Search

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.

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.

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., Competing Interests: The authors declare that there is no conflict of interests regarding the publication of this paper.

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., (© 2012 U.S. Government Photochemistry and Photobiology © 2012 The American Society of Photobiology.)

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.

Interest in airborne smallpox transmission has been renewed because of concerns regarding the potential use of smallpox virus as a biothreat agent. Air disinfection via upper-room 254-nm germicidal UV (UVC) light in public buildings may reduce the impact of primary agent releases, prevent secondary airborne transmission, and be effective prior to the time when public health authorities are aware of a smallpox outbreak. We characterized the susceptibility of vaccinia virus aerosols, as a surrogate for smallpox, to UVC light by using a benchtop, one-pass aerosol chamber. We evaluated virus susceptibility to UVC doses ranging from 0.1 to 3.2 J/m(2), three relative humidity (RH) levels (20%, 60%, and 80%), and suspensions of virus in either water or synthetic respiratory fluid. Dose-response plots show that vaccinia virus susceptibility increased with decreasing RH. These plots also show a significant nonlinear component and a poor fit when using a first-order decay model but show a reasonable fit when we assume that virus susceptibility follows a log-normal distribution. The overall effects of RH (P < 0.0001) and the suspending medium (P = 0.014) were statistically significant. When controlling for the suspending medium, the RH remained a significant factor (P < 0.0001) and the effect of the suspending medium was significant overall (P < 0.0001) after controlling for RH. Virus susceptibility did not appear to be a function of virus particle size. This work provides an essential scientific basis for the design of effective upper-room UVC installations for the prevention of airborne infection transmission of smallpox virus by characterizing the susceptibility of an important orthopoxvirus to UVC exposure.

Human sources of urokinase have led to the contamination of in-process lots of commercially available material with human pathogens. Effective pathogen inactivation of urokinase preparations can be achieved through the use of gamma-irradiation. Additionally, the presence of a free radical scavenger (ascorbate) and the control of temperature have resulted in maintenance of the enzymatic activity of urokinase without a significant effect on the pathogen inactivation properties of gamma-irradiation. In this study we have optimized the conditions during gamma-irradiation to achieve inactivation of porcine parvovirus by 5 logs and vaccinia virus to levels below the limits of detection, while maintaining 92% of urokinase activity. Product specific optimization of gamma-irradiation has the potential to provide effective pathogen inactivation while maintaining substantial functional activity for many therapeutic proteins.

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 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.

Cell-mediated immunity, especially the cytotoxic T lymphocyte (CTL), provides resistance to Epstein-Barr virus (EBV), as is demonstrated by the occurrence of posttransplant lymphoproliferative disease in immunosuppressed patients. We set out to use dendritic cells (DCs) to elicit anti-EBV-specific CTLs in culture. In unselected, HLA-B8(+) donors, monocyte-derived mature DCs were pulsed with the HLA-B8-restricted EBNA-3A peptide, FLRGRAYGL, and added to autologous T cells for 7 days at a DC:T ratio of 1:5 to 1:60. The cultured cells specifically lysed EBNA-3A peptide-pulsed, HLA-B8(+), B-lymphoblastoid cell lines in a 5-hour (51)Cr-release assay. The generation of CTLs did not require the addition of interleukin-2. In comparison, monocytes were weak antigen-presenting cells. DCs were then infected with recombinant vaccinia-EBNA-3A. Vaccinia infection significantly decreased the viability of immature DCs after 3 days of culture (to 25% to 45%) but had a smaller effect on mature DC recovery (40% to 70%). To decrease these cytopathic effects and to expand the potential use of vaccinia vectors for DC therapy in immunocompromised patients, we successfully used psoralen and UV-inactivated virus. Mature DCs pulsed with either live or inactivated vaccinia EBNA-3A virus could elicit strong EBNA-3A-specific CTLs. Therefore, mature DCs are powerful stimulators of EBV-specific CTLs and their major histocompatibility complex class I products can even be charged with UV-inactivated recombinant vaccinia.

Unlike most cell types examined, nonpermissive Chinese hamster ovary (CHO) cells readily underwent apoptosis upon infection with vaccinia virus (VV). Apoptosis was observed as early as 3 h postinfection with an electrophoretic assay of DNA fragmentation and by 8 h using an in situ (TUNEL) assay. The CHO hr gene from cowpox virus, which overcomes host range restriction of VV in CHO cells, merely delayed the onset of apoptosis by approximately 3 h. Intermediate and late viral protein synthesis were not necessary for apoptosis since these events do not proceed under nonpermissive conditions. Apoptosis also occurred in the presence of cytosine arabinoside or cycloheximide, which inhibits DNA or protein synthesis, respectively, and after infection with a mutant virus that is blocked in early transcription. We also demonstrated that viral early transcription was not required for induction of apoptosis by infecting CHO cells with psoralen/UV-inactivated virus. On the other hand, apoptosis was inhibited by a neutralizing antibody to the virion L1R protein added either before or after virus attachment to cells. These results indicate that a postbinding step associated with cell entry is sufficient and required for induction of apoptosis in CHO cells. Recent progress on apoptotic signaling pathways raises the possibility that a cellular receptor for VV may be involved in apoptosis.

Induction of the cytopathic effect (CPE) in cells infected with poxvirus seems ubiquitous in that it has been associated with all different strains and preparations of poxviruses, regardless of the replicating status of these viruses. The study of the mechanisms by which CPE is induced by nonreplicating poxviruses is hampered by the lack of any noncytopathic mutant strains and preparations. In this paper, we report on the patterns of gene expression and induction of CPE by vaccinia viruses treated by limited cross-linking with psoralen and long-wave UV light (PLWUV). We show that treatment of cell-free virus with PLWUV could inactivate viral replication without abolishing the ability of the virus to infect cells. Viral transcription as indicated by reporter genes was generally enhanced and prolonged under early viral promoters and abolished under late promoters. Furthermore, increasing the levels of cross-linking with PLWUV resulted in a decrease and abolishment of viral expression of a large reporter gene and a concomitant loss of the induction of CPE. Cells infected with such a virus were able to express the reporter genes and proliferate. The generation of nonreplicating and noncytopathic recombinant vaccinia viruses may help in studies of the mechanisms of CPE induction by poxvirus and may facilitate the use of poxviral vectors in broader areas of research and clinical applications.

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)

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.

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.

The fusion kinetics of cells expressing the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein with CD4 target cells was continuously monitored by image-enhanced Nomarski differential interference contrast optics. The analysis of the videotape recordings showed that (i) cells made contact relatively rapidly (within minutes), in many cases by using microspikes to "touch" and adhere to adjoining cells; (ii) the adhered cells fused after a relatively long waiting period, which varied from 15 min to hours; (iii) the morphological changes after membrane fusion, which led to disappearance of the interface separating the two cells, were rapid (less than 1 min); and (iv) the process of syncytium formation involved subsequent fusion with other cells and not simultaneous fusion of many cells. To measure the kinetics of early stages of cell fusion, we used the recently developed very stable membrane-soluble dye, PKH26, which redistributes between labeled and unlabeled membranes after fusion but does not exchange spontaneously between membranes for prolonged periods. We found that photoactivation of this dye by illumination with green light inhibits fusion of cell membranes as indicated by the lack of dye transfer from the labeled HIV-1 envelope-expressing cells to unlabeled CD4 cells. The inhibitory effect was localized in space and time, which allowed us to develop a new assay for measuring the kinetics of membrane fusion by illuminating the cell mixture at different times after coculture. This assay has also been used to monitor the fusion kinetics of HIV-1 and recombinant vaccinia virus. The photoactivation of nonexchangeable membrane-soluble fluorescent dyes may be useful for development of new assays for measuring the kinetics of membrane fusion and could also be important in designing new antiviral approaches.

A comparison of ultraviolet (UV) irradiation of two wavelength ranges UVB (280-320 nm) and UVC (lower than 280 nm) showed that UVC in particular could very effectively inactivate, in intravenous immunoglobulin (IVIG) and albumin preparations, non-enveloped and non-acid labile model viruses (i.e., Polio 2 and T4 phage) and dry heat-resistant viruses (vaccinia and T4 phage). This effective virucidal treatment (5 min, 5,000 J/m2 dose) was achieved before an unacceptable level of IVIG aggregates occurred. The use of UV irradiation to inactivate infectious agents could add safety and supplement current methods, e.g. solvent/detergent, low pH, which do not inactivate non-enveloped, non-acid labile or dry-heat-resistant viruses at present.

The selective inhibition of host-cell protein synthesis was studied in cells infected with vaccinia virus (VV) under aberrant conditions of transcription. Previous studies in our laboratory have correlated this selective inhibition with a class of short polyadenylated virus-directed RNAs (POLADS) which are synthesized in VV-infected cells during the early phase of transcription. Moreover, it was shown that infection of HeLa cells with UV-irradiated VV or infection in the presence of actinomycin D (ACD) amplifies the synthesis of POLADS compared to the amount produced in cells infected under normal conditions. To further study the role of POLADS in shut-off, we utilized a temperature sensitive mutant of VV which induces only marginal host shut-off at the restrictive temperature. POLADS were isolated from cells infected with either unirradiated or UV-irradiated VV ts mutant at the permissive and restrictive temperatures and their inhibitory activity on translation in vitro was assayed. The study yields further evidence associating excess of POLADS with greater inhibitory potential and supports the speculation that the increased production of POLADS is correlated with the inhibition of translation of both host-cell and viral polypeptides, albeit to different degrees. The results also demonstrate that a lack of POLADS accumulation is related to a corresponding reduction of shut-off.

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.

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.

Vaccinia virus-infected BSC 40 cells do not permit the replication of superinfecting vaccinia virus. The extent of superinfecting virus propagation depends on the time of superinfection; there is 90% exclusion by 4 hr after the initial infection, and more than 99% by 6 hr. When superinfection is attempted at 6 hr after infection, the superinfecting virus is incapable of carrying out DNA replication or early gene transcription, demonstrating that an early event in the virus life cycle is inhibited. The rate of adsorption of the superinfecting virus is unaltered which shows that exclusion is affected at a point between adsorption and early gene transcription. In order to exclude superinfection, the primary infecting virus does not require replication of its DNA or expression of its late genes but it must express one or more early genes.

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.

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.

Repair processes in blood lymphocytes of children with hereditary homocystinuria have been studied. The identical results on the defective repair processes of damaged DNA were received by the two methods used. The method of vaccinia virus reactivation registered total disturbance of the repair function in cells, while the method of alkaline elution revealed the defects in the enzymes providing resynthesis of DNA breaks in normal conditions.

As measured by in vitro, 51Cr-release assays, the expression on plasma membranes of two, immediate-early, vaccinia virus-specified cell-surface antigens, with mol wt of 25K-27K and 16K-17K, could be directly correlated with the susceptibility of target cells to lysis by vaccinia virus-specific cytotoxic T cells.

The antigenic and immunogenic properties of five ts mutants of neurovaccinia virus with markedly reduced pathogenicity for laboratory animals as compared with "wild" type virus were studied. The antigenic and immunogenic activities of the mutants correlated with their capacity to reproduce in the skin of the inoculated animals. When similar doses of UV-inactivated mutants were used for immunization of rabbits, significant differences (more than 100-fold) in their antigenic activity were found. When rabbits were immunized with active virus, neutralizing antibody in titer of 1:800 protected all the immunized animals against intracerebral challenge with log 4.0 LD50 of neurovaccinia virus. On the other hand, the neutralizing antibody formed in response to inoculation of UV-inactivated virus did not protect the immunized animals in titers exceeding 1:2560. A correlation between the antibody titer for "extracellular" virus and immunity in vaccinated rabbits has been demonstrated.

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.

Large numbers of lymphocytes are recruited from the blood into sites of cutaneous DTH reactions. Our goal was to investigate the factors controlling this recruitment. 111In-labeled peritoneal exudate lymphocytes were injected iv and the accumulation of these cells in skin sites injected with a variety of stimuli, was used to measure lymphocyte recruitment in rats. Large numbers of lymphocytes migrated into vaccinia- and KLH-injected sites in sensitized animals, but only into the viral and not the KLH lesions in non-immune animals. Lymphocytes also migrated efficiently into sites injected with the alpha-interferon (IFN) inducers, uv-inactivated vaccinia virus and poly I:C, as well as into sites injected with IFN. In each case there was a dose-response relationship. Analysis of the kinetics of lymphocyte recruitment demonstrated that the peak rate of migration occurred most rapidly after the injection of IFN, later after poly I:C, and was slowest to be reached after vaccinia virus. Rabbit anti-IFN blocked the recruitment of lymphocytes by uv-inactivated vaccinia and by IFN. Histologically, all of these sites demonstrated a dense mononuclear cell infiltrate in the dermis. It is suggested that IFN may be an important mediator in the recruitment of lymphocytes into inflammatory reactions.

Tumors were induced in adult and newborn rabbits by inoculation of fibroma virus. After 10 to 14 days, oil-induced peritoneal macrophages were harvested, purified, and tested in vitro for interferon synthesis after stimulation with specific and nonspecific viruses. Peritoneal macrophages from adult rabbits that had initiated tumor regression produced high levels of interferon (titers ranged from 160 to 640) after stimulation with fibroma virus, whereas macrophages from normal adult rabbits failed to produce significant levels of interferon under the same conditions (titers ranged from <10 to 10). Furthermore, fibroma-immune macrophages responded to vaccinia virus and Newcastle disease virus with higher levels of interferon than did normal macrophages. In contrast, macrophages from newborn tumor-bearing rabbits that showed no evidence of tumor regression failed to respond to fibroma virus stimulation with higher levels of interferon (titers ranged from <10 to 10). These macrophages did, however, yield significantly more interferon than newborn control macrophages when stimulated with a good interferon inducer, Newcastle disease virus (titers ranged from 10 to 80). These data suggest that interferon production may be an expression of macrophage activation to fibroma antigens and that macrophage activation is impaired in newborn rabbits with progressive growing tumors.

Although cell membranes have potent inhibitors which protect the activation of complement on the self cell membranes, some viruses have been shown to activate complement via the alternative pathway on the virus-infected cells. Tumour cells have been made reactive to homologous complement following treatment with such viruses and became highly immunogenic to syngeneic host guinea pigs and mice. Vaccinia virus (VV) made murine tumour cells highly immunogenic thus generating complement activating capacity on the infected cells. Since it has been suggested that VV can make some human tumour cells immunogenic to the cancer patients, we examined VV to see if the virus also has the capacity to make human tumour cells reactive with homologous human complement. Our present results indicate that not only is this the case but ultraviolet-treated VV also has the same effect.

The synthesis of vaccinia virus-induced thymidine kinase is normally arrested several hours after infection. In thymidine kinase-deficient LM cells infected with IHD strain of vaccinia virus, arrest occurs whether or not viral DNA synthesis is inhibited. With virus inactivated by UV irradiation, enzyme synthesis takes place, but arrest is abolished. It is suggested that an early viral genetic function is responsible for the cessation of thymidine kinase synthesis.

Abortive transcription products were synthesized in vitro by UV-irradiated vaccinia virus particles that were incubated with all four ribonucleoside triphosphates or by unirradiated particles that were incubated in reaction mixtures deficient in CTP or UTP. The RNA sedimented at 4 to 6S in sucrose gradients, suggesting that premature termination had occurred, presumably in one case because the DNA contained UV-induced pyrimidine dimers and in the other case because of ribonucleoside triphosphate was present at limiting concentration for transcription. Nevertheless, the short transcripts were capped, methylated, and polyadenylylated, indicating that neither completion of an RNA chain nor processing from a polycistronic precursor was required for modification of either end of the RNA. In addition, the finding of m7G(5')pppAm and m7G(5')pppGm at the 5' ends of the short RNA molecules implied that transcription was initiated with both ATP and GTP. The presence of the polyadenylic acid tract suggested that a slow-down or cessation of transcription, rather than a specific 3'-terminal sequence, served as a signal for polyadenylylation.

Purified preparations of molluscum contagiosum virus contain a DNA-dependent RNA polymerase (EC 2.7.7.6) with similar but not identical properties to those of the enzyme found in vaccinia virions. The ultraviolet inactivation kinetics of the RNA polymerase from both viruses were similar, displaying fast and slow components. Ultraviolet irradiation destroyed the interfering capacities of molluscum and inactivated vaccinia virions, and the interferon-inducing capacity of molluscum virus slowly and with first-order kinetics. Inactivation studies of the interferon-inducing capacity of vaccinia virus were complicated by cytotoxic effects. Electron microscopical studies showed all stages of virus growth in vaccinia-infected mouse embryo cells; molluscum virus appeared to be degraded in lysosome-like bodies. In preliminary studies, marked changes in cytoplasmic RNA synthesis and in patterns of polypeptide synthesis were found in vaccinia-infected but not in molluscum-infected mouse embryo cells.

Two-dimensional analysis of 32P-labelled ribosomal proteins revealed three proteins which are phosphorylated in vaccinia-virus-infected HeLa cells. All three proteins belong to the 40-S ribosomal subunits and were identified as S2, S6 and S16. The ribosomal protein S6 is phosphorylated also in uninfected HeLa cells. Phosphoserine was detected in all three proteins, phosphothreonine only in the protein S2. Phosphorylation of these ribosomal proteins in infected cells is dependent on the multiplicity of the viral infection and increases during the first six hours of infection. All three proteins are also phosphorylated in virus-infected cells treated with cycloheximide and in cells infected with ultraviolet-irradiated virus. This suggests that the phosphorylation reaction involves a vaccinia virion-associated protein kinase.

The effect of UV irradiation on the expression of the vaccinia virus genome was investigated in a cell-free system coupling transcription with translation. Exposure of vaccinia virus to an increasing dose of irradiation resulted in differential reduction in the syntheses of virus-specified polypeptides in the coupled system, with sensitivity being proportional to the size of the gene pro duct. This suggests that each translationally functional mRNA species produced in vitro by vaccinia virus cores is synthesized from an individual promoter site.

We compared the immunity induced by tumor cells modified with UV-inactivated purified vaccinia virus (UV-VV) and with live purified vaccinia virus (L-VV). C3H/HeN mice were inoculated i.p. with UV-VV or L-VV after whole-body irradiation with 150 rads of X-rays (priming). After 3 weeks the mice were immunized i.p. 3 times at weekly intervals with syngeneic X5563 or MH134 cells that had been absorbed in vitro with UV-VV or infected with L-VV and subsequently irradiated with 10(4) rads of X-rays. Then 1 week after the last immunization, the mice were challenged s.c. with X5563 viable tumor cells or challenged i.p. with MH134 viable tumor cells. The 50% lethal dose (TLD50) of X5563 in mice primed and immunized with UV-VV (UV-VV group) on s.c. challenge (10(6.06)) was the same as for mice treated with L-VV (L-VV group), whereas the TLD50 of unprimed or nonimmunized mice (control group) was 10(2.61). The TLD50 of MH134 in the UV-VV treated group on i.p. challenge (10(6.48)) was similar to that of the L-VV treated group (10(6.54)), while the TLD50 of the control group was 10(1.00). The difference between the TLD50 values of X5563 on s.c. challenge of mice primed and immunized with UV-VV or L-VV and control mice was 10(3.4). The difference between the TLD50 values of MH134 on i.p. challenge of primed and immunized mice and control mice was 10(5.5). These results indicate that the in vivo helper function of UV-VV is similar to that of L-VV and that the augmenting effect of this protocol depends on the kind of tumor.

The nature of the interaction between vaccinia virus (VAC) and fibroblastic cells that renders the latter capable of being recognized by virus-specific, H-2 identical murine T lymphocytes has been studied. L cells exposed for 10 min to VAC rendered noninfectious by exposure to ultraviolet light became susceptible targets for cytotoxic T lymphocytes (CTL) without the synthesis of new viral proteins. Susceptibility was retained even if cellular protein synthesis was irreversibly inhibited with pactamycin before virus exposure. Immobilization of cell-surface membranes by glutaraldehyde fixation before (but not after) exposure to virus severely impaired the formation of the "virus + self" complex that in vitro stimulated secondary CTL responses by H-2 identical virus-primed memory cells even though virus attachment to fixed cells were unaffected. This stimulatory complex, once formed, was maintained in membrane fragments prepared from cells previously exposed to VAC. These findings indicate that VAC-specific CTL or their immediate precursors can recognize only those viral envelope antigens that become membrane integrated and that this event requires neither host cell-specific nor virus-specific protein synthesis.