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Human fresh frozen plasma (FFP) was spiked with highly titered HIV-1 and illuminated with visible light in the presence of 1 microM of the photoactive dye methylene blue (MB). As shown by titration on MT-4 cells, the infectivity of the virus containing plasma was rapidly lost during illumination: after 5 min the infective titer was reduced by 4.3 and after 10 min by at least 6.32 log10, i.e. it was below the detection limit of the assay applied. Methylene blue without illumination and illumination alone had only a marginal effect on HIV-1 infectivity. Thus our data indicate that the MB/light treatment of FFP is an effective method to eliminate the risk of HIV-1 infection through use of the product. This is especially important for those cases in which the plasma is collected during the 'window period' between infection of the donor and the subsequent seroconversion.

The ability of the human immunodeficiency virus (HIV) to persist and replicate in human CD4+ T lymphocytes and mononuclear phagocytes is under the control of both virally encoded proteins and a variety of host-related factors. Ultraviolet (UV) light has been shown to induce transcription and expression of HIV. Both DNA damage and repair and DNA damage/repair-independent pathways caused by UV irradiation lead to expression of proviral HIV genomes via activation of the cellular transcription factor NF-kappa B. Transgenic mice that contain either long terminal repeat (LTR)-reporter genes or HIV genomes, either full length or deleted in the gag-pol region, express RNA and proteins at the epidermal level, particularly after UV irradiation. Furthermore, UV-triggered release of soluble factors capable of inducing expression of HIV in non-irradiated cells has been observed. Among other host factors, the functional network of pro-inflammatory and immunoregulatory cytokines has been demonstrated to act as a potent regulator of HIV replication, at least in different in vitro systems of infection.

Cytomegalovirus (CMV) infection constitutes a serious threat to patients with acquired immune deficiency syndrome. Recently we reported that human immunodeficiency virus (HIV) infection of CD4+ cells was associated with sustained elevation of cellular levels of cAMP. Moreover, cyclic nucleotide modulators enhanced HIV replication by increasing intracellular levels of cAMP. In this study, the effect of CMV on HIV replication in CMV/HIV mixed infection and its relationship to cAMP were examined. MT-4 cells, CMV strain AD169, and HIV strain IIIB were used. Optimal enhancement (4.4-fold increase) of HIV replication was observed when MT-4 cells were infected with CMV at Day 0 followed by HIV on Day 4 after infection, as determined by reverse transcriptase activity on Day 11 after infection. cAMP (measured by radioimmunoassay) levels in cells infected with CMV alone, HIV alone, or CMV/HIV together were 2-, 3-, and 5-fold above untreated cells, respectively. CMV also enhanced the replication of UV-irradiated HIV 4-fold and this was associated with a 2-fold increase in cAMP as well. Moreover, UV-irradiated CMV enhanced HIV replication 8.8-fold. The same dose of viable and UV-irradiated CMV used in the above experiments increased protein kinase C activity in these cells 3.0- and 8.0-fold, respectively. These findings might suggest that cAMP and protein kinase C are involved in CMV enhancement of HIV replication. These findings may have relevance to the identification of novel target sites for development of antiviral therapeutics.

Replication of herpes simplex virus type 1 was enhanced in fibroblast cultures when cells were ultraviolet irradiated 12 to 48 hours before infection. Maximum enhancement (340% increase) occurred at exposures of 9.6 J/m2 in DNA-repair competent fibroblasts. Repair deficient xeroderma pigmentosum cells responded to lower ultraviolet exposures (1.6 to 5.6 J/m2) with lower levels of enhancement (37% to 140%). Ultraviolet irradiation of host cells before infection increased viral DNA synthesis, but did not alter the ability of the cell to adsorb virus. Prostaglandins were not shown to contribute to the process. These results suggest that ultraviolet-enhanced replication of herpes simplex virus may be associated with DNA-repair activities, and that ultraviolet-enhanced herpes simplex virus replication should be further examined for its contribution to herpes simplex virus recurrence in vivo.

Photodynamic virus inactivation of human fresh plasma is achieved by illuminating single units of plasma in their plastic containers with light from fluorescent tubes in the presence of the phenothiazine dye methylene blue. The effective dye concentration is 1 microM, i.e. approx. 300 micrograms per litre of plasma. Under the conditions used, all enveloped viruses tested so far have been sensitive to photodynamic treatment, although some had to be illuminated for a longer time than others. For example, under production conditions the infectivity of SFV-containing plasma was reduced by more than 7 log10 within five minutes, whereas 30 minutes were required to reduce the infectivity of plasma containing VSV by c. 5 log10. Among the non-enveloped viruses tested some were sensitive to photodynamic treatment (e.g. SV40 and calicivirus) and some were not (e.g. encephalomyocarditis and polioviruses). Using the optimized procedure, plasma proteins remain largely unaffected. Photodynamically treated fresh frozen plasma (FFP) has been routinely produced and distributed since February 1992. Requested reporting of adverse drug reactions indicates that the virus inactivated product is a well tolerated as conventional FFP.

The plant trithiophene, alpha-terthienyl (alpha T), was evaluated for activity against the human immunodeficiency virus (HIV-1). Antiviral activity specifically required long wavelength light (UVA, 320-400 nm). The compound had little or no activity in visible light or in the dark. The anti-HIV effect was UVA-dose dependent and was proportional to the concentration of alpha T, according to several parameters of virus infectivity and replication. The efficacy was decreased to some extent by the presence of bovine serum in the reactions; but under optimal conditions 0.1 microgram/ml. alpha T (3 x 10(-7) M) could inactivate 10(4)-10(5) infectious particles. In contrast poliovirus and Coxsackievirus infectivity were relatively resistant to alpha T + UVA.

Cell proliferation is necessary for proviral integration and productive infection of most retroviruses. Nevertheless, the human immunodeficiency virus (HIV) can infect non-dividing macrophages. This ability to grow in non-dividing cells is not specific to macrophages because, as we show here, CD4+ HeLa cells arrested at stage G2 of the cell cycle can be infected by HIV-1. Proliferation is necessary for these same cells to be infected by a murine retrovirus, MuLV. HIV-1 integrates into the arrested cell DNA and produces viral RNA and protein in a pattern similar to that in normal cells. In addition, our data suggest that the ability to infect non-dividing cells is due to one of the HIV-1 core virion proteins. HIV infection of non-dividing cells distinguishes lentiviruses from other retroviruses and is likely to be important in the natural history of HIV infection.

Although systemic lupus erythematosus (SLE) and Sjögren's syndrome (SS) are distinct collagen vascular illnesses, they share certain features. Both have clinical manifestations involving skin and mucous membranes and characteristically have high titers of circulating autoantibodies to the cellular components Ro/SS-A, calreticulin/Ro, 52 kDa Ro and La/SS-B. Viruses have been postulated to be involved in the pathogenesis of both diseases. Sensitivity to sun is a cardinal feature of SLE, and UV light may be involved in its pathogenesis. Using human B-lymphoblastoid cell lines, the effect of the resident Epstein-Barr virus on the expression of the above cellular components was investigated by flow cytometry. Sublethal irradiation with ultraviolet B light appeared to diminish EBV antigen expression (gp350/220) during the first 48 to 72 hours in culture, whereas there was no change in the expression of MHC class I or immunoglobulin host cell proteins, and an apparent increase in the expression of host cell autoantigens. The virus appeared to be more sensitive to UVB-induced damage yet did appear to be able to undergo repair. No direct correlation could be made between the presence of the virus and the increase in autoantigen expression. La/SS-B and/or 52 kDa Ro antigen(s) were found to be present in the cytoplasm of the B lymphoblastoid cells at a higher base level in EBV-infected cell lines than in the EBV-negative cell lines.

Total body x-irradiation has been utilized in the treatment of several human diseases, including leukemia, where it is followed by bone marrow transplantation, and in some autoimmune disorders. Recently, it was reported that total body irradiation appeared useful in the treatment of Friend leukemia virus infection in mice. In this report, the effect of x-irradiation on the replication of human immunodeficiency virus (HIV) in vitro in CD4+ cells was examined. MT-4 cells and HIV strain human T cell lymphotropic virus Type IIIB were used to conduct this study. Infected MT-4 cells were irradiated at the time of infection or following infection with x-ray doses of 25-300 cGy. Doses of 50, 150, and 300 cGy enhanced HIV replication by 1.6-, 2-, and 4.8-fold, respectively. Irradiating the cells prior to infection also resulted in similar enhancement of HIV replication. This phenomenon was also observed with wild-type HIV isolates grown in peripheral blood mononuclear and in HIV chronically infected cells. In addition, the enhancement was associated with a radiation-induced increase in intracellular levels of cAMP. The use of the cAMP-dependent protein kinase A inhibitor, H-8, inhibited HIV replication by 65%. These data suggest that in vitro exposure to low doses of x-ray enhances HIV replication partially via a cAMP-dependent pathway.

Previous studies have revealed that the RE strain of HSV type 1 (HSV-1) induces a tissue-destructive inflammatory response in the mouse cornea that is mediated by CD4 T lymphocytes, whereas the KOS strain of HSV-1 preferentially activates CD8 T lymphocytes in the cornea. Langerhans cells (LC) normally reside only at the periphery of the cornea but can migrate centripetally after HSV-1 infection. We studied the relative contribution of LC to the corneal inflammation induced by the KOS and RE strains of HSV-1. Ten days after infection, the central one-third of RE HSV-1-infected corneas contained an average of 5.7 LC/high-power field compared with 0.6 LC/high-power field in KOS-infected corneas. We hypothesized that the increased density of LC in RE HSV-1-infected corneas at the time of T lymphocyte infiltration contributed to the preferential activation of CD4 T lymphocytes in these corneas. To test this hypothesis, we gave mice a low dose of UV-B corneal irradiation (150 mJ/cm2) 1 day before infection with HSV-1. UV-B irradiation effectively prevented the migration of LC into the central cornea when measured 10 or 21 days after corneal infection with either HSV-1 strain. UV-B corneal irradiation had no effect on the CTL response to HSV-1 Ag in the regional lymph nodes after corneal infection with KOS or RE HSV-1. The delayed-type hypersensitivity response induced by both strains of virus, when measured 8 and 14 days after corneal infection, was significantly reduced by UV-B irradiation. UV-B irradiation significantly reduced the incidence (p = 0.0023) and severity (p = 0.0008) of corneal stromal disease induced by RE HSV-1 but did not significantly affect the stromal disease induced by KOS HSV-1. To distinguish between the effect of UV-B treatment on the afferent and efferent arms of the Ir in mice, we administered UV-B treatment to one eye, followed 24 h later by RE HSV-1 infection of both eyes. These mice developed a normal delayed-type hypersensitivity response, and stromal inflammation developed normally in the untreated eye. However, stromal inflammation was significantly reduced in the treated eye. Our findings suggest that LC play a critical role in the activation of HSV-reactive CD4 T lymphocytes in the cornea. Moreover, the type of corneal inflammation induced by different strains of HSV-1 may reflect their differential capacity to induce LC migration into the central cornea.

Two viral inactivation methods suggested for use with cellular blood products have been evaluated as to their effects on platelets. In the first study, it was proposed that pulsed laser-ultraviolet radiation (UVB) at 308 nm could favor photodamage to UVB-sensitive viral nucleic acid with minimal effects on blood platelets. A "window of efficacy" was observed with UVB doses of 10.5-21.5 J/cm2 at which 4-6 log10 poliovirus were inactivated while platelets were relatively tolerant. However, this "window" occurred only with low-intensity UVB radiation (less than or equal to 0.25 MW/cm2). Damage to platelet proteins, evident at high laser intensities, was probably due to multiple photon excitation of amino acids. In the second study, platelets and viruses were treated with the photosensitizer, merocyanine 540 (MC 540) (less than or equal to 24 micrograms/ml), and visible light (450-600 nm) (less than or equal to J/cm2). Activation of washed platelets by dye/light treatment resulted in a spontaneous release of serotonin, spontaneous aggregation, and marked morphological changes. Increasing concentrations of albumin in the suspension medium protected against dye-mediated photodamage to platelets, but also significantly reduced the antiviral activity of MC 540 and light. These results illustrate the relative sensitivities of platelets and viruses to two inactivation methods and the difficulty in optimizing inactivation of viruses and preservation of platelet function in a protein-rich medium.

A wide variety of viruses are inactivated by psoralen compounds in the presence of ultraviolet A light (UVA). Use of aminomethyltrimethylpsoralen (AMT) and UVA is being evaluated as a method to inactivate viruses that may be present in platelet suspensions prepared for transfusion. Studies have been conducted to assess how variation in various environmental parameters influences the extent of viral inactivation and the retention of platelet properties. Most notably, it was determined that increasing levels of plasma progressively inhibited the inactivation of model viruses. As a result, experiments were routinely conducted at a plasma level of approximately 14.5%, using 40 micrograms/ml AMT, which was determined to be optimal when using this reduced plasma level. The reduced plasma level was achieved by dilution with a nonplasma medium that has been shown to be satisfactory for storage of platelets. Under these conditions, about 5 logs of vesicular stomatitis virus (VSV), pseudorabies, and phi 6 inactivation were achieved. Variation of platelet and leukocyte counts, within normal levels, had a minimal effect on extent of viral inactivation. Although oxygen level (mean levels, 97.9 mm Hg versus 19.2 mm Hg) had only a small influence on viral inactivation with 2.4, 4.8, and 7.2 J/cm2 of UVA (equivalent to 1-3 minutes of exposure), in vitro platelet properties, such as medium pH, morphology characteristics, and aggregation response, were better retained with a longer exposure time at the reduced oxygen level. With normal oxygen (97.9 mm Hg), platelet properties declined substantially relative to untreated controls (no UVA, no AMT) on exposure to 4.8 J/cm2. Our studies have identified two sets of conditions that provide about 5 logs of virus inactivation without extensively altering platelet in vitro properties.

A wide range of enveloped viruses, including human herpes simplex virus type 1, human cytomegalovirus, human T cell leukemia/lymphoma virus type I, human immunodeficiency virus type 1, Sindbis virus, and Friend erythroleukemia virus, are highly susceptible to merocyanine 540 (MC 540)-sensitized photoinactivation. By contrast, human pluripotent hematopoietic stem cells, red cells, factor VIII, and von Willebrand factor are much less sensitive. This suggests that MC 540 may be useful for the inactivation of enveloped viruses in blood and blood products. The dye has a low acute systemic toxicity, is rapidly eliminated from the blood stream, and has little or no mutagenic potential. The currently available data support the view that MC 540-sensitized photo-inactivation interferes with early events in the infectious process, notably the ability of the virus to adhere to and penetrate its host cell. The viral envelope is a major target of photodynamic damages which appear to be mediated at least in part by singlet molecular oxygen.

Transfusion-associated transmission of viral diseases remains a problem. A number of methods have been developed to inactivate viral pathogens in plasma and plasma fractions, including: dry heating, wet heating, solvent-detergent treatment, and immunoaffinity purification. While some of these methods successfully inactivate pathogenic viruses, inactivation may be incomplete or result in damage to labile plasma proteins. We have developed a method of photochemical decontamination (PCD) using psoralens and long wavelength ultraviolet light to inactivate pathogenic viruses. In the present study, a spectrum of model viruses have been added to plasma and plasma fractions to examine the efficiency of photochemical decontamination and the effects on labile plasma coagulation factors. Both RNA and DNA viruses have been inactivated under conditions which permit preservation of coagulation protein function. PCD technology appears to offer a promising solution to decontamination of blood products.

The transmission of human immunodeficiency virus (HIV-1) and other enveloped virus by blood transfusion is a major concern. Photosensitive dyes such as hematoporphyrin derivative (HPD), dihematoporphyrin ether (DHE), benzoporphyrin derivatives (BPD), extended ring porphyrins, sapphyrins and texaphyrins, and various cyanines were used with viral cultures to test the feasibility of using those light-excitable dyes to kill virus. A photodynamic flow cell was used to irradiate viral suspensions or viral infected cells in culture media or in whole blood. Herpes virus (HSV-1) was used to screen compounds. Effective compounds were subsequently tested for their ability to kill HIV-1, CMV, and SIV in culture medium and in blood and proved to effectively kill free virus and infected cells at significant viremias. Irradiation was achieved with a filtered xenon light source and/or tunable dye laser. Concentrations of dyes at 10 times viral kill dose were irradiated in blood which was tested for damage to erythrocytes (RBC), platelets, and blood proteins. No damage to RBC, complement factors, and immunoglobulins was evident immediately after photodynamic treatment. Platelet condition is minimally modified with time. Photodynamic treatment of blood appears to be a feasible means of eradicating virus and some protozoans from blood.

Using both the vesicular stomatitis virus (VSV) and feline leukemia virus (FeLV) as models we have shown that the photosensitizer benzoporphyrin derivative ring A (BPD), when activated with red light (600-700 nm), is effective in eliminating both free virus and virally infected cells from spiked blood products and whole blood drawn from viremic cats experimentally infected with FeLV, under conditions which appear to share red blood cells. The effect of photodynamic therapy on infected lymphocytes, as visualized by scanning electron microscopy, initially appeared as a limited area of tiny holes in the membrane. These holes were subsequently seen to increase in size until the membrane appeared completely decomposed. The red cell membranes however, seem to be undamaged by such photodynamic treatment.

Aluminum phthalocyanine tetrasulfonates (AIPcS) are photoactive compounds with absorption maxima at 665-675 nm. The inactivation of viruses (vesicular stomatitis virus, VSV; human immunodeficiency virus, HIV) added to either whole blood or red blood cell concentrates (RBCC) and platelet concentrates (PC) on treatment with tetrasulfonated AIPc (AIPcS4) was evaluated. Treatment of RBCC with 10 microM AIPcS4 and 44 J/cm2 visible light resulted in the inactivation of greater than or equal to 10(5.5) infectious doses (TCID50) of cell-free VSV, greater than or equal to 10(5.6) TCID50 of cell-associated VSV, and greater than or equal to 10(4.7) TCID50 of cell-free sindbis virus. Both greater than or equal to 10(4.2) TCID50 of cell-free and greater than or equal to 10(3.6) TCID50 of cell-associated forms of HIV were also shown to be inactivated. Encephalomyocarditis virus, used as a model for nonenveloped viruses, was not inactivated. Equivalent virus kill with Photofrin II required a substantially higher concentration of dye and longer exposure to visible light. Following AIPcS4 treatment, red cell integrity was well maintained as judged by the low level (less than 2%) of hemoglobin release immediately following treatment and on subsequent storage, by measurements of erythrocyte osmotic fragility, and by the normal recovery and circulatory survival on infusion of treated, autologous red blood cells in baboons. Treatment of PC with 10 microM AIPcS4 and 44 J/cm2 visible light also resulted in effective virus kill (greater than or equal to 10(5.5) TCID50) of VSV; however, both the rate and extent of platelet aggregation in response to collagen addition declined by at least 50%. Based on these results, further characterization of AIPcS4-treated RBCC is justified.

Murine acquired immunodeficiency syndrome (MAIDS) develops when C57B1/6 mice are inoculated with LP-BM5 murine leukemia viruses. Disease progression in these animals is characterized by lymphadenopathy, polyclonal B-cell activation, severe immunodeficiency, and death. Mice with MAIDS have been used to examine the efficacy of antiretroviral therapies for possible use in AIDS patients. In the present work, MAIDS mice were employed to test the hypothesis that established retroviral infection might be cured by the combined use of a cytotoxic agent (cyclophosphamide) and total body irradiation--a regimen reported to have successfully cured HIV-1 infection in one AIDS patient. Results indicate that the ablation of retrovirus-infected lymphoid cells reduced but did not eliminate LP-BM5 infection. Moreover, this regimen was no more effective at controlling virus proliferation or preventing the polyclonal IgG activation characteristic of murine AIDS than was AZT alone.

Transmission of viral diseases through blood products remains a problem in transfusion medicine. We have developed a photochemical decontamination system (PCD) for platelet concentrates (PC) utilizing treatment with long wavelength ultraviolet radiation (UVA, 320-400 nm) and 8-methoxypsorlan (8-MOP). This system is capable of inactivating 25-30 logs/hour of bacteria E. coli or S. aureus, 6 logs/hour of bacteriophage fd, 0.9 log/hour of bacteriophage R17, and 1.1 logs/hour of feline leukemia virus (FeLV) in PC. Immediately following 6 hours of PCD treatment, platelet integrity and function of PCD-treated and control PC were equivalent. After overnight storage, PCD-treated and control PC platelet properties were equal, but there was a slight reduction in TXB-2 production of PCD-treated PC compared to controls. Following PCD treatment, PC were stored for 48 to 96 hours. Platelet counts, morphology scores, extracellular LDH levels, aggregation response, dense body (db) content, and alpha granule (alpha g) content of PCD-treated and control PC were comparable. We assessed the ability of the PCD technique to inactivate intracellular and extracellular virus, quantified the degree of DNA adduct formation in contaminating lymphocytes, and measured the inhibition of polymerase chain reaction (PCR) mediated amplification of intracellular DNA. High titers of cell-free murine cytomegalovirus added to human platelet concentrates (final concentration 10(6)) were inactivated by PCD within 30 minutes. Cat renal fibroblasts infected at high levels with feline rhinotracheitis virus (FeRTV) were seeded into PC followed by PCD treatment with inactivation of 4.8 logs of FeRTV within 10 minutes. Purified human lymphocytes were seeded into PC and treated with PCD in the presence of 3H 8-MOP. Six hours of PCD treatment resulted in the formation of 9.3 to 12.8 8-MOP adducts per 1000 base pairs (bp) of DNA. PCR amplification of a 242 bp segment at the HLA-DQ alpha locus was examined. Inhibition of PCR DNA amplification was dependent on the numbers of 8-MOP adducts formed, and no amplification was present when greater than 12 adducts per 1000 bp were formed. These studies indicate that PCD can effectively inactivate high titers of cell-associated and cell-free virus seeded into standard human PC. The efficiency of DNA adduct formation can be quantitated, and the level of 8-MOP adduct formation in lymphocytes contaminating PC is comparable to the level of adduct formation in cellular DNA reported in the absence of platelets.

In the presence of longwave ultraviolet light, psoralen derivatives photoreact with the nucleic acids within intact viruses and cells. This photoreaction can leave protein antigens and other surface components relatively unmodified, while eliminating the infectivity of a wide range of infectious agents. The kinetics of inactivation differ among RNA and DNA viruses photoreacted with different derivatives of psoralen. The inactivation kinetics are nonlinear as a result of photodegradation of psoralens and the unexplained biphasic inactivation of some viruses. In spite of these complexities, the photoreaction is capable of generating broad safety margins in the disinfection of microbial products under gentle, physiologic conditions. The psoralen photoreaction provides a potential method for inactivating both known and unknown viruses in active blood products. Psoralen-inactivated viruses have already proven useful as noninfectious antigens for use in immunoassays and as successful experimental vaccines.

Titration experiments in swine macrophages have shown that African swine fever virus infectivity was not enhanced in the presence of antiviral antibodies. The early viral protein synthesis and the viral DNA replication in swine macrophages infected with virus-antibody complexes were inhibited in the presence of high doses of uv-inactivated virus, which saturated specific virus receptors, but not when Fc receptors were saturated with antibodies. These results indicate that African swine fever virus does not infect swine macrophages through Fc receptors and that the normal entry pathway through virus receptors is not bypassed by the virus-antibody complexes.

Psoralen and UVA radiation inactivate human immunodeficiency virus (HIV) in vitro whereas UVB and UVC radiation under experimental conditions transactivate HIV. We studied the effect of systemic PUVA treatment on immunologic and virologic findings in five HIV-infected patients. Systemic PUVA was given in two-4-week periods, 2 months apart. The total irradiation ranged from 30 to 262 joules/cm2. All skin lesions, including therapy-resistant psoriasis vulgaris, seborrheic dermatitis, folliculitis, and chronic urticaria, cleared during the first weeks of PUVA. A slight increase in the CD4 lymphocyte numbers was seen in two patients. Serum beta 2-microglobulin values and urine neopterin values remained steady, and the elevated serum immunoglobulin values became normal in all patients. The PUVA treatment did not induce appearance of HIV antigen in serum and HIV isolation was repeatedly negative in all patients whose cultures were initially negative. Lymphocyte recall responses to purified protein derivative (tuberculin) became positive in three and to HIV-specific antigens in two patients. These responses, however, were transient. All patients except one, who was positive for HIV antigen at entry, have remained well 1 year after PUVA therapy.

The influence of gamma radiation, X-rays, UV-light at 254 nm and 365 nm, the latter combined with furocoumarin sensitizers has been studied on plaque forming ability, phage adsorption, DNA injection, and replication processes. UV-light (365 nm) plus furocoumarin treatment of phage particles gave rise to two types of DNA crosslinks. Type I crosslink corresponded to furocoumarin mediated covalent linkage between adjacent sites in opposite strands of the double helix. Crosslink type II (hairpin crosslink) required a highly condensed DNA and corresponded to the covalent linking of adjacent sites in double helical segments of a folded DNA molecule. The relationships of the type I crosslinks to inhibition of DNA replication and of the type II crosslinks to suppression of the DNA injection process are discussed. Pronounced deviations in phage inactivation have been obtained by X-ray radiation alone compared with UV-light (254 nm) pretreated and subsequently X-ray irradiated probes. The observed protective effect of the latter was described in terms of an inducible repair mechanism. The same protection has been observed by combination of gamma radiation with a sublethal UV-light (254 nm) dose.

Peripheral blood monocytes and lymphocytes isolated from most humans are resistant to HSV infection in vitro. Viral replication is inhibited very early in the cycle, prior to the onset of alpha-protein synthesis; no viral protein or DNA synthesis is detectable even up to 1 week later. The enhanced expression of two 62-kDa and 57-kDa cellular proteins, however, is induced in the lymphocyte population within 3 to 5 h after infection. A 30-kDa protein is induced in the monocyte population immediately after infection. The induced expression of 62-kDa and 57-kDa lymphocyte proteins appears to be virus-mediated because: a) HSV and pseudorabies virus (although not vaccinia virus) induce the expression of 62-kDa and 57-kDa proteins, b) heat shock or exposure of lymphocytes to uninfected cell extracts does not induce expression of either protein, c) 62-kDa protein is not induced in lymphocytes stimulated with a mitogenic concentration of PHA. UV-inactivated HSV induces expression of 62-kDa and 57-kDa proteins in a manner similar to that observed with untreated virus. In contrast, expression of 30-kDa monocyte protein is induced nonspecifically by either uninfected cell extracts or cell extracts containing virus. Sixty-two-kilodalton and 57-kDa protein induction appears to be a marker for human lymphocytes that express profound intracellular resistance to infection with HSV. Induced expression of these proteins occurs only in lymphocytes that inhibit viral replication very early in the growth cycle, prior to the onset of alpha-protein synthesis. Expression of 62-kDa and 57-kDa proteins is not induced in lymphocytes that are permissive or partially permissive to infection with HSV.

The hazards of ultraviolet radiation (UVR) include immunosuppression, activation of human immunodeficiency virus (HIV) type 1 expression, and photocarcinogenesis particularly in immunosuppressed individuals. Fifty-eight male homosexuals positive for HIV antibody and 61 controls not at risk for HIV infection answered a questionnaire on their attitudes and exposure to natural and artificial sources of UVR. Controls were matched for sex but were not from an at-risk group for HIV infection. Mean ages were similar for both groups. HIV seropositives had greater recreational UVR exposure than controls: 12/58 versus 4/61 had regular use of a sunbed (P less than 0.05), and experienced 11.6 weeks versus 9.5 weeks of prolonged natural UVR exposure (P = 0.056) over a four-year period. One reason for this difference may be the misconception present in two-thirds of the HIV seropositive group that a suntan would improve their health and the outcome of their HIV infection. Those with HIV infection must be made aware that there is a potential for further immunosuppression and viral activation from UVR and they should be advised to avoid undue recreational exposure.

The induction of focus formation in low serum and of neoplastic transformation of Syrian hamster embryo cells was examined after the expression of herpes simplex virus type 2 functions. Syrian hamster embryo cells infected at a high multiplicity (5 PFU/cell) with 5-bromo-2'-deoxyuridine-labeled herpes simplex virus type 2 (11% substitution of thymidine residues) were exposed to near UV light irradiation at various times postinfection. This procedure specifically inactivated the viral genome, while having little, if any, effect on the unlabeled cellular DNA. Focus formation in 1% serum and neoplastic transformation were observed in cells exposed to virus inactivated before infection, but the frequency was enhanced (15- to 27-fold) in cells in which the virus was inactivated at 4 to 8 h postinfection. Only 2 to 45 independently isolated foci were capable of establishing tumorigenic lines. The established lines exhibited phenotypic alterations characteristic of a transformed state, including reduced serum requirement, anchorage-independent growth, and tumorigenicity. They retained viral DNA sequences and, even at relatively late passage, expressed viral antigens, including ICP 10.

Gardner's syndrome is an autosomal dominant disorder that predisposes to cancer of the large intestine and to other tumors. We have previously demonstrated that fibroblasts from a patient with this disease are hypersensitive to the cytotoxic effects of ultraviolet light. In this report we have measured several parameters of the repair of ultraviolet light-induced DNA damage in an attempt to identify a defect responsible for the hypersensitivity. We have found the excision rate of pyrimidine dimers, the host cell reactivation of UV-irradiated herpes simplex virus, the induction and rejoining of DNA single strand breaks and the response of semi-conservative DNA replication to UV-irradiation to be in all cases indistinguishable from such phenomena in a variety of normal cells.

The product of gene 31 is normally required for assembly of the T4 capsid. Two mutations that each bypass that requirement are shown to be located at separate sites in gene 23, which encodes the major structural protein of the capsid. A second phenotypic effect that characterizes both bypass31 mutant strains is the ability to multiply in host-defective strains, such as hdB3-1 and groEL mutants, on which wild-type T4 is unable to assemble capsids. The genetic data indicate that both phenotypic effects are due to the bypass31 mutation. Elimination of the requirement for both the phage protein, gp31, and the host protein, GroEL, by either of two single mutations in gene 23 indicates that GroEL and gp31 are normally needed to interact with gp23 in capsid assembly of wild-type T4.

The bacteriophage lambda repressor, a protein that maintains the lysogenic state of a bacterium containing a lambda prophage, is cleaved when the lysogen is induced by mitomycin C or ultraviolet light. This cleavage does not occur when induction is prevented by mutational alteration either of the phage repressor or of the host recA gene product. Proteolytic cleavage may be the primary mechanism of repressor inactivation in this induction pathway, or it may follow a different event which causes the initial inactivation.

The effects of temperature and of ultraviolet radiation on the multiplication of bacteriophage phi29 were studied. Samples of phi29 that had been irradiated to surviving fractions of 0.44 or 0.10 were propagated at 37 degrees C, 42 degrees C and 43.5 degrees C. Latent periods and burst sizes were obtained from one-step growth curves. At a particular temperature, as the dose delivered to the virus was increased, the latent period was extended and the burst size was decreased. For unirradiated virus, the burst size was the same at 42 degrees C as at 37 degrees C, but decreased dramatically at 43.5 degrees C. For virus subjected to a particular dose, the burst size decreased as the temperature was raised. A statistical technique for improving the reliability of parameters obtained from one-step growth curves is presented.

Photodynamic treatment of herpes simplex virus type 1-infected hamster embryo fibroblasts (LSH strain) with a low concentration of proflavine (0.08 mug/10(5) cells per ml), a 3-9-diamine acridine dye, inhibited production not only of infectious progeny but also of virion particles. However, there was no appreciable inhibition of viral or cellular DNA synthesis, even when the infected cells were repeatedly exposed to this low concentration of dye and light during the replication cycle of the virus. It thus appears that photodynamic treatment of infected cells interferes with the processes involved in virus maturation.

Experiments reported here show that some UV-irradiated wild-type T7 phage markers can be rescued efficiently by coinfection with T7 amber mutant phage in a permissive host. Other results show that the segments of a UV-irradiated genome that replicate efficiently are those that also are rescued efficiently during a marker rescue experiment. At higher doses, fewer markers are rescued efficiently and fewer segments of the genome replicate efficiently. The results clearly indicate that the probability of marker rescue is correlated with the ability of the DNA containing the marker to replicate. Sucrose density gradient analysis shows that UV irradiation does not produce double-strand scissions in T7 DNA at doses used here. Therefore, the partial replication and rescue of markers from the left end of the genome is not due simply to injection of only the left end of the T7 DNA.

The effects of ultraviolet light (uv) upon SV40 DNA synthesis in monkey cells were examined to determine whether replication forks were halted upon encountering lesions in the DNA, or alternatively whether lesions were rapidly bypassed. Ultraviolet light inhibits elongation of nascent DNA strands; the extent of incorporation of [3H]deoxythymidine ( [3H]dT) into DNA decreases with increasing uv fluence. Inhibition begins within minutes of irradiation, and becomes more pronounced with increasing time after irradiation. The synthesis of form I (covalently closed) molecules is inhibited even more severely than is total incorporation: post-uv incorporation is predominantly into replication intermediates. In contrast to previous reports, we find that replication intermediates labeled after uv resemble those in unirradiated cells, and contain covalently closed parental strands. DNA strands made after uv are approximately the size of parental DNA which has been cleaved at pyrimidine dimers by a uv endonuclease, indicating that they do not extend past dimers. The hypothesis that replication forks are halted upon encountering pyrimidine dimers in the template strand is consistent with these data.

A partial release of K-specific restriction of phage lambda grown in Escherichia coli C was observed when E. coli K strains AB1157 (having wild-type repair of UV-produced DNA damage) and AB1886 (uvrA) were irradiated with UV light before infection. The effect occurred in AB1886 at lower UV fluences than it did in AB1157. Little or no release of restriction was observed when AB2463 (recA) or AB2494 (lex-1) was used. Such release of restriction appears to be another of the UV-induced phenomena associated with "SOS" repair.

In order to investigate the nature of tubular structures specifically found in herpes simplex virus type 2 (HSV-2)-infected cells, the multiplication of HSV-2 was studied in Vero cells cultured in the presence of varying concentrations of cytosine arabinoside (Ara-C) and cycloheximide (CH), inhibitors of DNA synthesis and protein synthesis respectively. Ara-C, at a concentration of 60 micrograms/ml, inhibited the multiplication of HSV-2 by more than 99% and also prevented the appearance of tubular structures and virus particles in the nuclei of infected cells. Nevertheless, the synthesis of virus specific surface antigens of HSV-2-infected Vero cells was not reduced, as revealed by the fluorescent antibody technique. On the other hand, 10 micrograms/ml of CH inhibited both the appearance of tubular structures and virus particles and the synthesis of virus specific surface antigens by more than 99%. These observations strongly suggest that the appearance of tubular structures is one of the late events in the process of virus multiplication. To measure the comparative genome size needed to produce membrane antigens, tubular structures and infectious centres, the effect of u.v.-inactivation of HSV-2 on these processes was studied. After u.v.-irradiation, the capacity to induce tubular structures was inactivated at a slower rate than the capacity to form infectious centres, but at a faster rate than the induction of surface antigens. Furthermore, more tubular structures could be induced by u.v.-inactivated virus than by the non-irradiated virus which was diluted to the same infectivity as the u.v.-irradiated virus. These results indicate that expression of the entire genome is not required for the production of tubular structures.

We describe experiments which demonstrate that shortly after infection of Friend erythroleukemia cells with herpes simplex virus (HSV), polyribosomes dissociate and cellular mRNA degrades. Analysis of infected cell extracts on sucrose density gradients demonstrates that the majority of the polyribosomes have dissociated to monoribosomes at 2 h postinfection. Physical measurements of infected-cell RNAs support this conclusion and demonstrate that the polyadenylated RNAs decrease in size. The degradation of mRNA is apparently a stochastic process as judged by the failure to detect a shift in the Crt1/2 when polyadenylated RNA extracted from infected cells at different times is hybridized to globin complementary DNA. In experiments designed to determine whether dissociation of polyribosomes is sufficient to cause degradation of globin mRNA, the amount of globin mRNA in uninfected cells did not change when cells were treated with NaF or pactamycin at concentrations sufficient to dissociate all polyribosomes. In cells infected with UV-irradiated virus polyribosomes dissociate but globin mRNA does not degrade, suggesting that it is possible to separate dissociation from degradation.

A study of virus-specific protein synthesis in infectious pancreatic necrosis virus-infected RTG-2 cells was undertaken to find a relationship between the coding capacity of virus genome (two segments of double-stranded RNA of 2.5 X10(6) and 2.3 X 10(6) molecular weight) and the sizes and relative amounts of polypeptides in the virion and in infected cells. The time course of virus-specific protein synthesis was followed by pulse labeling infected UV-irradiated cells with [35S]methionine and analyzing the labeled proteins by polyacrylamide gel electrophoresis followed by autoradiography. Three size classes of virus-specific polypeptides were synthesized, in the same relative proportion, throughout the infectious cycle, beginning 3 h postinfection. Their designation and molecular weight was as follows: alpha1, 1000,000; alpha2, 90,000; beta1, 59,000; beta2, 56,000; gamma1, 32,000; gamma2, 30,000; and gamma3, 28,000. Experiments using amino acid analogues, protease inhibitors, ZnCl2, and supraoptimal temperatures showed that polypeptides of the beta and gamma families did not arise from the alpha polypeptides by post-translational cleavage. Slow cleavage late in the infectious cycle could be demonstrated, since during 12-h period radioactivity was chased from beta1 via beta3 to beta4 (molecular weight 50,000) and beta5 (molecular weight, 49,000). During the chase most of gamma2 was degraded, whereas radioactivity could not be chased from the remaining virus-specific polypeptides. Purified virus contained polypeptides alpha1, alpha2, beta4, beta5, and gamma1. The beta polypeptides made up over 60% of the virion proteins. The results suggest that infectious pancreatic necrosis vibrus possesses a unique mechanism for synthesis of three size-classes of proteins using mRNA transcripts from two high-molecular-weight double-stranded RNA genome segments.

P-815 mastocytoma cells developed susceptibility to immune T-cell-mediated cytolysis shortly after infection by ectromelia virus. Intracellular viral replication and late protein synthesis seem to bu unnecessary events. Interference with early protein synthesis, however, inhibits the development of susceptibility to lysis. The important intracellular events necessary for subsequent cytolysis appear to occur within 1 hour of infection. Virus rendered non-infectious by ultraviolet irradiation but not by gamma irradiation is able to induce these changes. By determining the minimum and essential events of the infectious process which result in T-cell-mediated cytolysis, the task of establishing the molecular changes occurring in the target cell surface membrane necessary for immune T-cell recognition should be simplified.

The plaque development of Herpes simplex virus type 1 (HSV) is slower for viruses treated with two anti-DNA agents: ultraviolet radiation (UV) or n-acetoxy-2-acetyl-aminofluorene. For HSV treated with three antimembrane agents--butylated hydroxytoluene, acridine plus near UV radiation, or ether--the plaque development time is the same as for untreated viruses. These differences hold even for viruses that survived treatment that lowered viability below the 1% level. Gamma ray inactivation of HSV produces no change in plaque development even though this agent is believed to preferentially affect viral DNA.

Lymphocyte transforming properties of B95-8 strain Epstein-Barr virus (EBV) are very sensitive to inactivation by either UV or X irradiation. No dose of irradiation increases the transforming capacity of EBV. The X-ray dose needed for inactivation of EBV transformation (dose that results in 37% survival, 60,000 rads) is similar to the dose required for inactivation of plaque formation by herpes simplex virus type 1 (Fischer strain). Although herpes simplex virus is more sensitive than EBV to UV irradiation, this difference is most likely due to differences in the kinetics or mechanisms of repair of UV damage to the two viruses. The results lead to the hypothesis that a large part, or perhaps all, of the EBV genome is in some way needed to initiate transformation. The abilities of EBV to stimulate host cell DNA synthesis, to induce nuclear antigen, and to immortalize are inactivated in parallel. All clones of marmoset cells transformed by irradiated virus produce extracellular transforming virus. These findings suggest that the abilities of the virus to transform and to replicate complete progeny are inactivated together. The amounts of UV and X irradiation that inactivate transformation by B95-8 virus are less than the dose needed to inactivate early antigen induction by the nontransforming P(3)HR-1 strain of EBV. Based on radiobiological inactivation, 10 to 50% of the genome is needed for early antigen induction. Inactivation of early antigen induction is influenced by the cells in which the assay is performed. Inactivation proceeds more rapidly in EBV genome-free cells than in genome carrier Raji or in P(3)HR-1 converted EBV genome-free cells clone B(1). These results indicate that the resident EBV genome participates in the early antigen induction process. Variation in radio-biological killing of B95-8 and P(3)HR-1 EBV is not attributable to variations in the repair capacities of the cells in which the viruses were assayed, since inactivation of HSV was the same in primary lymphocytes and in all lymphoid cell lines tested.

We are examining the effects of preirradiation of host (monkey) cells upon the replication of UV-damaged SV40. Control cells and cells preirradiated with low fluences (5 or 10 J/m2) of UV were infected with undamaged SV40, and the immediate effects of a subsequent irradiation were determined. UV inhibited total SV 40 DNA synthesis (incorporation of thymidine into viral DNA) in both preirradiated and control cells, but the extent of inhibition was less in the preirradiated cells. A test fluence of 60 J/m2 to SV40 replicating in preirradiated cells reduced synthesis only as much as a test fluence of 25 J/m2 in control cells. The fraction of recently replicated SV40 molecules that re-entered the replication pool and subsequently completed one round of replication in the first 2 h after UV was also decreased less in the preirradiated cells. Thus preirradiation of the host cell mitigates the immediate inhibitory effects of a subsequent UV exposure upon SV40 replication.

Simian virus 40-transformed hamster cells were induced to produce infectious virus by treatment with mitomycin C or gamma-irradiation. A portion of the simian virus-40 DNA, which is integrated into the host cell genome in uninduced cells, was recovered in a pool of relatively low-molecular-weight DNA early after induction treatment in the absence of DNA replication. The data indicte that excision of the viral genome occurs subsequent to the induction stimulus.

The induction by ultraviolet light of simian virus 40 (SV40) from two SV40--transformed hamster kidney cell lines is enhanced by caffeine. In order to investigate the mechanism responsible for this enhancement, the effect of caffeine on postreplication repair of DNA damaged by UV light was studied utilizing alkaline sucrose-gradient sedimentation. Caffeine at concentrations of 0.5, 1.0 or 2.0 mM inhibited the filling of gaps during postreplication repair. In addition, caffeine was found to potentiate cell killing by mitomycin C, an alkylating agent, and to enhance SV40 induction by mitomycin C. We postulate that the persistence of gaps in DNA, caused by the presence of caffeine, results in the enhancement of SV40 virus induction.