HOST CELL REACTIVATION AND UV-ENHANCED REACTIVATION IN SYNCHRONIZED MAMMALIAN CELLS

Abstract— Does host cell reactivation (HCR) or UV-enhanced reactivation (UVER) of UV-irradiated Herpes simplex virus (UV-HSV) vary during the host mammalian cell cycle? The answer could be useful for interpreting UVER and/or the two-component nature of the UV-HSV survival curve. Procedures were developed for infection of mitotically-synchronized CV-1 monkey kidney cells. All virus survival curves determined at different cell cycle stages had two components with similar D₀'s and intercepts of the second components. Thus, no single stage of the host cell cycle was responsible for the second component of the virus survival curve. When the cells were UV-irradiated immediately prior to infection, enhanced survival of UV-HSV occurred for cell irradiation and virus infection initiated during late G₁/early S phase or late S/early G₂ phase but not during early G₁ phase. For infection delayed by 24 h after cell irradiation, UVER was found at all investigated times. These results indicate that: (1) HCR is similar at all stages of the host cell cycle; and (2) the “induction” of UVER is not as rapid for cell-irradiation in early G₁ phase. This latter observation may be one reason why normal, contact-inhibited cells do not express UVER as rapidly as faster growing, less contact-inhibited cells.     

UV-ENHANCED REACTIVATION IN MAMMALIAN CELLS: INCREASE BY CAFFEINE

Abstract— It has been reported that caffeine decreases UV-enhanced reactivation of UV-irradiated Herpes simplex virus in CV-1 monkey kidney cells. That occurred when there was no delay between cell irradiation and virus infection. In the present study, virus infection was delayed following cell irradiation to allow an 'induction'period separate from the 'expression'period which occurs during the virus infection. Thus, the effects of caffeine on 'induction'and 'expression'could be determined separately. Caffeine increased the expression of UV-enhanced reactivation, while causing a small decrease in the 'induction'of enhanced reactivation.     

UV-ENHANCED VIRUS REACTIVATION IN MAMMALIAN CELLS: EFFECTS OF METABOLIC INHIBITORS*

Abstract—The induction process of UV-enhanced reactivation of UV-irradiated herpes simplex virus was investigated in CV-1 monkey kidney cells. A protein synthesis inhibitor, cycloheximide (0.5–5 μg/m/), present in the culture medium For 24 h between cell irradiation and virus infection decreased the enhanced virus survival normally found in UV-irradiated cultures. The enhanced virus reactivation became essentially resistant to the addition of cycloheximide by 6–8 h after cell irradiation, indicating that the cycloheximide-sensitive process necessary for enhanced reactivation was complete by that time. Since cycloheximide not only inhibits protein synthesis, but DNA synthesis as well, we investigated the effect of a DNA synthesis inhibitor, hydroxyurea. Hydroxyurea did not decrease UV-enhanced virus survival, but resulted in enhanced virus survival even in unirradiated cells. Therefore, the cycloheximide-caused inhibition of UV-enhanced reactivation did not arise from inhibition of DNA synthesis. The combined results indicate that (1) UV-enhanced virus reactivation in monkey kidney cells requires de novo protein synthesis during the first 6–8 h after cell irradiation and that (2) DNA synthesis inhibition may be the initiating event.     

REACTIVATION OF UV- AND γ-IRRADIATED HERPES VIRUS IN UV- AND X-IRRADIATEDCV–1 CELLS

Abstract— Enhanced reactivation of UV- and y-irradiated herpes virus was investigated by the plaque assay onCV–1 monkey kidney monolayer cells irradiated with UV light or X-rays. Both UV- and X-irradiatedCV–1 cells showed enhancement of survival of UV-irradiated virus, while little or no enhancement was detected for y-irradiated virus assayed on UV- or X-irradiated cells. The enhanced reactivation of UV-irradiated virus was greater when virus infection was delayed 24 or 48 h, than for infection immediately following the irradiation of cells. It is demonstrable that the UV- or X-irradiatedCV–1 cells are able to enhance the repair of UV damaged herpes virus DNA, but not of y-ray damaged ones.     

ENHANCED REPLICATION OF DAMAGED SV40 DNA IN CARCINOGEN-TREATED MONKEY CELLS

Abstract Treatment of mammalian cells with certain chemical or physical carcinogens prior to infection with ultraviolet-irradiated virus results in enhanced survival or reactivation of the damaged virus. To investigate the molecular basis of this enhanced reactivation (ER), we have examined Simian virus 40 DNA replication in carcinogen-treated cells. We find that treatment of monkey kidney cells with N-acetoxy-2-acetylamino-fluorene or UV radiation 24 h prior to infection with ultraviolet-irradiated Simian virus 40 leads to enhancement of viral DNA replication measured at 36 h after infection by [³H]thymidine incorporation or hybridization. The enhancement of DNA replication is observed when cells are treated from 1 to 60 h before infection or 1 to 16 h after infection. The fact that enhancement is observed also when cells are treated after infection rules out the possibility that enhancement occurs at the level of adsorption or penetration of the virus. Measurements of the time course of viral DNA replication indicate that pretreatment of cells does not alter the time of onset of viral DNA replication. We conclude from these studies that ER of Simian virus 40 occurs at the level of viral DNA replication.     

PHOTODYNAMIC TREATMENT OF HERPES SIMPLEX VIRUS INFECTION IN VITRO

Abstract— The effects of photodynamic action on in vitro herpes simplex virus infections of CV-1 monkey kidney fibroblasts or human skin fibroblasts were determined using proflavine sulfate and white fluorescent lamps. Photodynamic treatment of confluent cell monolayers prior to virus infection inactivated cell capacity, i.e. the capacity of the treated cells to support subsequent virus growth as measured by plaque formation. The capacity of human cells was more sensitive to inactivation than the capacity of monkey cells when 6 μM proflavine was used. Treated cell monolayers recovered the capacity to support virus plaque formation when virus infection was delayed four days after the treatment. Experiments in which the photodynamically treated monolayers were infected with UV-irradiated virus demonstrated that this treatment induced Weigle reactivation in both types of cells. This reactivation occurred for virus infection just after treatment or 4 days later. A Luria-Latarjet-type experiment was also performed in which cultures infected with unirradiated virus were photodynamically treated at different times after the start of infection. The results showed that for the first several hours of the virus infection the infected cultures were more sensitive to inactivation by photodynamic treatment than cell capacity. By the end of the eclipse period the infected cultures were less sensitive to inactivation than cell capacity. Results from extracellular inactivation of virus grown in monkey cells at 6 μM proflavine indicated that at physiological pH the virus has a sensitivity to photodynamic inactivation similar to that for inactivation of cell capacity. The combined data indicated that photodynamic treatment of the cell before or after virus infection could prevent virus growth. Thus, photodynamic inactivation of infected and uninfected cells may be as important as inactivation of virus particles when considering possible mechanisms in clinical photodynamic therapy for herpes simplex.     

UV-ENHANCED REACTIVATION OF MINUTE-VIRUS-OF-MICE: STIMULATION OF A LATE STEP IN THE VIRAL LIFE CYCLE

Abstract— UV-enhanced reactivation of minute-virus-of-mice (MVM), an autonomous parvovirus, was studied in parasynchronous mouse A9 cells. The survival of UV-irradiated MVM is increased in cells which have been UV-irradiated prior to infection. UV-enhanced reactivation can be explained neither by facilitated plaque detection on UV-treated indicator cells, nor by altered kinetics of virus production by UV-irradiated cells. No effect of the multiplicity of infection on virus survival was detected in unirra-diated or irradiated cells. The magnitude of UV-enhanced reactivation is a direct exponential function of the UV dose administered to the virus while virus survival is inversely proportional to the UV dosage. The expression of UV-enhanced reactivation can be activated in cells arrested in G₀, it requires de novo protein synthesis and it is maximal when cells are irradiated 30 h before the onset of viral DNA replication. Early phases of the viral cycle, such as adsorption to cellular receptors, migration to the nucleus and uncoating, were not affected by cell irradiation and are unlikely targets of the UV-enhanced reactivation function(s). These results, together with the single-strandedness of the viral genome, strongly suggest that the step stimulated in UV-irradiated cells functions concomitant with, or subsequent to, viral DNA replication.     

THE EFFECT OF METABOLIC INHIBITORS ON THE LARGE PLAQUE EFFECT WITH HERPES SIMPLEX VIRUS

Abstract— Herpes simplex virus (HSV) macroplaque strain plaque development is faster on ultraviolet (UV)-irradiated African green monkey kidney cells if viral infection is delayed for 12 h or more after cell irradiation (Coohill et al. , 1980). This phenomenon has been termed the large plaque effect (LPE). Here we show that treatment of UV-irradiated cells with cycloheximide inhibits the LPE. Pretreatment of unirradiated cells with hydroxyurea, caffeine, or acetoxy-acetylaminofluorene results in faster plaque development. Treatment of UV-irradiated cells with either hydroxyurea or caffeine gave a LPE of the same magnitude as UV alone. In addition, the LPE was observed with other HSV strains—microplaque, syn-20, and KOS. Our results are consistent with the interpretation that the LPE is 'inducible' in African green monkey kidney cells and that inhibition of DNA synthesis is the inducing event. Possible causes of the LPE and similarities between the LPE and enhanced viral reactivation are discussed.     

SV40 INDUCTION FROM A MAMMALIAN CELL LINE BY ULTRAVIOLET RADIATION and THE PHOTOSENSITIZERS 8-METHOXYPSORALEN and ANGELICIN

Abstract SV40-transformed weanling Syrian hamster kidney cells were exposed to long wavelength ultraviolet radiation in the presence of 8-methoxypsoralen or angelicin. The number of viruses induced as a result of this treatment was quantitated on CV-1P cells. The results indicate that angelicin, which is generally believed to form only monoadducts, is a potent viral inducing agent even when compared with 8-methoxypsoralen (8-MOP), which forms both monoadducts and crosslinks. At low radiation exposures 8-MOP is more effective than angelicin; as the radiation exposure is increased angelicin is capable of inducing the same peak level of virus expression as was 8-MOP. It appears that the induction of SV40 is a phenomenon possibly related to the repair of monoadducts.     

THE EFFECTS OF ULTRAVIOLET LIGHT ON HOST CELL REACTIVATION AND PLAQUE SIZE OF Herpes simplex VIRUS TYPE I IN C3H/lOT1/2 MOUSE CELLS

— Herpes simplex virus — type 1 (HSV-I) plaque-forming ability and plaque size were measured on C3H/1OT1/2 cell monolayers as functions of pretreatment dose with UV light at different times before inoculation with virus, in order to determine if UV-enhanced reactivation (ER) of UV-irradiated virus. as well as associated phenomena, could be obtained in this cell system. The number of virus plaques observed (i.e. the capacity of the cells to support virus growth) and the size of the plaques were found to increase substantially with pretreatment of the cells with UV light. However, no significant ER was observed. Therefore, the mechanisms responsible for the increases in plaque size and cell capacity seem to be independent of those responsible for ER. In work by others. C3H/l0T1/2 cells have hcen transformed by UV light at doses similar to those used in this study; the absence of ER of UV-irradiated virus in this study indicates that the mechanism underlying ER is not required for transformation.     

Regulation of SV40-induced cell division and tumor antigen by dibutyryl adenosine 3'-5'-monophosphate.

Simian virus 40 (SV40) induces cell division in microcultures of sparsely plated nongrowing mouse BALB/3T3 cells during acute infection at moderate multiplicities of infection (MOI = 10-100). The infected cells are killed when a MOI of 1,000 is used. SV40 tumor (T) antigen is synthesized in the infected cells, but viral DNA, virion antigen, and progeny virions are not synthesized (abortive infection). The addition of exogenous dibutyryl adenosine 3'-5'-monophosphate (dbcAMP) at the time of infection stimulates the SV40-induced cell division at all MOI and inhibits SV40-induced cell death at high MOI. The percentage of T antigen-positive cells, as monitored by immunofluorescence, is also increased by the addition of dbcAMP at the time of infection. This regulation of SV40-induced cell division and T antigen formation by exogenous dbcAMP occurs within the first 6 hr after infection at 37 degrees C and is dependent upon both the MOI and the concentration of added dbcAMP. The addition of dbcAMP to productively infected TC7 monkey cells has litte effect on the SV40-induced cell death or T antigen formation.     

Comparison of electrical properties of viruses studied by AC capacitance scanning probe microscopy

Capacitances of five types of viruses, adenovirus type 5 (AV5), herpes simplex virus type 1 (HSV1), simian virus 40 (SV40), vaccinia (MVA), and cowpea mosaic virus (CPMV), were compared by AC capacitance scanning probe microscopy. This technique, using a Pt-coated AFM tip as an electrode to probe capacitance of materials between the tip and a bottom electrode, has been applied to study surface structures of semiconductors and polymers with nanometer spatial resolution; however, biological samples at the nanoscale have not been explored by this technique yet. Because most biological cells are poor conductors, this approach to probe electric properties of cells by capacitance is logical. This scanning probe technique showed that each virus has distinguishable and characteristic capacitance. A series of control experiments were carried out using mutant viruses to validate the origin of the characteristic capacitance responses for different viruses. A mutation on the capsid in HSV1 with green fluorescence proteins increased capacitance from 9 x 10(-6) to 1 x 10(-5) F/cm2 at the frequency of 10(4) Hz. Herpes simplex virus type 2 (HSV2) decreased capacitance when its envelope and glycoproteins were chemically extracted. These control experiments indicate that dielectric properties of capsid proteins and envelope glycoproteins significantly influence overall dielectric constants of viruses. Because those capsid proteins and glycoproteins are characteristic of the virus strain, this technique could be applied to detect and identify viruses at the single viron level using their distinct capacitance spectra as fingerprints without labeling.     

HOST CELL REACTIVATION IN MAMMALIAN CELLS-V. PHOTOREACTIVATION STUDIES WITH HERPES VIRUS IN MARSUPIAL AND HUMAN CELLS

Abstract— The survival of UV-irradiated herpes simplex virus was determined in cultured Potoroo (a marsupial) and human cells under lighting conditions which promote photoreactivation. Photoreactivation was readily demonstrated for herpes virus in two lines of Potoroo cells with dose reduction factors of 0.7-0.8 for ovan cells and 0.5-0.7 for kidney cells. Light from Blacklite (near UV) lamps was more effective than from Daylight (mostly visible) lamps, suggesting that near UV radiation was more efficient for photoreactivation in Potoroo cells. The quantitative and qualitative aspects of this photoreactivation were similar to those reported for a similar virus infecting chick embryo cells. UV-survhal curves for herpes virus in Potoroo cells indicated a high level of "dark" host cell reactivation. No photoreactivation was found for UV-irradiated vaccinia virus in Potoroo cells. A similar photoreactivation study was done using special control lighting (Λ > 600 nm) and human cells with normal repair and with ceils deficient in excision repair (XP). No photoreactivation was found for UV-irradiated herpes virus in either human cell with either Blacklite or Daylight lamps as the sources of photoreacti-vating light. This result contrasts with a report of photoreactivation for a herpes virus in the same XP cells using incandescent lamps.     

THE WAVELENGTH DEPENDENCE OF ULTRAVIOLET INACTIVATION OF HOST CAPACITY IN A MAMMALIAN CELL-VIRUS SYSTEM

Abstract. The ability of UV-irradiated African green monkey kidney cells (CV-1) to support the growth of unirradiated herpes simplex virus type 1 as measured by plaque forming ability has been investigated. The lowering of plaque formation by the virus when the host cell was irradiated was examined at thirteen different wavelengths. An action spectrum for this cellular parameter (capacity) was obtained in the wavelength region of 235–302 nm. This action spectrum points to nucleic acid as the critical target molecule for this effect.     

Flow cytometry analysis of early DNA content changes in human and monkey cells following infection with Simian Virus 40.

Simian virus 40 (SV40) is capable of inducing cellular DNA synthesis in permissive and nonpermissive cells. Utilizing flow cytometry, we analyzed the DNA content changes in two diploid human cell strains and two monkey cell lines. The osteogenesis imperfecta (OI) human skin fibroblasts were induced into DNA synthesis, and within one to two cell generations, a polyploid cell population was produced. With WI-38 phase II cells, a similar pattern of increased cycling of cells into DNA synthesis was observed; however, the majority (approximately 60%) of the cells were blocked in the G2 + M phase of the cell cycle. At later time intervals, an increase in the G1 population was demonstrated. The two monkey cell lines responded to SV40 virus with an accumulation of cells in the G2 + M phase of the cell cycle. Thus, two diploid human cell strains exhibited different cell cycle kinetics early after infection with SV40 virus. The one strain (WI-38) behaved similarly to the two monkey cell lines studied. The other strain (OI) responded similarly to nonpermissive (transforming) cells infected with SV40 virus.     

THE WAVELENGTH DEPENDENCE OF ULTRAVIOLET ENHANCED REACTIVATION IN A MAMMALIAN CELL-VIRUS SYSTEM

Abstract— The effect of UV radiation in the wavelength region 230 nm to 302 nm on the ability of an irradiated mammalian cell to reactivate UV-irradiated mammalian virus was tested. An action spectrum for radiation enhanced reactivation (RER) is presented. The shape of the action spectrum points to a combined nucleic acid-protein target for UV radiation effects on this cellular parameter. An analysis of the results of others involving the biochemical and photobiological events involved in RER does not allow us to distinguish which macromolecule is the major contributor to this effect. Studies involving an analogous phenomenon in bacteria (Weigle reactivation) imply that RER and WR may involve similar mechanisms.     

Erythemal Ultraviolet Exposure of Cyclists in Valencia,Spain

UV exposure is considered to be one of the most important risk factors in skin cancers, mainly in outdoor occupational or recreational activities. Outdoor athletes such as cyclists receive regular and significant solar UV erythemal radiation (UVER). The aim of this work was to quantify UVER exposure of amateur cyclists over the course of several days in their training schedules. To quantify UVER exposure of this group, dosimeters (Viospor) were attached at the top of the helmet in the course of their training. The study took place in Valencia, Spain, in June to July 2008 and February to March 2009, and involved a group of five cyclists over a period of 4 days for each period. The mean 2-day personal UV exposure was 32.24 ± 4.14 SED (standard erythema dose) in summer and 11.30 ± 5.36 SED in the winter period. One SED is defined as an effective radiant exposure of 100 J m⁻² when weighted with the International Commission on Illumination (CIE) erythemal response function. The mean exposure ratio (ER) of cyclists was 0.37 ± 0.04 in summer and 0.40 ± 0.11 in winter. The cyclists received the highest UVER exposure in the summer period, but in both training periods UVER exposure was in excess of occupational and recreational guidelines, indicating that protective measures are very necessary.