Research Institute (NMRI) suckling mice infectivity model. Methods and Results: Six/seven‐day‐old mice were administered orally 2–10 × 10⁴Cryptosporidium parvum oocysts. Compared with nonirradiated oocysts, 40 mJ cm^?2 UV irradiation of ingested oocysts resulted 7 days later in a 3·4–4·0 log₁₀ reduction in the counts of small intestine oocysts, using a fluorescent flow cytometry assay. Conclusion: Present data extend to industrial conditions previous observations of the efficiency of UV irradiation against Cryptosporidium parvum oocyst in vivo development. Significance and Impact of the study: Present results suggest that in WTP conditions, a medium‐pressure UV reactor is efficient in reducing the infectivity of Cryptosporidium parvum oocysts, one of the most resistant micro‐organisms present in environmental waters.     

Efficacy of UV Irradiation in Inactivating Cryptosporidiumparvum Oocysts

To evaluate the effectiveness of UV irradiation in inactivating Cryptosporidium parvum oocysts, the animal infectivities and excystation abilities of oocysts that had been exposed to various UV doses were determined. Infectivity decreased exponentially as the UV dose increased, and the required dose for a 2-log₁₀ reduction in infectivity (99% inactivation) was approximately 1.0 mWs/cm² at 20°C. However, C. parvum oocysts exhibited high resistance to UV irradiation, requiring an extremely high dose of 230 mWs/cm² for a 2-log₁₀ reduction in excystation, which was used to assess viability. Moreover, the excystation ability exhibited only slight decreases at UV doses below 100 mWs/cm². Thus, UV treatment resulted in oocysts that were able to excyst but not infect. The effects of temperature and UV intensity on the UV dose requirement were also studied. The results showed that for every 10°C reduction in water temperature, the increase in the UV irradiation dose required for a 2-log₁₀ reduction in infectivity was only 7%, and for every 10-fold increase in intensity, the dose increase was only 8%. In addition, the potential of oocysts to recover infectivity and to repair UV-induced injury (pyrimidine dimers) in DNA by photoreactivation and dark repair was investigated. There was no recovery in infectivity following treatment by fluorescent-light irradiation or storage in darkness. In contrast, UV-induced pyrimidine dimers in the DNA were apparently repaired by both photoreactivation and dark repair, as determined by endonuclease-sensitive site assay. However, the recovery rate was different in each process. Given these results, the effects of UV irradiation on C. parvum oocysts as determined by animal infectivity can conclusively be considered irreversible.     

The effect of UV light and mitomycin C on the transfection ofBacillus subtilis 168 I−

An investigation was made of the effect of UV light and mitomycin C on the transfection process inBacillus subtilis 168 I⁻ and phage SP 50. It was found that irradiation with a total dose of 470 ergs/mm²/sec and treatment with 0.4 μg/ml of mitomycin C resulted in a 19-fold and 3.6-fold increase in the transfection frequency.     

Influence of the viral regulatory region on tumor induction by simian virus 40 in hamsters

Most of the simian virus 40 (SV40) genome is conserved among isolates, but the noncoding regulatory region and the genomic region encoding the large T-antigen C terminus (T-ag-C) may exhibit considerable variation. We demonstrate here that SV40 isolates differ in their oncogenic potentials in Syrian golden hamsters. Experimental animals were inoculated intraperitoneally with 10⁷ PFU of parental or recombinant SV40 viruses and were observed for 12 months to identify genetic determinants of oncogenicity. The viral regulatory region was found to exert a statistically significant influence on tumor incidence, whereas the T-ag-C played a minor role. Viruses with a single enhancer (1E) were more oncogenic than those with a two-enhancer (2E) structure. Rearrangements in the 1E viral regulatory region were detected in 4 of 60 (6.7%) tumors. Viral loads in tumors varied, with a median of 5.4 SV40 genome copies per cell. Infectious SV40 was rescued from 15 of 37 (40%) cell lines established from tumors. Most hamsters with tumors and many without tumors produced antibodies to T antigen. All viruses displayed similar transforming frequencies in vitro, suggesting that differences in oncogenic potential in vivo were due to host responses to viral infection. This study shows that SV40 strains differ in their biological properties, suggests that SV40 replicates to some level in hamsters, and indicates that the outcome of an SV40 infection may depend on the viral strain present.     

Insights into the Initiation of JC Virus DNA Replication Derived from the Crystal Structure of the T-Antigen Origin Binding Domain

JC virus is a member of the Polyomavirus family of DNA tumor viruses and the causative agent of progressive multifocal leukoencephalopathy (PML). PML is a disease that occurs primarily in people who are immunocompromised and is usually fatal. As with other Polyomavirus family members, the replication of JC virus (JCV) DNA is dependent upon the virally encoded protein T-antigen. To further our understanding of JCV replication, we have determined the crystal structure of the origin-binding domain (OBD) of JCV T-antigen. This structure provides the first molecular understanding of JCV T-ag replication functions; for example, it suggests how the JCV T-ag OBD site-specifically binds to the major groove of GAGGC sequences in the origin. Furthermore, these studies suggest how the JCV OBDs interact during subsequent oligomerization events. We also report that the OBD contains a novel “pocket”; which sequesters the A1 & B2 loops of neighboring molecules. Mutagenesis of a residue in the pocket associated with the JCV T-ag OBD interfered with viral replication. Finally, we report that relative to the SV40 OBD, the surface of the JCV OBD contains one hemisphere that is highly conserved and one that is highly variable.     

Virion Assembly Factories in the Nucleus of Polyomavirus-Infected Cells

Most DNA viruses replicate in the cell nucleus, although the specific sites of virion assembly are as yet poorly defined. Electron microscopy on freeze-substituted, plastic-embedded sections of murine polyomavirus (PyV)-infected 3T3 mouse fibroblasts or mouse embryonic fibroblasts (MEFs) revealed tubular structures in the nucleus adjacent to clusters of assembled virions, with virions apparently “shed” or “budding” from their ends. Promyelocytic leukemia nuclear bodies (PML-NBs) have been suggested as possible sites for viral replication of polyomaviruses (BKV and SV40), herpes simplex virus (HSV), and adenovirus (Ad). Immunohistochemistry and FISH demonstrated co-localization of the viral T-antigen (Tag), PyV DNA, and the host DNA repair protein MRE11, adjacent to the PML-NBs. In PML^−/− MEFs the co-localization of MRE11, Tag, and PyV DNA remained unchanged, suggesting that the PML protein itself was not responsible for their association. Furthermore, PyV-infected PML^−/− MEFs and PML^−/− mice replicated wild-type levels of infectious virus. Therefore, although the PML protein may identify sites of PyV replication, neither the observed “virus factories” nor virus assembly were dependent on PML. The ultrastructure of the tubes suggests a new model for the encapsidation of small DNA viruses.