UV Irradiation of Vaccinia Virus-Infected Cells Impairs Cellular Functions, Introduces Lesions into the Viral Genome, and Uncovers Repair Capabilities for the Viral Replication Machinery.

Vaccinia virus (VV), the prototypic poxvirus, encodes a repertoire of proteins responsible for the metabolism of its large dsDNA genome. Previous work has furthered our understanding of how poxviruses replicate and recombine their genomes, but little is known about whether the poxvirus genome undergoes DNA repair. Our studies here are aimed at understanding how VV responds to exogenous DNA damage introduced by UV irradiation. Irradiation of cells prior to infection decreased protein synthesis and led to an ∼12-fold reduction in viral yield. On top of these cell-specific insults, irradiation of VV infections at 4 h postinfection (hpi) introduced both cyclobutene pyrimidine dimer (CPD) and 6,4-photoproduct (6,4-PP) lesions into the viral genome led to a nearly complete halt to further DNA synthesis and to a further reduction in viral yield (∼35-fold). DNA lesions persisted throughout infection and were indeed present in the genomes encapsidated into nascent virions. Depletion of several cellular proteins that mediate nucleotide excision repair (XP-A, -F, and -G) did not render viral infections hypersensitive to UV. We next investigated whether viral proteins were involved in combatting DNA damage. Infections performed with a virus lacking the A50 DNA ligase were moderately hypersensitive to UV irradiation (∼3-fold). More strikingly, when the DNA polymerase inhibitor cytosine arabinoside (araC) was added to wild-type infections at the time of UV irradiation (4 hpi), an even greater hypersensitivity to UV irradiation was seen (∼11-fold). Virions produced under the latter condition contained elevated levels of CPD adducts, strongly suggesting that the viral polymerase contributes to the repair of UV lesions introduced into the viral genome. IMPORTANCE Poxviruses remain of significant interest because of their continuing clinical relevance, their utility for the development of vaccines and oncolytic therapies, and their illustration of fundamental principles of viral replication and virus/cell interactions. These viruses are unique in that they replicate exclusively in the cytoplasm of infected mammalian cells, providing novel challenges for DNA viruses. How poxviruses replicate, recombine, and possibly repair their genomes is still only partially understood. Using UV irradiation as a form of exogenous DNA damage, we have examined how vaccinia virus metabolizes its genome following insult. We show that even UV irradiation of cells prior to infection diminishes viral yield, while UV irradiation during infection damages the genome, causes a halt in DNA accumulation, and reduces the viral yield more severely. Furthermore, we show that viral proteins, but not the cellular machinery, contribute to a partial repair of the viral genome following UV irradiation.

Virus inactivation under the photodynamic effect of phthalocyanine zinc(II) complexes.

Various metal phthalocyanines have been studied for their capacity for photodynamic effects on viruses. Two newly synthesized water-soluble phthalocyanine Zn(II) complexes with different charges, cationic methylpyridyloxy-substituted Zn(II)- phthalocyanine (ZnPcMe) and anionic sulfophenoxy-substituted Zn(II)-phthalocyanine (ZnPcS), were used for photoinactivation of two DNA-containing enveloped viruses (herpes simplex virus type 1 and vaccinia virus), two RNA-containing enveloped viruses (bovine viral diarrhea virus and Newcastle disease virus) and two nude viruses (the enterovirus Coxsackie B1, a RNA-containing virus, and human adenovirus 5, a DNA virus). These two differently charged phthalocyanine complexes showed an identical marked virucidal effect against herpes simplex virus type 1, which was one and the same at an irradiation lasting 5 or 20 min (Δlog=3.0 and 4.0, respectively). Towards vaccinia virus this effect was lower, Δlog=1.8 under the effect of ZnPcMe and 2.0 for ZnPcS. Bovine viral diarrhea virus manifested a moderate sensitivity to ZnPcMe (Δlog=1.8) and a pronounced one to ZnPcS at 5- and 20-min irradiation (Δlog=5.8 and 5.3, respectively). The complexes were unable to inactivate Newcastle disease virus, Coxsackievirus B1 and human adenovirus type 5.

Sensitivity to ultraviolet radiation of Lassa, vaccinia, and Ebola viruses dried on surfaces.

Germicidal UV (also known as UVC) provides a means to decontaminate infected environments as well as a measure of viral sensitivity to sunlight. The present study determined UVC inactivation slopes (and derived D(37) values) of viruses dried onto nonporous (glass) surfaces. The data obtained indicate that the UV resistance of Lassa virus is higher than that of Ebola virus. The UV sensitivity of vaccinia virus (a surrogate for variola virus) appeared intermediate between that of the two virulent viruses studied. In addition, the three viruses dried on surfaces showed a relatively small but significant population of virions (from 3 to 10 % of virus in the inoculum) that appeared substantially more protected by their environment from the effect of UV than the majority of virions tested. The findings reported in this study should assist in estimating the threat posed by the persistence of virus in environments contaminated during epidemics or after an accidental or intentional release.

RNA-Seq Based Transcriptome Analysis of the Type I Interferon Host Response upon Vaccinia Virus Infection of Mouse Cells.

Vaccinia virus (VACV) encodes the soluble type I interferon (IFN) binding protein B18 that is secreted from infected cells and also attaches to the cell surface, as an immunomodulatory strategy to inhibit the host IFN response. By using next generation sequencing technologies, we performed a detailed RNA-seq study to dissect at the transcriptional level the modulation of the IFN based host response by VACV and B18. Transcriptome profiling of L929 cells after incubation with purified recombinant B18 protein showed that attachment of B18 to the cell surface does not trigger cell signalling leading to transcriptional activation. Consistent with its ability to bind type I IFN, B18 completely inhibited the IFN-mediated modulation of host gene expression. Addition of UV-inactivated virus particles to cell cultures altered the expression of a set of 53 cellular genes, including genes involved in innate immunity. Differential gene expression analyses of cells infected with replication competent VACV identified the activation of a broad range of host genes involved in multiple cellular pathways. Interestingly, we did not detect an IFN-mediated response among the transcriptional changes induced by VACV, even after the addition of IFN to cells infected with a mutant VACV lacking B18. This is consistent with additional viral mechanisms acting at different levels to block IFN responses during VACV infection.

Ultraviolet-irradiated vaccinia virus recombinants, exposing HIV-envelope on their outer membrane, induce antibodies against this antigen in rabbits.

The construction and isolation of recombinants of vaccinia virus (IHD-J strain), bearing on their outer membrane a chimeric protein consisting of the cytoplasmic and transmembrane domains of vaccinia B5R protein and the external domain of HIV envelope, has been previously described by us. The present study aimed to investigate the potential use of such recombinants as a vaccine, following inactivation of their infectivity by ultraviolet (UV) irradiation. The minimal dose of UV irradiation, required for the complete inactivation of the infectivity of these recombinants, was determined. Injections of rabbits with the irradiated noninfectious recombinant viruses successfully induced specific antibodies against the HIV envelope antigen, in addition to those against the poxvirus.

The antiviral activity exerted by vaccinia virus on the growth of herpes simplex virus in BS-C-1 cells.

The growth of herpes simplex virus type 2 (HSV-2) in BS-C-1 cells, was inhibited following super-infection with vaccinia virus. This inhibition was efficiently induced by both the intracellular mature virus (IMV) form of vaccinia virus and the extracellular enveloped virus (EEV), containing an additional external viral membrane. Treatment of vaccinia IMV with the detergents NP-40, Brij-58 or n-octyl-alpha-D-glucopyranoside, abolished its ability to inhibit the growth of HSV-2. Ultraviolet irradiation of vaccinia virus, that completely inactivated the infectivity of the virus, resulted in partial loss of the capability to inhibit the growth of HSV-2: 16-fold more irradiated virus was needed for the inhibition. Electron microscopy showed that the irradiated vaccinia virus adsorbed and penetrated into the HSV-infected cells but remained morphologically intact within the cells for at least 22 h. When the steps in the growth of HSV affected by the irradiated vaccinia virus were followed, it was found that while the synthesis of HSV DNA was partially decreased, the synthesis of HSV proteins was very strongly inhibited and virus particles were not formed.

Killing of Burkitt-lymphoma-derived Daudi cells by ultraviolet-inactivated vaccinia virus.

Interaction of active and UV-inactivated vaccinia virus at high multiplicity caused cytological changes and inhibition in cellular protein and DNA synthesis, thus arresting the multiplication of Burkitt-lymphoma-derived Daudi cells and eventually killing the cells. Adsorption to the cells but the lack of penetration was evident by immunofluorescence, electron microscopy and [3H]thymidine-labeled virus incorporation. Viral DNA synthesis or virus replication was not demonstrated. Thus, it appears that the massive adsorption of viral particles, active or UV-inactivated, or possibly a "toxic" component that resides in the virion, damages the plasma membrane and may be responsible for killing the cells by a mechanism of lysis from without.

Photoinactivation (365 nm) of vaccinia and herpes simplex viruses induced by a new built-in DNA photosensitizer: 4-thiothymidine.

The thymidine analogue 4-thiothymidine (s4T) strongly absorbs light at wavelengths in the UVA range (lambda max 335 nm) and we have examined the photoinactivation of vaccinia and herpes simplex viruses grown in the presence of this nucleoside. The cells used in this study (Vero, mouse 1D-TK+) were able to grow at the same rate when cultured in the presence of 2 mM s4T or 2 mM thymidine, albeit at a slower rate than control cells. Consistent with this finding, viruses grown in the presence of 1-4 mM s4T were obtained in reduced yield but retained full infectivity. Both viruses were specifically inactivated by irradiation with 365 nm light and their photosensitivity, as measured by the initial slope of the inactivation curve, increased in parallel with the concentration of s4T added to the culture medium. More than 90% of vaccinia virus grown in the presence of 4 mM s4T was inactivated. Organomercurial agarose chromatography of sheared DNA isolated from vaccinia virus grown in the presence of 2 mM s4T showed that approximately 2.5% of DNA fragments were specifically retained, as compared to 0.2% for control DNA. This value corresponds to at least one s4T residue incorporated per 30,000 nucleotides of vaccinia virus DNA. In fact, it is likely that this ratio is actually approximately 10 times higher because of the incomplete retention of control thiolated oligodeoxynucleotides.(ABSTRACT TRUNCATED AT 250 WORDS)

Human cell clones, RSa and UVr-1, differing in their capability for UV-induced virus reactivation and phenotypic mutation.

UVr-1 is a human cell clone established as a variant with increased resistance to cell killing by ultraviolet light (UV, principally 254 nm wavelength) from a UV-sensitive cell clone, RSa. Both cells have been characterized to have much the same capacity of UV-induced DNA repair synthesis in whole cells, and the parent RSa cells were recently found to be hypermutable. In the present study UVr-1 cells were characterized in comparison RSa cells with respect to UV-induced virus reactivation and phenotypic mutation. Survival levels of UV-irradiated vaccinia virus and herpes simplex virus type 1 (HSV-1) were much the same in logarithmically proliferating UVr-1 and RSa cells. Correlated with these host cell reactivation levels, the same extent of UV-induced DNA repair replication synthesis was observed in isolated nuclei of the two cell clones. Enhancement of survival levels of UV-irradiated HSV-1 was detected when proliferating RSa cells were irradiated with UV prior to the virus infection. In contrast, this enhanced virus reactivation (EVR) was not detected in similarly irradiated and infected UVr-1 cells. As for phenotypic mutation frequencies assessed by the cloning efficiency of cells with increased resistance to ouabain cell killing (OuaR), OuaR mutants were not obtained from UVr-1 cells either with or without UV irradiation. When the proliferation of cells was synchronized, both EVR and OuaR mutations were detected in RSa cells irradiated with UV at any cell cycle phase, being greatest in the later half of the G1 phase. However, there was no detectable EVR or mutation in any phase of synchronous UVr-1 cells. The hypomutability of UVr-1 cells and hypermutability of RSa cells in a G1 cell cycle phase was also found even if 4-nitroquinoline 1-oxide was used as a mutagen or mutant cells with increased resistance to 6-thioguanine cell killing were estimated.

Survival of UV-irradiated vaccinia virus in normal and xeroderma pigmentosum fibroblasts; evidence for repair of UV-damaged viral DNA.

Vaccinia virus replicates in the cytoplasm of cells from a large number of vertebrates and is independent of most or all cellular enzymes and factors needed for DNA replication and gene transcription. To investigate whether vaccinia virus is also independent of nucleotide excision-repair enzymes present in the nucleus, we have investigated the host-cell reactivation of UV-irradiated virus in normal human fibroblasts and fibroblasts from various xeroderma pigmentosum (XP) complementation groups (A, C, D, G and XP-variant). It was found that the survival of UV-damaged vaccinia virus is the same in the normal and all UV-sensitive cell strains tested, suggesting it is independent of host-cell excision-repair enzymes. This agrees with results of Lytle et al. (1972), but is in conflict with data from Závadová (1971). The D37 of vaccinia virus survival is approximately 7 J/m2 in all cells tested, indicating that in normal cells vaccinia virus is very sensitive to ultraviolet light. We also found that cyclobutane pyrimidine dimers disappear from parental viral DNA strands, suggesting that vaccinia DNA is subject to some form of DNA repair. The implications of these results are discussed.

[Survival curve during virus inactivation]. / K voprosu o forme krivykh vyzhivaemosti pri inaktivatsii virusov.

The survival curves for bacteriophage lambda and vaccinia virus were shown theoretically and in experiments to have a plateau at prolonged inactivation by UV-irradiation or 8-methoxypsoralen. The level of the plateau is dependent of the accuracy of the repair process. The method for extrapolation of the survival curves is proposed.

[Chromosomal instability related to disordered replication and repair processes of DNA synthesis in the cells of patients with gouty nephropathy]. / Khromosomnaia nestabil'nost', sviazannaia s narusheniem protsessov replikatsii i reparatsii sinteza DNK, v kletkakh bol'nykh podagricheskoi nefropatiei.

Lymphocytes of patients with gouty nephropathy were investigated using the criteria of sister chromatid exchanges (SCE) formation, rapidity of generation, virus reactivation, detection of the level of virus mutagenesis and DNA repair and replication synthesis in the experiments with some mutagens. Disorders, according to these criteria, were observed in the cells of all the patients. Cells of patients with gouty nephropathy may be used as a model to study DNA repair and replication mechanisms.

[Approach to the development of an actovaccine using vaccinia virus as a model]. / O podkhode k pazrabotke aktovaktsiny na modeli virusa ospovaktsiny

Injection of living vaccinia virus to the mesenteric vein of rabbits leads to its accumulation in liver in high concentration. In the other series of experiments vaccinia virus inactivated with gamma-radiation was inoculated into the mesenteric vein. Later on the animals were sacrificed at various dates. Homogenates were prepared from their liver. Comparative study of these homogenates made it possible to reveal increased immunogenic activity of homogenate obtained 24 hours after intravenous inoculation of inactivated vaccinia virus into animals. Immunogenic activity of this homogenate was more clearly manifested than immunogenic activity of the antigen itself--inactivated vaccinia vorus. Neither original inactivated vaccinia virus nor inactivated virus absorbed by Kupffer cells induced antibody formation. It is supposed that vaccines may be prepared from various viruses or tumor cells treated with macrophages with subsequent chemical extraction of the most immunogenic fraction.

[The preservation of a DNA repair disorder in continuous-line fibroblasts obtained from gout patients]. / Sokhranenie narushemiia reparatsii DNK v perevivaemykh fibroblastakh, poluchennykh ot bol'nykh podagroi.

DNA repair was explored in continuous cells withdrawn from gout patients. The data obtained were compared to those on primary cells (lymphocytes) from the same patients. Two continuous lines of fibroblasts obtained from the biopsy material of patients suffering from gout were examined for stability of reparation defects on long cell passage. The studies were made with 4 to 12 passages of patients' fibroblasts. The use of criteria reflecting certain stages of DNA repair (reparative synthesis of DNA, formation of induced DNA ruptures and their resynthesis during cell postincubation, reactivation and induced mutagenesis of measles vaccine virus in patients' cells) allowed confirmation of repair defect stability in gout patients' cells on their long passage. Based on the data on preservation of the repair defect on cell passage it is concluded that gout patients demonstrate the genetically determined impairment of the synthesis of DNA repair enzymes participating in the recovery of DNA impairments induced by UV radiation or UV mimetics.

Inactivation of vaccinia virus by natural sunlight and by artificial UVB radiation.

This study determined the sensitivity of vaccinia virus, an orthopox virus commonly used as a surrogate for variola virus (etiological agent of smallpox), exposed to UVB radiation emitted by a solar simulator, or to direct natural sunlight. The data obtained indicate that: (1) the virucidal effect of natural sunlight can be mimicked adequately by an artificial light source with similar spectral characteristics in the UVB, (2) viral sensitivity to UVB or to solar radiation can be correlated with experimental data previously obtained with UVC, (3) the correlation factor between virus inactivation by solar radiation (measured at 300 ± 5 nm) and by UVC (254 nm) is between 33 and 60, and (4) the sensitivity of viruses either dry on glass surfaces or in liquid suspension is similar when in the presence of similar amounts of cellular debris and growth media. The findings reported in this study should assist in estimating the threat posed by the persistence of virus during epidemics or after an accidental or intentional release.