Radiation-induced upregulation of gene expression from adenoviral vectors mediated by DNA damage repair and regulation.

PURPOSE: In the present study, we evaluated the combination of replication-deficient adenoviruses and radiotherapy in vitro. The purpose of the present study was to analyze the mechanism of radiation-mediated upregulation of adenoviral transgene expression. METHODS AND MATERIALS: Adenoviral transgene expression (luciferase or green fluorescent protein) was studied with and without radiation in three cell lines: breast cancer M4A4-LM3, prostate cancer PC-3MM2, and lung cancer LNM35/enhanced green fluorescent protein. The effect of the radiation dose, modification of the viral capsid, and five different transgene promoters were studied. The cellular responses were studied using mass spectrometry and immunofluorescence analysis. Double strand break repair was modulated by inhibitors of heat shock protein 90, topoisomerase-I, and DNA protein kinase, and transgene expression was measured. RESULTS: We found that a wide range of radiation doses increased adenoviral transgene expression regardless of the cell line, transgene, promoter, or viral capsid modification. Treatment with adenovirus, radiation, and double strand break repair inhibitors resulted in persistence of double strand breaks and subsequent increases in adenovirus transgene expression. CONCLUSIONS: Radiation-induced enhancement of adenoviral transgene expression is linked to DNA damage recognition and repair. Radiation induces a global cellular response that results in increased production of RNA and proteins, including adenoviral transgene products. This study provides a mechanistic rationale for combining radiation with adenoviral gene delivery.

LacZ gene transfer to skeletal muscle using a replication-defective herpes simplex virus type 1 mutant vector.

Herpes simplex virus type 1 (HSV-1) represents a promising new viral vector capable of efficient transduction of myofibers in vivo. Here we report on the use of a replication-defective HSV-1 mutant vector (DZ) deleted for the essential immediate early (IE) gene ICP4 for studies of reporter gene transfer and expression following direct inoculation of mouse skeletal muscle. The recombinant vector was engineered to contain the Escherichia coli lacZ gene under transcriptional control of the strong human cytomegalovirus (HCMV) IE promoter. The effect of vector cytotoxicity on the durability of transgene expression following infection of muscle cells in culture and myofibers in vivo revealed that this first-generation HSV vector was cytopathic, limiting the persistence of vector-transduced cells. UV irradiation of vector preparations reduced viral cytotoxicity for myoblasts in culture without reducing significantly beta-galactosidase production. Moreover, muscle cell viability and the durability of transgene expression was enhanced by several days following UV inactivated-vector infection in vivo. Nevertheless, the viral DNA was subsequently lost from vector-inoculated muscle tissue within 2 weeks. This observation indicated that vector toxicity alone did not account for the lack of persistent transgene expression. Longer-term vector transduction and transgene expression was observed, however, following inoculation of immunodeficient SCID mice, indicating that host immunocompetence played an important role in determining the duration of transgene expression in animals. To support this hypothesis, cells expressing CD4 and CD8 antigens have been found in the HSV-1 injected muscle of immunocompetent mice. These data demonstrated that both vector toxicity and vector-induced immunity are significant obstacles to the use of HSV-1 vectors for muscle gene transfer. These impediments must be overcome to further develop HSV vectors for muscle gene therapy applications.

Antiretroviral activity of furocoumarins plus UVA light detected by a replication-defective retrovirus.

The replication defective retrovirus, pXM5(N2), was used for an easy, safe and reproducible test for the screening of furocoumarins with antiretroviral activity. High titer viral supernatants have been photomodified by UVA light (20 kJ m-2) in the presence of different concentrations of two psolarens (8-methoxypsoralen, 8-MOP and 4,5',8-trimethylpsoralen, TMP) and one angelicin (4,6,4'-trimethylangelicin, TMA). At low concentrations (100-250 ng ml-1) 8-MOP and TMA did not show any significant antiviral activity, while TMP demonstrated a reduction of virus infectivity by one log at 250 ng ml-1. At the highest concentration (5 micrograms ml-1), TMA and TMP reduced the virus titer by one and more than two logs, respectively, being, therefore, two and four times more active than 8-MOP. The most active compound, TMP, was further tested on HIV-1 viral supernatants. Total inactivation of the HIV-1 (200 SFU) was obtained in the presence of 1 microgram ml-1 of TMP and 20 kJ m-2 of UVA light. Our results support the validity of the N2 system to detect the antiretroviral activity of furocoumarins and suggest the potential of TMP in combination with UVA light against HIV-1.

Protection of three strains of mice against lethal influenza in vivo by defective interfering virus.

This report examines the protective effects of defective interfering (DI) WSN on three strains of mice (C3H/He-mg (H-2k), C57BL/6 (H-2b) and BALB/c (H-2d)) infected with various doses of A/WSN influenza virus. All three strains were protected in terms of morbidity and mortality, to varying extents, DI WSN protected optimally against a low but lethal dose of A/WSN in C3H/He-mg mice, but also protected this and other strains against very high doses of A/WSN. Intermediate sized inocula gave little, if any, protection. In all cases protection required an active DI genome since inactivation with beta-propiolactone abrogated any sparing effect. Consolidation of the lungs was reduced by treatment with active DI virus, but at some doses of inoculum there was reduction in lung pathology without reduction of mortality. Treatment of infected mice with DI virus did not reduce the lung virus titre, but in C3H/He-mg mice resulted in recovery of infectious virus from other tissues, notably the heart, where it was not normally found. No infectivity was recovered from brain, liver or serum. Haemagglutination-inhibiting (HI) antibody could not be detected in the lungs of any of the infected mice co-inoculated with the control BPL-inactivated DI WSN but was present in considerable amounts in all three strains when these were co-inoculated with DI virus. These and previous data (Morgan and Dimmock, 1992) suggested that influenza virus was immunosuppressive and that active DI virus abrogated these suppressive effects.(ABSTRACT TRUNCATED AT 250 WORDS)

Induction, purification and some properties of phage tail-like particles from Myxococcus coralloides D.

Myxococcus coralloides D was lysogenic for a defective prophage. The particles of the defective bacteriophage could be induced by ultraviolet light and mitomycin C, but the particles did not appear in the supernatants, unless the cells were lysed with chloroform. The phage tails were purified by using a two-phase separation method, ultracentrifugation, chromatography through Sepharose 4B, treatment with chloroform, dialysis and centrifugation on a sucrose gradient. The chemical analysis of the purified samples revealed that the phage tails contained only proteins, neither DNA nor RNA. The different parts of the phage tails (sheath, core and baseplate) did not have the same sensitivity to the chemical and physical agents which were assayed.

[Molecular aspects of interferon induction by adenoviruses]. / Molekuliarnye aspekty induktsii interferona adenovirusami.

The capacity of adenovirus to induce interferon in the infected cells was studied. The examined adenovirus strains of early types were grouped in 2 groups according to their capacity to induce interferon and to the sensitivity of the infecting and interferon-inducing activity to UV-irradiation. A common property of adenoviruses, potent interferon inducers, is their high sensitivity to UV-irradiation.

UV irradiation impairs in vivo encapsidation of bacteriophage T4 DNA.

T4 DNA structural requirements for encapsidation in vivo were investigated, using thin-section electron microscopy to quantitate the kinetics and yields of head intermediates after synchronous DNA packaging into accumulated processed proheads. UV irradiation (254 nm) of T4-infected bacteria just before initiation of encapsidation resulted in a reduction in the rate of DNA packaged measured by electron microscopy and in the yield of viable phage progeny. In UV-irradiated infections with excision-deficient mutants (denV-), the extent of packaging decline was proportional to the UV dose and phage yields were lower than expected based on the packaging levels observed by microscopy. Rescue analysis of progeny from such infections revealed elevated levels of nonviable virions. Pyrimidine dimers were encapsidated in denV- infections, but in excision-competent infections (denV+) dimers were not packaged. A UV-independent, 15 to 20% packaging arrest was also observed when denV endonuclease was inactive during encapsidation, indicating a denV requirement to achieve normal T4 packaging levels. Pyrimidine dimers apparently represent or induce transient blockage of DNA encapsidation or both, causing a decline in the rate. This is in contrast to other DNA structural blocks to packaging induced by mutations in T4 genes 30 and 49, which appear to arrest the process.

A colorimetric assay for quantification of defective interfering particles of respiratory syncytial virus.

A colorimetric assay for defective interfering (DI) particles of respiratory syncytial (RS) virus was developed. This quantitative biological assay is based on neutral red dye uptake by DI particle-protected cells that survive standard virus challenge. This assay was more sensitive than the reduction of infectious yield (RIY) assay and was capable of detecting 1 X 10(4) to 2 X 10(4) DI particles/ml. The coefficient of variation for parallel, simultaneous replicates (n = 10) was 23%. Cell-protecting activity in the colorimetric assay appeared simultaneously with activity in the RIY assay on undiluted passage of plaque-purified virus. Both activities were particulate, were inactivated by RS virus antiserum and exhibited similar ultraviolet-inactivation kinetics. The absolute values of the slopes of dilution curves for both assays were similar, and using regression analysis both assays enabled estimation of similar numbers of active particles. These results suggest that both activities are mediated by the same DI particle. The mechanism of cell protection does not appear to involve extracellular interferon because the inclusion of interferon antibody in the assay did not diminish DI particle cell protection. Finally, the colorimetric assay was used to reveal alternating cycles of infectious and DI virus production on serial undiluted passage.

Interferon induction by viruses. IV. Sindbis virus: early passage defective-interfering particles induce interferon.

We have shown that a single defective-interfering (DI) particle of early (5th) passage Sindbis virus induces maximal amounts of interferon in an 'aged' primary chick embryo cell. The capacity of such DI particles to induce interferon is inactivated by small amounts of u.v. radiation (i/e dose = 232 ergs/mm2). The I/e dose for inactivation of the interferon-inducing capacity of infectious virus particles is 399 ergs/mm2 and for infectivity is 101 ergs/mm2. Pre-treatment with interferon blocks formation of interferon in response to either DI or infectious virus particles. Our results suggest that Sindbis virus genes must be expressed to form the interferon inducer, which is presumably a molecule of double-stranded (ds)RNA. We postulate that for interferon induction, the genomic RNA which codes for genes G and A must be translated into products whose concerted action produces a dsRNA molecule upon synthesis of a segment of RNA complementary to the genome. The RNA from early passage DI particles is sufficiently large (25S, 1.6 x 10(6) mol. wt.) to accommodate these genes, whereas the RNA from the late passage DI particles (20S, 1.0 x 10(6) mol. wt.) is not. Late (15th) passage DI particles do not induce interferon formation.

Incomplete influenza virus: partial functional complementation as revealed by hemadsorbing cell count test.

In MDCK cells inoculated with an appropriate dilution of influenza virus, single hemadsorbing cells could be counted 8 h postinfection against a background of nonadsorbing cells. Standard virus preparation exhibited a linear relationship between the virus dilution and the number of hemadsorbing cells. With incomplete virus preparations obtained by passages of undiluted virus in chicken embryo, the dependence was nonlinear. A ts mutant (ts-29) of A/FPV/Weybridge (Hav1 Neq1) failed to convert MDCK cells into a hemadsorbing state at 42 degrees C. The ability of ts-29 to produce hemadsorbing cells could be rescued by incomplete wild-type virus. The capacity of incomplete virus for this partial functional complementation was inactivated by UV irradiation with one-hit kinetics. The size of the target was estimated to be 5.5 times smaller than that of the virus genome. The results suggest that at least some of the influenza virus genes in defective interfering particles are functional.

Inactivation by u.v.-irradiation of interfering herpes simplex virus particles: interference requires a functional genome.

The sensitivity of interfering herpes simplex virus (HSV) particles to u.v.-irradiation was studied in a virus stock of HSV-1 strain ANG that contained an excess of interfering over infectious particles. Following u.v.-irradiation, samples of this virus stock were assayed for their plaque-forming capacity and their capacity to interfere with the replication of unirradiated standard virus. Depending on the assay conditions, interfering particles appeared to be less, equally, or more sensitive to u.v. light than infectious particles. It is concluded that interference is a gene function of interfering particles rather than being exerted directly by structural constituents of these particles.

[Interrelationships between the interfering capacity of an "incomplete" virus and its infectivity]. / Vzaimootnosheniia mezhdu interferiruiushchei sposobnost'iu "nepolnogo" virusa grippa i infektsionnost'iu.

A comparative analysis of UV inactivation curves of the interfering activity of "incomplete" influenza virus and infectivity showed certain differences in the structures responsible for these functions. All the data exclude the role of virus protein and virus-induced interferon of "incomplete" influenza virus and suggest that RNA is responsible for this interference. The size of the "target" of the "incomplete" virus interfering capacity calculated on the basis of sensitivity to UV-light is approximately 40 times as small as that of the "target" responsible for infectivity. The analogous pattern of UV inactivation in the standard influenza virus and the so-called Magnus virus suggests that in the latter the infectivity is due to the presence of complete virions in the preparation.

Synthesis of a small RNA in cells coinfected by standard and defective interfering particles of vesicular stomatitis virus.

A small RNA, containing approximately 50 nucleotides, is synthesized by cells coinfected with standard vesicular stomatitis virus and its defective interfering (DI) particles. Infection of cells by standard virus or DI particles alone does not lead to synthesis of significant amounts of small RNA. The RNA is initiated at its 5' end with (p)ppXp and is not polyadenylylated at the 3' end despite a content of 51% adenosine. It has sequences complementary to the genome of a DI particle. The synthesis of the small RNA correlates with the replication of the genome of DI particles with molar ratio small RNA/genome RNA of DI particles greater than 50. When replication of DI genomes is prevented by the addition of cycloheximide or prior UV irradiation of DI particles, small RNA is not synthesized in coinfected cells. These results indicate that the small RNA is not the result of transcriptional initiation and that it may relate to interference mediated by DI particles.

Prevention of death in Semliki Forest virus-infected mice by administration of defective-interfering Semliki Forest virus.

Adult mice inoculated with Semliki Forest virus (SFV) were protected from a lethal infection of the central nervous system by intranasal administration of defective-interfering (DI) SFV. DI SFV was prepared by eight passages at high m.o.i. in BHK 21 cells. Mice were treated with unpurified, unconcentrated tissue culture fluid which had been u.v.-irradiated to inactivate the infective virus present. Prevention of death was maximal when the DI virus was administered simultaneously with the infecting inoculum, and under the same conditions multiplication of infective virus in the brains of treated mice was reduced by 10(5)-fold. It was shown that DI SFV was propagated in mouse brains followed intranasal inoculation and it was concluded that protection was brought about through the intrinsic interfering capacity of the DI virus.

Induction of cellular DNA synthesis by defective human cytomegalovirus.

The results presented in this paper show that virus stocks enriched for defective particles are more effective in stimulating cellular DNA synthesis than are stocks relatively free of defective particles. Several lines of evidence support this conclusion: (1) Low levels of UV irradiation enhance the ability of standard virus stocks to stimulate cellular DNA synthesis. (2) Three different stocks derived from individual plaques (which presumably contain very few defective particles) and passaged only twice at low multiplicity, were found to be poor inducers of cellular DNA synthesis. (3) Stocks of virions obtained after serial undiluted passage which were shown to be enriched for defective particles were quite effective in stimulating cellular DNA synthesis.

Defective interfering particles with covalently linked [+/-]RNA induce interferon.

Defective interfering (DI) particles of vesicular stomatitis virus which contain covalently linked complementary [+]message and [-]anti-message RNA as a single-stranded ribonucleoprotein complex within the particle, are extremely efficient inducers of interferon. A single particle can induce a quantum yield of interferon. A single molecule of double-stranded RNA presumed to form, at least in part, on entry into the cell is thought to induce interferon synthesis. Conventional [-]RNA DI particles with the same polypeptide composition as [+/-]RNA DI particles fail to induce interferon.

Inhibition of pseudorabies virus replication by vesicular stomatitis virus. II Activity of defective interfering particles.

Purified defective interfering (DI) particles of vesicular stomatitis virus (VSV) inhibit the replication of a heterologous virus, pseudorabies virus (PSR), in hamster (BHK-21) and rabbit (RC-60) cell lines. In contrast to infectious B particles of VSV, UV irradiation of DI particles does not reduce their ability to inhibit PSR replication. However, UV irradiation progressively reduces the ability of DI particles to cause homologous interference with B particle replication. Pretreatment with interferon does not affect the ability of DI particles to inhibit PSR replication in a rabbit cell line (RC-60) in which RNA, but not DNA, viruses are sensitive to the action of interferon. Under similar conditions of interferon pretreatment, the inhibition of PSR by B particles is blocked. These data suggest that de novo VSV RNA or protein synthesis is not required for the inhibition of PSR replication by DI particles. DI particles that inhibit PSR replication also inhibit host RNA and protein synthesis in BHK-21 and RC-60 cells. Based on the results described and data in the literature, it is proposed that the same component of VSV B and DI particles is responsible for most, if not all, of the inhibitory activities of VSV, except homologous interference.