Department of Pathological Physiology, Charles University, 1st Medical Faculty, U nemocnice 5, 128 53, Prague 2, Czech Republic
Centro Nacional de Biotecnología, CSIC, Campus Universidad Autónoma, 28049 Madrid, Spain2
Author for correspondence: Zora Mlková. Present address: Department of Immunology and Microbiology, Charles University, 1st Medical Faculty, Studni
kova 7, 128 00, Prague 2, Czech Republic. Fax +420 2 2491 3110. e-mail zmelk{at}lf1.cuni.cz
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Programmed cell death, or apoptosis, is a frequent result of virus infection. As part of the host-cell defence mechanisms, it may reduce virus growth as well as its spread and dissemination within the organism. However, viruses modulate apoptosis in both directions: they block or delay apoptosis by specific virus-encoded factors in order to get sufficient virus progeny produced, or they use it as a strategy to get released from the cell. Sometimes, the host cell inhibits the virus-induced apoptosis (Teodoro & Branton, 1997 ; Barry & McFadden, 1998
; Roulston et al., 1999
; Fujimoto et al., 2000
).
Poxviruses have a special ability to survive and replicate within a host organism, despite a strong immune response. This ability is due to the expression of various virus-encoded genes that modulate or counteract the host response at both extracellular and intracellular levels (Buller & Palumbo, 1991 ; Haig, 1998
). Poxviruses usually cause lysis (i.e. necrosis) of the infected cell, and they encode several factors opposing apoptosis directly or indirectly: factors that inhibit cytokine processing and proteolytic activation of caspases, soluble receptors neutralizing the effects of various cytokines, factors that inactivate IFN-inducible antiviral enzyme activities, and analogues of growth factors and hormones (Gagliardini et al., 1994
; Smith et al., 1997
, 1999
; Bird, 1998
; Nash et al., 1999
; Alcamí & Koszinowski, 2000
; Everett et al., 2000
). Reports of apoptosis caused by poxviruses are rather rare, except those describing apoptosis induced by mutants deficient in some apoptosis-preventing factor (Kibler et al., 1997
) or by a recombinant protein expressed using vaccinia virus (VV) as a vector (Ronen et al., 1996
; Gil et al., 1999
; Timiryasova et al., 1999
). The examples of poxvirus-induced apoptosis include canarypoxvirus-infected primate dendritic cells (Ignatius et al., 2000
) and rabbitpox virus-infected pig kidney LLC-PK1 cells (Macen et al., 1998
). VV infection has also been observed to cause apoptosis in certain cells. VV induced apoptosis of Chinese hamster ovary (CHO) cells after virion binding to the cell surface, supposedly with no further requirements for VV early genes expression (Ramsey-Ewing & Moss, 1998
). In contrast, VV early proteins were expressed in an immature B lymphocyte cell line WEHI-231 in which the virus caused apoptosis but did not replicate (Baixeras et al., 1998
). Similarly, VV caused apoptosis of human dendritic cells with only VV early proteins being expressed (Engelmayer et al., 1999
). Additionally, VV infection in mice was shown to lead to a lymphopenia, which has been ascribed to programmed cell death (Gonzalo et al., 1994
).
Phagocytic cells of the monocyte/macrophage lineage play a central role in virushost interactions. Their responses to VV infection have been studied in several species. In rabbit blood monocytes and macrophages, VV replication was found to be slow and the virus matured asynchronously (Jelinkova et al., 1975 ; McLaren et al., 1976
). In mouse peritoneal macrophages, the VV replication cycle was found to be abortive with only early VV proteins expressed, neither VV DNA synthesis nor VV late proteins detected, and with no assembly of progeny virions (Natuk & Holowczak, 1985
). Similarly, a study of the in vitro interactions between VV and monocyte-derived human dendritic cells revealed that only early virus-encoded proteins were expressed, while viral DNA synthesis and virus late protein expression did not occur (Drillien et al., 2000
). In contrast, in activated rabbit peritoneal macrophages, a block in a late step of the virus replication cycle, after DNA synthesis, caused abortive replication of VV (Buchmeier et al., 1979
). However, there have been no reports of monocyte or macrophage apoptosis induced by wild-type VV.
Cytotoxic and apoptosis-inducing properties of macrophages, as well as many of their antimicrobial and antiviral effects, are dependent on the activation of macrophages by IFN- and on induction of nitric oxide (NO) formation (Stuehr & Nathan, 1989
; Nathan & Hibs, 1991
; Nathan, 1992
; Sarih et al., 1993
; Reiss & Komatsu, 1998
). Viruses themselves can be considered poor inducers of inducible NO synthase (iNOS) expression and NO production, but iNOS expression can be induced by other stimuli concomitantly with virus infection. iNOS expression appears to be mediated through induction of cytokines in a variety of experimental infections with viruses in rats and mice (Akaike et al., 1998
), including neuroviruses (Zheng et al., 1993
), pneumotropic viruses (Akaike et al., 1996
) and cardiotropic viruses (Mikami et al., 1996
). However, iNOS has also been reported to be induced directly by a virus structural component, a viral envelope glycoprotein of human immunodeficiency virus type 1, gp120 (Dawson et al., 1993
).
Alteration of host-cell protein synthesis caused by viruses with a cytoplasmic site of replication, like VV, influence viability of the cell. During VV infection, many aspects of cellular processes are perturbed: nuclear DNA and RNA synthesis are inhibited (Kit & Dubbs, 1962 ; Becker & Joklik, 1964
), the overall amount of protein synthesis is reduced (Shatkin, 1963
) and viral polypeptides are exclusively synthesized (Esteban & Metz, 1973
). This selective inhibition of host-cell protein synthesis (shut-off phenomenon) is thought to be mediated by VV virion-associated protein kinase (Buendia et al., 1987
), as well as by virus-induced, untranslated, polyadenylated short RNA sequences (POLADS; Su & Bablanian, 1990
; Cacoullos & Bablanian, 1991
).
Viability of the cell is controlled by many different and distinct signals. The expression of anti-apoptotic proteins, such as Bcl-2 or Bcl-xL, can inhibit apoptosis, while expression of pro-apoptotic proteins, such as Bax or Bak, can accelerate cell death. It has been shown that Bcl-2 family proteins form homo-and heterodimers (Oltvai et al., 1993 ) and that the relative levels of the anti- and pro-apoptotic members of the Bcl-2 family appear to be a key determinant of the fate of cells when confronted with an apoptotic stimulus (Korsmeyer, 1999
). It is generally accepted that Bcl-2 family proteins exert their effects mainly by controlling mitochondrial permeability transition, but the exact mechanism of their function remains unknown.
Here, we demonstrate for the first time that cells of a monocyte/macrophage lineage undergo apoptosis when infected with VV. We have observed that NO is not responsible for apoptosis in macrophages infected by VV. However, our results suggest that VV early gene expression triggers the induction phase of apoptosis, while decreased levels of the anti-apoptotic Bcl-xL mediate the effector phase. The significance of the loss of Bcl-xL was established by inhibition of VV-induced apoptosis on expression of Bcl-2 using a recombinant VV.
![]() |
Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cells.
The mouse monocyte/macrophage cell line J774.G8 was grown in Dulbeccos modified Eagles medium (DMEM), with 4·5 g/l glucose, supplemented with 10% heat-inactivated fetal calf serum and antibiotics (1x105 U/l penicillin, 100 mg/l streptomycin) (10% FCS DMEM). In some experiments, cells were pretreated for 18 h with mouse recombinant IFN- (Genzyme), as specified below. The African green monkey kidney cell line BSC-40 was grown in DMEM supplemented with 10% neonatal calf serum and antibiotics (10% NCS DMEM). The cells were maintained at 37 °C in a 5% CO2 atmosphere with 95% humidity.
Viruses.
VVs used included wild-type (WT) VV, strain Western Reserve (WR; ATCC VR-119), recombinant VV expressing luciferase under the control of the VV early/late promoter p7.5 (VVLUC; Rodriguez et al., 1989 ) used as a control for other recombinant viruses, and analogous recombinant VVs expressing human proto-oncogene Bcl-2 in sense (VVBcl2+) or antisense (VVBcl2-) orientations. Recombinant viruses with the Bcl-2 gene were prepared by homologous recombination into the thymidine kinase (TK) region of WT VV (Mackett et al., 1982
) using VV insertion vectors pSC11-Bcl2+ or pSC11-Bcl2- generated and kindly provided by S. B. Lee (Lee, 1994
). The insertion and Bcl-2 expression were confirmed by Southern, Northern and Western blot analyses (not shown). Viruses were propagated in BSC-40 cells and purified by sucrose gradient sedimentation (Joklik, 1962
). Virus titres were determined by serial dilutions and plaque assays in BSC-40 cells. For virus infection of macrophages, purified viruses were added at m.o.i.s specified in each experiment and allowed to adsorb to cells for 1 h. After removal of inoculum, cells were supplemented with 10% FCS DMEM. At various times after infection, the cells were collected and subjected to further analysis. For virus growth determination, cells were grown in 24-well plates, with 0·5x106 cells in 1 ml in each well, infected at an m.o.i. of 1, and virus titres were determined using 100 µl of the original sample volume of 1 ml. Inactivated viruses were prepared by boiling for 5 min or by treatment with psoralen and UV. For UV-inactivation, viruses (WT 2·7x108 p.f.u./ml, VVLUC 3·78x108 p.f.u./ml) were suspended in Hanks' balanced salt solution supplemented with 0·1% BSA and psoralen at a final concentration 2 µg/ml. The virus (0·5 ml) was incubated in a 35 mm well at room temperature for 10 min and then irradiated for 5 min with 365 nm UV (Ramsey-Ewing & Moss, 1998
). The titres of inactivated viruses were determined on a BSC-40 cell monolayer. After this treatment, no plaque-forming activity was found.
Treatment with inhibitors.
iNOS inhibitor, aminoguanidine (AG; 1 mM), cytosine arabinoside (AraC; 44 µg/ml), actinomycin D (AcD; 5 µg/ml) or cycloheximide (CHX; 100 µg/ml) were present during virus inoculation as well as at later times after infection.
Flow cytometry.
Cell viability and apoptosis were determined by flow cytometry using a FACScan (Becton Dickinson) equipped with the software Lysis II or Cell Quest. Macrophages were vitally stained for 15 min with the potentiometric dyes 3,3'-dihexyloxacarbocyanine iodide [DiOC6(3)], rhodamine 123 (Rh 123), or tetramethylrhodamine methyl ester (TMRM) at final concentrations of 20 nM, 80 nM and 100 nM, respectively; propidium iodide (PI; final concentration 2 µg/ml) was then added shortly before measurement to distinguish between live and dead cells. The specificity of fluorescence of the potentiometric dyes was confirmed by inhibition of their accumulation by an uncoupler of the respiratory chain, carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP). A DiOC6(3)-negative or low positive and PI-negative population was considered as apoptotic, a DiOC6(3)-positive and PI-negative population was considered as live cells, and a DiOC6(3)-negative and PI-positive population represented necrotic or late apoptotic cells. The number of apoptotic cells was expressed as a percentage of DiOC6(3)-negative or low positive events in a PI-negative population. Apoptotic cells were also detected using the Annexin-V-FLUOS Staining kit (Roche Diagnostics). The percentage of infected macrophages was determined using FITC-labelled rabbit polyclonal IgG against wild-type VV, strain WR (Seva-Imuno Praha, Czech Republic). Macrophages were collected, washed with PBS and stained with FITC-labelled rabbit polyclonal IgG (dilution 1:500). Cells were incubated for 30 min on ice, washed with PBS, stained with PI and processed by FACS analysis.
DNA ladder.
Low molecular mass DNA was isolated as previously described (Lee & Esteban, 1994 ). Briefly, at the indicated times after infection, the cells were collected in medium, lysed in buffer containing 20 mM Tris, 10 mM EDTA and 1% Triton X-100, and high molecular mass DNA was removed by centrifugation at 10000 g for 10 min. Supernatant containing low molecular mass DNA was extracted with phenolchloroformisoamyl alcohol (25:24:1), and low molecular mass DNA was precipitated with ethanol, resuspended in 10 mM TrisHCl (pH 8·1), 1 mM EDTA, and treated with 5 µg/ml of RNase A at 37 °C for 1 h. DNA was resolved by 2% agarose gel electrophoresis in 1x TBE and visualized by UV after ethidium bromide staining.
Western blot analysis.
SDSPAGE and Western blot analysis were performed as previously described (Laemmli, 1970 ; Harlow & Lane, 1988
), using enhanced chemiluminescence (ECL). Bcl-2, Bcl-xL and Bax were detected with rabbit polyclonal antibodies specific for the individual proteins (dilution 1:500; Santa Cruz Biotechnology); peroxidase-conjugated goat anti-rabbit IgG was used as a secondary antibody (dilution 1:5000; Cappel Research Products).
-Actin was detected with goat polyclonal antibody (dilution 1:100; Santa Cruz Biotechnology) and peroxidase-conjugated donkey anti-goat IgG as a secondary antibody (dilution 1:5000; Jackson ImmunoResearch Laboratories). Densitometric analysis of the Western blots was performed using image analysis software Lucia 4.6 (Laboratory Imaging Ltd).
NO determination.
Production of NO was characterized by measuring the accumulation of nitrite, a NO oxidation product. Nitrite was determined in the culture medium by a diazotation assay with Griess reagent (Griess, 1879 ; Bogle et al., 1992
). Briefly, a 100 µl aliquot of each sample was mixed with an equal volume of Griess reagent (0·5% sulfanilamide, 0·05% naphthylethylenediamine and 2·5% H3PO4), and absorbance at 550 nm was determined after a 1015 min incubation at room temperature. Sodium nitrite was used as a standard.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
VV induces a decrease in mitochondrial membrane potential (m) of infected macrophages
DNA fragmentation by endonucleases is a typical but rather late sign of apoptosis. In contrast, changes in mitochondrial membrane potential, m, are considered as early events in the effector phase of apoptosis although they reveal lower specificity.
m was estimated using flow cytometry analysis after vital staining of the cells with the cationic lipophilic fluorochromes Rh 123, DiOC6(3) and TMRM. These potentiometric dyes distribute to the mitochondrial matrix as a function of the Nernst equation, thus correlating with
m (Darzynkiewicz et al., 1982
). Cells undergoing apoptosis typically reveal a reduction in incorporation of
m-sensitive dyes; cellular membrane remains intact (PI-impermeable, reflected as a PI-negative population in flow cytometry) in early phases of apoptosis, while it becomes permeable (PI-positive) during secondary necrosis in later phases. Therefore, we characterized early stages of apoptosis of VV-infected macrophages by changes of
m using DiOC6(3) (Figs 1B
and 2A
), Rh 123 and TMRM (data not shown). The decrease in
m could be observed at 6 h p.i. and became more apparent at 12 and 18 h p.i. (Fig. 1B
). Fig. 2(A)
represents the result of a dot plot analysis of cells stained with DiOC6(3) and PI. The percentage of apoptotic cells (DiOC6(3)-negative or low positive and PI-negative population) increases in a time-dependent manner in both WT VV-infected and VVLUC-infected cells, the latter being used as a control for other recombinant viruses. Similar results were observed using Rh123 or TMRM. The percentage of VV-infected macrophages determined using FITC-labelled rabbit polyclonal antibody against VV and flow cytometry (Fig. 2B
) was compared with the percentage of apoptotic cells (Fig. 2A
; both characteristics determined in a live, PI-negative population), and at individual intervals p.i. more cells were found to be infected than apoptotic. This result is in agreement with the hypothesis that VV infection of the individual cells precedes the decrease in their
m and apoptosis.
|
|
VV replication is a cascade event, completion of each step being a pre-requisite for the following one (Moss, 1990 ). In order to distinguish which VV gene products might induce apoptosis, we treated macrophages with AraC to inhibit VV DNA synthesis and, consequently, expression of VV late genes. Table 2
represents a flow cytometric analysis of the effects of AraC on macrophage apoptosis. In the uninfected cells, AraC did not affect the background, low percentage of apoptosis; however, in cells infected in the presence of AraC, apoptosis occurred in a similar percentage to untreated infected cells. Therefore, apoptosis in VV-infected macrophages is an early event in virus infection.
|
VV induces a marked decrease of the anti-apoptotic Bcl-xL and a limited increase of the pro-apoptotic Bax
Members of the Bcl-2 family regulate the process of apoptosis at the level of mitochondria, and changes in their relative levels are known to promote cell survival or induce cell death. Therefore, using Western blot analysis, we first determined the effect of VV infection on levels of the major anti-apoptotic proteins, Bcl-2 and Bcl-xL. While we did not detect any endogenous Bcl-2 protein, even in uninfected cells, Bcl-xL levels were found to be decreased at 18 h p.i. in cells infected by both WT VV or another control virus, VVLUC (Fig. 3). When performing a time-course experiment using WT VV as well as various VV recombinants, the levels of Bcl-xL were found to decrease in a time-dependent manner relative to uninfected controls (Fig. 4A
). In contrast, levels of pro-apoptotic Bax were found somewhat increased in most of the infected samples when compared with the appropriate uninfected controls (Fig. 4B
). Fig. 4(C)
shows relatively comparable levels of the house-keeping
-actin in the infected and uninfected samples. Densitometric analysis of the Western blots revealed a fourfold decrease of Bcl-xL and a 1·7-fold increase of Bax proteins at 18 h p.i. (mean of three independent experiments). Standardization of the relative densities of Bcl-xL or Bax to the relative densities of
-actin in each individual sample also revealed decreasing or increasing tendencies, respectively (not shown).
|
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Our results suggest that apoptosis of VV-infected macrophages is mediated by VV early gene products. It occurs in the absence of VV DNA synthesis and/or expression of VV late genes in IFN--pretreated macrophages or in the presence of AraC. It has been previously shown that apoptosis of HeLa cells infected with VV lacking the E3L gene was prevented on inhibition of VV DNA synthesis and late genes expression by AraC; this apoptosis was, however, due to activation of IFN-induced dsRNA-activated protein kinase (PKR) by dsRNA accumulated during expression of VV late genes (Colby et al., 1971
; Kibler et al., 1997
). In contrast, WT VV as well as the recombinant VVs used in this study encode the early genes E3L and K3L, the products of which counteract IFN-inducible dsRNA-activated PKR and 2'-5' oligoadenylate synthetase (2-5A synthetase)/RNaseL system (Chang et al., 1992
; Davies et al., 1992
; Rivas et al., 1998
). Therefore, it is not clear if these systems could be involved in the apoptosis of VV-infected macrophages described here, despite the fact that both the 2-5A synthetase/RNaseL system and PKR are able to trigger apoptosis (Lee & Esteban, 1994
; Diaz-Guerra et al., 1997
; Zhou et al., 1997
). Possibly, high endogenous levels of the dsRNA-activated 2-5A synthetase/RNaseL system or PKR in macrophages might overcome the VV-encoded early proteins E3L and K3L that should normally counteract them.
Treatment of macrophages with the RNA or protein synthesis inhibitors, AcD or CHX, respectively, induced apoptosis of uninfected macrophages. Infection by VV in the presence of these inhibitors did not further increase the percentages of apoptotic cells, possibly suggesting that an additional VV-induced apoptosis was prevented by the lack of expression of VV early genes. Also, AcD- and CHX-mediated inhibition of protein synthesis might mimic VV-mediated shut-off of host-cell protein synthesis (Kit & Dubbs, 1962 ; Shatkin, 1963
; Becker & Joklik, 1964
). Since the shut-off is mediated by VV early gene products or by VV virion proteins (Buendia et al., 1987
; Bablanian et al., 1993
), such a mechanism would be compatible with apoptosis induced in infected macrophages. VV-induced apoptosis was shown to be mediated by VV early gene products or by virion proteins in other systems as well. In VV-induced apoptosis of the immature B-lymphocyte cell line, WEHI-231, several VV-specific proteins were expressed, but no viral DNA synthesis or virus progeny were detected (Baixeras et al., 1998
). In contrast, in CHO cells, apoptosis was induced after binding of VV to the cell without any requirement for virus gene expression (Ramsey-Ewing & Moss, 1998
). Our results with heat- or UV-inactivated VV suggest that VV early gene expression is necessary for apoptosis. However, a VV deletion mutant deficient, for example, in virion-associated protein kinase (Buendia et al., 1987
) would better clarify this point.
We have previously demonstrated that VV inhibited host-cell protein synthesis in J774.G8 macrophages (Mlková & Esteban, 1994
), and in this study, inhibition of RNA and protein synthesis by AcD and CHX, respectively, mimicked the induction of apoptosis by VV. Therefore, we assumed that levels of certain proteins critical for the control of apoptosis could be affected. Specifically, we have focused on the levels of the proto-oncogenes Bcl-2, Bcl-xL and Bax, the ratios of which are considered to control the effector phase of apoptosis (Kroemer, 1997
; Korsmeyer, 1999
). We were not able to detect any endogenous levels of Bcl-2 in J774.G8 macrophages; instead, Bcl-xL was present, and infection with VV induced a decrease in its level, while levels of the pro-apoptotic homologue Bax were increased in most of the infected samples (results of three independent experiments). Induction of expression of Bax simultaneously with down-regulation of Bcl-2 in response to various apoptotic stimuli has been reported previously (Gillardon et al., 1995
). Additionally, Bcl-xL protein could be preferentially degraded on infection with VV in a way similar to glutathione depletion-induced degradation of Bcl-2 and apoptosis in cholangiocytes (Celli et al., 1998
).
We demonstrated the role Bcl-xL plays in VV-induced macrophage apoptosis by substituting for its function with expression of its homologue, Bcl-2, using a recombinant VV. Bcl-2 preserved mitochondrial membrane potential, prevented exposure of phosphatidyl serine on the cell surface and preserved normal cellular morphology despite relatively increased levels of Bax. The exact mode of action of Bcl-2 homologues still remains elusive, but most hypotheses stress their effects on mitochondrial function (Kroemer, 1997 ; Reed et al., 1998
). Since VV growth was comparable in the presence as well as in the absence of Bcl-2 expression, it seems unlikely that Bcl-2 could fundamentally change the properties of VV-expressed proteins or the VV growth cycle. It might rather be predicted that Bcl-2 could modify the metabolic consequences imposed on the host cell by VV growth. For example, Bcl-2 might prevent changes of the intracellular milieu induced by VV or could affect the extent of VV-mediated shut-off of host-cell macromolecular synthesis. These and other questions remain to be explored.
In conclusion, we have demonstrated for the first time that VV induces apoptosis of infected macrophages. We did not find any role of NO in this VV-induced apoptosis. However, VV induced a marked decrease of cellular levels of Bcl-xL simultaneously with a limited increase in levels of Bax. The importance of the loss of Bcl-xL was demonstrated by prevention of apoptosis on expression of Bcl-2, a functional homologue of Bcl-xL.
![]() |
Acknowledgments |
---|
![]() |
Footnotes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Akaike, T., Suga, M. & Maeda, H. (1998). Free radicals in viral pathogenesis: molecular mechanisms involving superoxide and NO. Proceedings of the Society for Experimental Biology and Medicine 217, 64-73.[Abstract]
Alcamí, A. & Koszinowski, U. H. (2000). Viral mechanisms of immune evasion. Molecular Medicine Today 6, 365-372.[Medline]
Bablanian, R., Scribani, S. & Esteban, M. (1993). Amplification of polyadenylated nontranslated small RNA sequences (POLADS) during superinfection correlates with the inhibition of viral and cellular protein synthesis. Cellular & Molecular Biology Research 39, 243-255.[Medline]
Baixeras, E., Cebrian, A., Albar, J. P., Salas, J., Martinez-A, C., Vinuela, E. & Revilla, Y. (1998). Vaccinia virus-induced apoptosis in immature B lymphocytes: role of cellular Bcl-2. Virus Research 58, 107-113.[Medline]
Barry, M. & McFadden, G. (1998). Apoptosis regulators from DNA viruses. Current Opinion in Immunology 10, 422-430.[Medline]
Becker, Y. & Joklik, W. K. (1964). Messenger RNA in cells infected with vaccinia virus. Proceedings of the National Academy of Sciences, USA 51, 577-585.[Medline]
Bird, P. I. (1998). Serpins and regulation of cell death. Results and Problems in Cell Differentiation 24, 63-89.[Medline]
Bogle, R. G., Baydoun, A. R., Pearson, J. D., Moncada, S. & Mann, G. E. (1992). L-Arginine transport is increased in macrophages generating nitric oxide. Biochemical Journal 284, 15-18.[Medline]
Buchmeier, N. A., Gee, S. R., Murphy, F. A. & Rawls, W. E. (1979). Abortive replication of vaccinia virus in activated rabbit macrophages. Infection and Immunity 26, 328-338.[Medline]
Buendia, B., Person-Fernandez, A., Beaud, G. & Madjar, J. (1987). Ribosomal protein phosphorylation in vivo and in vitro by vaccinia virus. European Journal of Biochemistry 162, 95-103.[Abstract]
Buller, R. M. L. & Palumbo, G. J. (1991). Poxvirus pathogenesis. Microbiological Reviews 55, 80-122.
Cacoullos, N. & Bablanian, R. (1991). Polyadenylated RNA sequences produced in vaccinia virus-infected cells under aberrant conditions inhibit protein synthesis in vitro. Virology 184, 747-751.[Medline]
Celli, A., Que, F. G., Gores, G. J. & LaRusso, N. F. (1998). Glutathione depletion is associated with decreased Bcl-2 expression and increased apoptosis in cholangiocytes. American Journal of Physiology 275, 749-757.
Chang, H. W., Watson, J. C. & Jacobs, B. L. (1992). The E3L gene of vaccinia virus encodes an inhibitor of the interferon-induced, double-stranded RNA-dependent protein kinase. Proceedings of the National Academy of Sciences, USA 89, 4825-4829.[Abstract]
Colby, C., Jurale, C. & Kates, J. R. (1971). Mechanism of synthesis of vaccinia virus double-stranded ribonucleic acid in vivo and in vitro. Journal of Virology 71, 71-76.
Corbett, J. A., Tilton, R. G., Chang, K., Hasan, K. S., Ido, Y., Wang, J. L., Sweetland, M. A., Lancaster, J. R.Jr, Williamson, J. R. & McDaniel, M. L. (1992). Aminoguanidine, a novel inhibitor of nitric oxide formation, prevents diabetic vascular dysfunction. Diabetes 41, 552-556.[Abstract]
Darzynkiewicz, Z., Traganos, F., Staiano-Coico, L., Kapuscinski, J. & Melamed, M. R. (1982). Interaction of rhodamine 123 with living cells studied by flow cytometry. Cancer Research 42, 799-806.[Abstract]
Davies, M. V., Elroy-Stein, O., Jagus, R., Moss, B. & Kaufman, R. J. (1992). The vaccinia virus K3L gene product potentiates translation by inhibiting double-stranded-RNA-activated protein kinase and phosphorylation of the alpha subunit of eukaryotic initiation factor 2. Journal of Virology 66, 1943-1950.[Abstract]
Dawson, V. L., Dawson, T. M., Uhl, G. R. & Snyder, S. H. (1993). Human immunodeficiency virus type 1 coat protein neurotoxicity mediated by nitric oxide in primary cortical cultures. Proceedings of the National Academy of Sciences, USA 90, 3256-3259.[Abstract]
Diaz-Guerra, M., Rivas, C. & Esteban, M. (1997). Activation of the IFN-inducible enzyme RNase L causes apoptosis of animal cells. Virology 236, 354-363.[Medline]
Drillien, R., Spehner, D., Bohbot, A. & Hanau, D. (2000). Vaccinia virus-related events and phenotypic changes after infection of dendritic cells derived from human monocytes. Virology 268, 471-481.[Medline]
Engelmayer, J., Larsson, M., Subklewe, M., Chahroudi, A., Cox, W. I., Steinman, R. M. & Bhardwaj, N. (1999). Vaccinia virus inhibits the maturation of human dendritic cells: a novel mechanism of immune evasion. Journal of Immunology 163, 6762-6768.
Esteban, M. & Metz, D. H. (1973). Early virus protein synthesis in vaccinia infected cells. Journal of General Virology 19, 201-216.[Medline]
Everett, H., Barry, M., Lee, S. F., Sun, X., Graham, K., Stone, J., Bleackley, R. C. & McFadden, G. (2000). M11L. A novel mitochondria-localized protein of myxoma virus that blocks apoptosis of infected leukocytes. Journal of Experimental Medicine 191, 1487-1498.
Fujimoto, I., Pan, J., Takizawa, T. & Nakanishi, Y. (2000). Virus clearance through apoptosis-dependent phagocytosis of influenza A virus-infected cells by macrophages. Journal of Virology 74, 3399-3403.
Gagliardini, V., Fernandez, P. A., Lee, R. K., Drexler, H. C., Rotello, R. J., Fishman, M. C. & Yuan, J. (1994). Prevention of vertebrate neuronal death by the crmA gene. Science 263, 826-828.[Medline]
Gil, J., Alcami, J. & Esteban, M. (1999). Induction of apoptosis by double-stranded-RNA-dependent protein kinase (PKR) involves the alpha subunit of eukaryotic translation initiation factor 2 and NF-kappaB. Molecular and Cellular Biology 19, 4653-4663.
Gillardon, F., Wickert, H. & Zimmermann, M. (1995). Up-regulation of bax and down-regulation of bcl-2 is associated with kainate-induced apoptosis in mouse brain. Neuroscience Letters 192, 85-88.[Medline]
Gonzalo, J. A., Gonzalez-Garcia, A., Kalland, T., Hedlung, G., Martinez, C. & Kroemer, G. (1994). Linomide inhibits programmed cell death of peripheral T cells in vivo. European Journal of Immunology 24, 48-52.[Medline]
Griess, P. (1879). Bemergungen zu der Abhandlung der HH. Weselsky und Benedikt Ueber einige Azoverbindungen. Chemische Berichte 12, 426-428.
Haig, D. M. (1998). Poxvirus interference with the host cytokine response. Veterinary Immunology and Immunopathology 63, 149-156.[Medline]
Harlow, E. & Lane, D. (1988). Antibodies: a Laboratory Manual, pp. 471510. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Ignatius, R., Marovich, M., Mehlhop, E., Villamide, L., Mahnke, K., Cox, W. I., Isdell, F., Frankel, S. S., Mascola, J. R., Steinman, R. M. & Pope, M. (2000). Canarypox virus-induced maturation of dendritic cells is mediated by apoptotic cell death and tumor necrosis factor alpha secretion. Journal of Virology 74, 11329-11338.
Jelinkova, A., Benda, R. & Novak, M. (1975). Electron microscopy study of the development of neurovaccinia virus in rabbit blood leucocytes cultures. Journal of Hygiene, Epidemiology, Microbiology, and Immunology 19, 321-328.
Joklik, W. K. (1962). The purification of four strains of poxviruses. Virology 18, 9-18.
Karupiah, G., Xie, Q., Buller, R. M. L., Nathan, C., Duarte, C. & MacMicking, J. D. (1993). Inhibition of viral replication by interferon-gamma-induced nitric oxide synthase. Science 261, 1445-1448.[Medline]
Kibler, K. V., Shors, T., Perkins, K. B., Zeman, C. C., Banaszak, M. P., Biesterfeldt, J., Langland, J. O. & Jacobs, B. L. (1997). Double-stranded RNA is a trigger for apoptosis in vaccinia virus-infected cells. Journal of Virology 71, 1992-2003.[Abstract]
Kit, S. & Dubbs, D. R. (1962). Biochemistry of vaccinia-infected mouse fibroblasts (strain L-M). Virology 18, 274-285.[Medline]
Korsmeyer, S. J. (1999). BCL-2 gene family and the regulation of programmed cell death. Cancer Research 59, 1693-1700.
Kroemer, G. (1997). The proto-oncogene Bcl-2 and its role in regulating apoptosis. Nature Medicine 3, 614-620.[Medline]
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685.[Medline]
Lee, S. B. (1994). Characterization of interferon-induced double-stranded RNA-activated protein kinase. Thesis, School of Graduate Studies, State University of New York, Health Science Center at Brooklyn.
Lee, S. B. & Esteban, M. (1994). The interferon-induced double-stranded RNA-activated protein kinase induces apoptosis. Virology 199, 491-496.[Medline]
Macen, J., Takahashi, A., Moon, K. B., Nathaniel, R., Turner, P. C. & Moyer, R. W. (1998). Activation of caspases in pig kidney cells infected with wild-type and CrmA/SPI-2 mutants of cowpox and rabbitpox viruses. Journal of Virology 72, 3524-3533.
Mackett, M., Smith, G. L. & Moss, B. (1982). Vaccinia virus: a selectable eukaryotic cloning and expression vector. Proceedings of the National Academy of Sciences, USA 79, 7415-7419.[Abstract]
McLaren, C., Cheng, H., Spicer, D. L. & Tompkins, W. A. (1976). Lymphocyte and macrophage responses after vaccinia virus infections. Infection and Immunity 14, 1014-1021.[Medline]
Mlková, Z. (1995). Macrophage antiviral activity: role of IFN
and nitric oxide in the inhibition of vaccinia virus growth in macrophages. Thesis, School of Graduate Studies, State University of New York, Health Science Center at Brooklyn.
Mlková, Z. & Esteban, M. (1994). Interferon-gamma severely inhibits DNA synthesis of vaccinia virus in a macrophage cell line. Virology 198, 731-735.[Medline]
Mikami, S., Kawashima, S., Kanazawa, K., Hirata, K., Katayama, Y., Hotta, H., Hayashi, Y., Ito, H. & Yokoyama, M. (1996). Expression of nitric oxide synthase in a murine model of viral myocarditis induced by coxsackievirus B3. Biochemical and Biophysical Research Communications 220, 983-989.[Medline]
Moss, B. (1990). Regulation of vaccinia virus transcription. Annual Review of Biochemistry 59, 661-688.[Medline]
Nash, P., Barrett, J., Cao, J. X., Hota-Mitchell, S., Lalani, A. S., Everett, H., Xu, X. M., Robichaud, J., Hnatiuk, S., Ainslie, C., Seet, B. T. & McFadden, G. (1999). Immunomodulation by viruses: the myxoma virus story. Immunological Reviews 168, 103-120.[Medline]
Nathan, C. (1992). Nitric oxide as a secretory product of mammalian cells. FASEB Journal 6, 3051-3064.
Nathan, C. F. & Hibs, J. B. (1991). Role of nitric oxide synthesis in macrophage antimicrobial activity. Current Opinion in Immunology 3, 65-70.[Medline]
Natuk, R. J. & Holowczak, J. A. (1985). Vaccinia virus proteins on the plasma membrane of infected cells. III. Infection of peritoneal macrophages. Virology 147, 354-372.[Medline]
Oltvai, Z. N., Milliman, C. L. & Korsmeyer, S. J. (1993). Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell death. Cell 74, 609-619.[Medline]
Paez, E. & Esteban, M. (1984). Resistance of vaccinia virus to interferon is related to an interference phenomenon between the virus and the interferon system. Virology 134, 12-28.[Medline]
Ramsey-Ewing, A. & Moss, B. (1998). Apoptosis induced by a postbinding step of vaccinia virus entry into Chinese hamster ovary cells. Virology 242, 138-149.[Medline]
Reed, J. C., Jurgensmeier, J. M. & Matsuyama, S. (1998). Bcl-2 family proteins and mitochondria. Biochimica et Biophysica Acta 1366, 127-137.[Medline]
Reiss, C. S. & Komatsu, T. J. (1998). Does nitric oxide play a critical role in viral infections? Journal of Virology 72, 4547-4551.
Rivas, C., Gil, J., Melkova, Z., Esteban, M. & Diaz-Guerra, M. (1998). Vaccinia virus E3L protein is an inhibitor of the interferon (IFN)-induced 2-5A synthetase enzyme. Virology 243, 406-414.[Medline]
Rodriguez, J. F., Rodriguez, D., Rodriguez, J. R., McGowan, E. & Esteban, M. (1989). Expression of the firefly luciferase gene in vaccinia virus: a highly sensitive gene marker to follow virus dissemination in tissues of infected animals. Proceedings of the National Academy of Sciences, USA 85, 1667-1671.
Rodriguez, J. R., Rodriguez, D. & Esteban, M. (1991). Interferon treatment inhibits early events in vaccinia virus gene expression in infected mice. Virology 185, 929-933.[Medline]
Ronen, D., Schwartz, D., Teitz, Y., Goldfinger, N. & Rotter, V. (1996). Induction of HL-60 cells to undergo apoptosis is determined by high levels of wild-type p53 protein whereas differentiation of the cells is mediated by lower p53 levels. Cell Growth & Differentiation 7, 21-30.[Abstract]
Roulston, A., Marcellus, R. C. & Branton, P. E. (1999). Viruses and apoptosis. Annual Review of Microbiology 53, 577-628.[Medline]
Sarih, M., Souvannavong, V. & Adam, A. (1993). Nitric oxide synthase induces macrophage death by apoptosis. Biochemical and Biophysical Research Communications 191, 503-508.[Medline]
Shatkin, A. J. (1963). Actinomycin D and vaccinia virus infection of HeLa cells. Nature 199, 357-358.[Medline]
Smith, G. L., Symons, J. A., Khanna, A., Vanderplasschen, A. & Alcami, A. (1997). Vaccinia virus immune evasion. Immunological Reviews 159, 137-154.[Medline]
Smith, G. L., Symons, J. A. & Alcami, A. (1999). Immune modulation by proteins secreted from cells infected by vaccinia virus. Archives of Virology Supplement 15, 111-129.[Medline]
Stuehr, D. J. & Nathan, C. F. (1989). Nitric oxide: a macrophage product responsible for cytostasis and respiratory inhibition in tumor target cells. Journal of Experimental Medicine 169, 1543-1545.[Abstract]
Su, M. J. & Bablanian, R. (1990). Polyadenylated RNA sequences from vaccinia virus-infected cells selectively inhibit translation in a cell-free system: structural properties and mechanism of inhibition. Virology 179, 679-693.[Medline]
Teodoro, J. G. & Branton, P. E. (1997). Regulation of apoptosis by viral gene products. Journal of Virology 71, 1739-1746.
Timiryasova, T. M., Li, J., Chen, B., Chong, D., Langridge, W. H., Gridley, D. S. & Fodor, I. (1999). Antitumor effect of vaccinia virus in glioma model. Oncology Research 11, 133-144.[Medline]
van den Broek, M. F., Muller, U., Huang, S., Aguet, M. & Zinkernagel, R. M. (1995). Antiviral defense in mice lacking both alpha/beta and gamma interferon receptors. Journal of Virology 69, 4792-4796.[Abstract]
Zheng, Z. M., Schöfer, M. K. H., Weihe, E., Sheng, H., Corisdeo, S., Fu, Z. F., Koprowski, H. & Dietzschold, B. (1993). In vivo expression of inducible nitric oxide synthase in experimentally induced neurologic diseases. Proceedings of the National Academy of Sciences, USA 90, 3024-3027.[Abstract]
Zhou, A., Paranjape, J., Brown, T. L., Nie, H., Naik, S., Dong, B., Chang, A., Trapp, B., Fairchild, R., Colmenares, C. & Silverman, R. H. (1997). Interferon action and apoptosis are defective in mice devoid of 2',5'-oligoadenylate-dependent RNase L. EMBO Journal 16, 6355-6363.
Received 22 February 2002;
accepted 3 July 2002.