Gamma interferon-induced, nitric oxide-mediated inhibition of vaccinia virus replication.

Gamma interferon (IFN-gamma)-induced nitric oxide synthase (iNOS) and nitric oxide (NO) production in the murine macrophage-like RAW 264.7 cells were previously shown to inhibit the replication of the poxviruses vaccinia virus (VV) and ectromelia virus and herpes simplex virus type 1. In the current study, we performed biochemical analyses to determine the stage in the viral life cycle blocked by IFN-gamma-induced NO. Antibodies specific for temporally expressed viral proteins, a VV-specific DNA probe, and transmission electron microscopy were used to show that the cytokine-induced NO inhibited late protein synthesis, DNA replication, and virus particle formation but not expression of the early proteins analyzed. Essentially similar results were obtained with hydroxyurea and cytosine arabinoside, inhibitors of DNA replication. Enzymatically active iNOS was detected in the lysates of IFN-gamma-treated but not in untreated RAW 264.7 cells. The IFN-gamma-treated RAW 264.7 cells which express iNOS not only were resistant to productive infection but also efficiently blocked the replication of VV in infected bystander cells of epithelial origin. This inhibition was arginine dependent, correlated with nitric production in cultures, and was reversible by the NOS inhibitor N omega-monomethyl-L-arginine.     

Induction of nitric oxide synthase during Japanese encephalitis virus infection: evidence of protective role.

The ability of Japanese encephalitis virus (JEV) and JEV-induced macrophage-derived factor (MDF) to modulate nitric oxide synthase (NOS) activity in brain and tumor necrosis factor-alpha (TNF-alpha) and the possible antiviral role of NOS during JEV infection were investigated. NOS activity and particularly that of the inducible form of NOS (iNOS) was significantly enhanced in JEV or JEV-induced MDF-treated mice. Following JEV infection, total NOS activity in brain was gradually increased from Day 3 and reached a peak on Day 6. MDF-induced NOS activity and iNOS activity were dose dependent and maximum activity was observed at 1 h after treatment. The response was sensitive to anti-MDF antibody treatment and N(G)-monomethyl-L-arginine (L-NMMA), an inhibitor of NOS. Pretreatment of JEV-infected mice with L-NMMA increased the mortality as evident from reduced mean survival time (MST, 11.8 days) compared to placebo treated JEV-infected mice (MST, 17 days). The enhanced level of TNF-alpha observed in the early phase of JEV infection correlated well with the enhanced activity of iNOS. These observations thus provide evidence of the protective role of iNOS during JEV infection and indicate that iNOS may be a key mediator in host innate immune response to infection.     

Inhibition of vesicular stomatitis virus infection by nitric oxide.

Inhibitory effects of nitric oxide (NO) on vesicular stomatitis virus (VSV) infection were investigated by using a VSV-susceptible mouse neuroblastoma cell line, NB41A3. Productive VSV infection of NB41A3 cells was significantly inhibited by an organic NO donor, S-nitro-N-acetylpenicillamine (SNAP), while the control compound N-acetylpenicillamine (NAP) had no effect. Survival rate of VSV-infected cells was greatly increased by the treatment with SNAP, while the NAP treatment did not have any effect. Adding SNAP 30 min prior to infection resulted in complete inhibition of viral production when a low multiplicity of infection (MOI) was used. Substantial inhibition of viral production was also obtained with treating cells 6 h earlier before infection with a higher MOI. Activating the neuronal NO synthase by treating cells with N-methyl-D-aspartate (NMDA) led to significant inhibition of viral production by cells infected at the three doses of virus tested (MOIs of 0.1, 1, and 5). The inhibitory effect of NMDA on viral infection was totally blocked by the NO synthase inhibitor N-methyl-L-arginine. However, adding hemoglobin, a strong NO-binding protein and thus an inactivator of NO activity, did not reverse the NMDA-induced inhibition of viral production, suggesting that NO might exert its antiviral effects inside the NO-producing cells. Collectively, these data support the anti-VSV effects of NO, which might be one of the important factors of natural immunity in controlling the initial stages of VSV infection in the central nervous system.     

Processing of capsid protein by cathepsin L plays a crucial role in replication of Japanese encephalitis virus in neural and macrophage cells

The flavivirus capsid protein not only is a component of nucleocapsids but also plays a role in viral replication. In this study, we found a small capsid protein in cells infected with Japanese encephalitis virus (JEV) but not in the viral particles. The small capsid protein was shown to be generated by processing with host cysteine protease cathepsin L. An in vitro cleavage assay revealed that cathepsin L cleaves the capsid protein between amino acid residues Lys(18) and Arg(19), which are well conserved among the mosquito-borne flaviviruses. A mutant JEV resistant to the cleavage of the capsid protein by cathepsin L was generated from an infectious cDNA clone of JEV by introducing a substitution in the cleavage site. The mutant JEV exhibited growth kinetics similar to those of the wild-type JEV in monkey (Vero), mosquito (C6/36), and porcine (PK15) cell lines, whereas replication of the mutant JEV in mouse macrophage (RAW264.7) and neuroblastoma (N18) cells was impaired. Furthermore, the neurovirulence and neuroinvasiveness of the mutant JEV to mice were lower than those of the wild-type JEV. These results suggest that the processing of the JEV capsid protein by cathepsin L plays a crucial role in the replication of JEV in neural and macrophage cells, which leads to the pathogenesis of JEV infection.     

Role of nitric oxide synthase type 2 in acute infection with murine cytomegalovirus

Whether or not NO plays a critical role in murine CMV (MCMV) infection has yet to be elucidated. In this study, we examined the role of NO in acute infection with MCMV using NO synthase type 2 (NOS2)-deficient mice. NOS2(-/-) mice were more susceptible to lethal infection with MCMV than NOS2(+/+) mice and generated a much higher peak virus titer in the salivary gland after acute infection. A moderate increase in the MCMV titer was also observed in other organs of NOS2(-/-) mice such as the spleen, lung, and liver. The immune responses to MCMV infection including NK cell cytotoxicity and CTL response in NOS2(-/-) mice were comparable with those of NOS2(+/+) mice. Moreover, the ability to produce IFN-gamma is not impaired in NOS2(-/-) mice after MCMV infection. The peritoneal macrophages from NOS2(-/-) mice, however, exhibited a lower antiviral activity than those from NOS2(+/+) mice, resulting in an enhanced viral replication in macrophages themselves. Treatment of these cells from NOS2(+/+) mice with a selective NOS2 inhibitor decreased the antiviral activity to a level below that obtained with NOS2(-/-) mice. In addition, the absence of NOS2 and NOS2-mediated antiviral activity of macrophages resulted in not only an enhanced MCMV replication and a high mortality but also a consequent risk of the latency. It was thus concluded that the NOS2-mediated antiviral activity of macrophages via NO plays a protective role against MCMV infection at an early and late stage of the infection.     

Inhibitory effect of nitric oxide on the replication of a murine retrovirus in vitro and in vivo.

Nitric oxide (NO) exerts microbicidal effects on a broad spectrum of pathogens, including viruses, but its antiretrovirus properties have not yet been described. The purpose of this study was to determine whether NO inhibits murine Friend leukemia virus (FV) replication in vitro and to what extent NO may play a role in defenses against FV infection in mice. Three NO-generating compounds were studied: 3-morpholino-sydononimine (SIN-1), sodium nitroprusside (SNP), and S-nitroso-N-acetylpenicillamine (SNAP). The effects of these three compounds were compared with those of their controls (SIN-1C, potassium ferricyanide, and N-acetylpenicillamine, respectively), which do not generate NO and with that of sodium nitrite (NaNO2). SIN-1, SNP, and SNAP inhibited FV replication in dunni cells in a concentration-dependent manner. In contrast, no significant inhibitory effect was observed with the three controls or NaNO2. Furthermore, the addition of superoxide dismutase did not alter the inhibitory effect of SIN-1, which is also known to generate superoxide anions. No dunni cell toxicity was observed in the range of concentrations tested. We also assessed the effect of NO produced by activated macrophages on FV replication. Macrophages activated by gamma interferon and lipopolysaccharide inhibited FV replication in a concentration-dependent manner. This inhibition was due in part to NO production, since it was reversed by NG-monomethyl L-arginine, a competitive inhibitor of NO synthase. In vivo administration of NG-nitro-L-arginine methyl ester, a competitive inhibitor of NO synthase, significantly increased the viral load in spleen cells of FV-infected mice. These results suggested that NO may play a role in defenses against the murine Friend leukemia retrovirus.     

Japanese Encephalitis Virus Disrupts Cell-Cell Junctions and Affects the Epithelial Permeability Barrier Functions

Japanese encephalitis virus (JEV) is a neurotropic flavivirus, which causes viral encephalitis leading to death in about 20–30% of severely-infected people. Although JEV is known to be a neurotropic virus its replication in non-neuronal cells in peripheral tissues is likely to play a key role in viral dissemination and pathogenesis. We have investigated the effect of JEV infection on cellular junctions in a number of non-neuronal cells. We show that JEV affects the permeability barrier functions in polarized epithelial cells at later stages of infection. The levels of some of the tight and adherens junction proteins were reduced in epithelial and endothelial cells and also in hepatocytes. Despite the induction of antiviral response, barrier disruption was not mediated by secreted factors from the infected cells. Localization of tight junction protein claudin-1 was severely perturbed in JEV-infected cells and claudin-1 partially colocalized with JEV in intracellular compartments and targeted for lysosomal degradation. Expression of JEV-capsid alone significantly affected the permeability barrier functions in these cells. Our results suggest that JEV infection modulates cellular junctions in non-neuronal cells and compromises the permeability barrier of epithelial and endothelial cells which may play a role in viral dissemination in peripheral tissues.     

Inhibition of nitric oxide synthesis increases mortality in Sindbis virus encephalitis.

Sindbis virus (SV) is an alphavirus that causes acute encephalomyelitis in mice. The outcome is determined by the strain of virus and by the age and genetic background of the host. The mortality rates after infection with NSV, a neurovirulent strain of SV, were as follows v: 81% (17 of 21) in BALB/cJ mice; 20% (4 of 20) in BALB/cByJ mice (P < 0.001); 100% in A/J, C57BL/6J, SJL, and DBA mice; and 79% (11 of 14) in immunodeficient scid/CB17 mice. Treatment with Nomega-nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthetase (NOS) inhibitor, increased mortality to 100% (P < 0.05) in NSV-infected BALB/cJ mice, to 95% (P < 0.001) in BALB/cByJ mice, and to 100% in scid/CB17 mice. BALB/cJ and BALB/cByJ mice had similar levels of inducible NOS mRNA in their brains, which were not affected by L-NAME or NSV infection. Brain NOS activity was similar in BALB/cJ and BALB/cByJ mice before and after infection and was markedly inhibited by L-NAME. NSV replication in the brains of BALB/cJ mice, BALB/cByJ mice, and mice treated with L-NAME was similar. Treatment of N18 neuroblastoma cells with NO donors S-nitroso-N-acetylpenicillamine or sodium nitroprusside in vitro before infection increased cell viability at 42 to 48 h compared with untreated NSV-infected N18 cells with little effect on virus replication. These data suggest that NO protects mice from fatal encephalitis by a mechanism that does not directly involve the immune response or inhibition of virus growth but rather may enhance survival of the infected neuron until the immune response can control virus replication.     

Inhibition of protein synthesis by nitric oxide correlates with cytostatic activity: nitric oxide induces phosphorylation of initiation factor eIF-2 alpha.

BACKGROUND: Nitric oxide (NO) is cytostatic for proliferating cells, inhibits microbial growth, and down-regulates the synthesis of specific proteins. Studies were undertaken to determine the mechanism by which NO inhibits total protein synthesis and whether the inhibition correlates with established cytostatic activities of NO. MATERIALS AND METHODS: In in vitro experiments, various cell types were exposed to NO using either donors or expression of inducible NO synthase (iNOS). The capacity of NO to suppress total protein synthesis, measured by incorporation of 35S-methionine into protein, was correlated with the capacity of NO to suppress cell proliferation, viral replication, or iNOS expression. Phosphorylation of eIF-2 alpha was examined as a possible mechanism for the suppressed protein synthesis by NO. RESULTS: Both NO donors and expression of the iNOS suppressed total protein synthesis in L929 cells and A2008 human ovarian tumor cells in parallel with decreased cell proliferation. Suppressed protein synthesis was also shown to correlate with decreased vaccinia virus proliferation in murine peritoneal macrophages in an iNOS-dependent manner. Furthermore, iNOS expression in pancreatic islets or RAW264.7 cells almost completely inhibited total protein synthesis, suggesting that nonspecific inhibition of protein synthesis may be the mechanism by which NO inhibited the synthesis of specific proteins such as insulin or iNOS itself. This possibility was confirmed in RAW264.7 cells where the inhibition of total protein synthesis correlated with the decreased iNOS protein. The decrease in protein levels occurred without changes in iNOS mRNA levels, implicating an inhibition of translation. Mechanistic studies revealed that iNOS expression in RAW264.7 cells resulted in the phosphorylation of eIF-2 alpha and inhibition of the 80S ribosomal complex formation. CONCLUSIONS: These results suggest that NO suppresses protein synthesis by stimulating the phosphorylation of eIF-2 alpha. Furthermore, our observations indicate that nonspecific inhibition of protein synthesis may be a generalized response of cells exposed to high levels of NO and that inhibition of protein synthesis may contribute to many of the described cytostatic actions of NO.     

The cellular antiviral protein viperin is attenuated by proteasome-mediated protein degradation in Japanese encephalitis virus-infected cells

Viperin is identified as an antiviral protein induced by interferon (IFN), viral infections, and pathogen-associated molecules. In this study, we found that viperin is highly induced at the RNA level by Japanese encephalitis virus (JEV) and Sindbis virus (SIN) and that viperin protein is degraded in JEV-infected cells through a proteasome-dependent mechanism. Promoter analysis revealed that SIN induces viperin expression in an IFN-dependent manner but that JEV by itself activates the viperin promoter through IFN regulatory factor-3 and AP-1. The overexpression of viperin significantly decreased the production of SIN, but not of JEV, whereas the proteasome inhibitor MG132 sustained the protein level and antiviral effect of viperin in JEV-infected cells. Knockdown of viperin expression by RNA interference also enhanced the replication of SIN, but not that of JEV. Our results suggest that even though viperin gene expression is highly induced by JEV, it is negatively regulated at the protein level to counteract its antiviral effect. In contrast, SIN induces viperin through the action of IFN, and viperin exhibits potent antiviral activity against SIN.     

Antioxidant enzymes suppress nitric oxide production through the inhibition of NF-kappa B activation: role of H(2)O(2) and nitric oxide in inducible nitric oxide synthase expression in macrophages.

Reactive molecules O(-)(2), H(2)O(2), and nitrogen monoxide (NO) are produced from macrophages following exposure to lipopolysaccharide (LPS) and involved in cellular signaling for gene expression. Experiments were carried out to determine whether these molecules regulate inducible nitric oxide synthase (iNOS) gene expression in RAW264.7 macrophages exposed to LPS. NO production was inhibited by the antioxidative enzymes catalase, horseradish peroxidase, and myeloperoxidase but not by superoxide dismutase (SOD). In contrast, the NO-producing activity of LPS-stimulated RAW264.7 cells was enhanced by the NO scavengers hemoglobin (Hb) and myoglobin. The antioxidant enzymes decreased levels of iNOS mRNA and protein in LPS-stimulated RAW264.7 cells, whereas the NOS inhibitor N(G)-monomethyl-L-arginine as well as Hb increased the level of iNOS protein but not mRNA, indicating that NO inhibits iNOS protein expression. NF-kappa B was activated in LPS-stimulated RAW264.7 cells and the activation was significantly inhibited by antioxidant enzymes, but not by Hb. Similar results were obtained using LPS-stimulated rodent peritoneal macrophages. Extracellular O(-)(2) generation by LPS-stimulated macrophages was suppressed by SOD, but not by antioxidative enzymes, while accumulation of intracellular reactive oxygen species was inhibited by antioxidative enzymes, but not by SOD. Exogenous H(2)O(2) induced NF-kappa B activation in macrophages, which was inhibited by catalase and pyrroline dithiocarbamate (PDTC). H(2)O(2) enhanced iNOS expression and NO production in peritoneal macrophages when added with interferon-gamma, and the effect of H(2)O(2) was inhibited by catalase and PDTC. These findings suggest that H(2)O(2) production from LPS-stimulated macrophages participates in the upregulation of iNOS expression via NF-kappa B activation and that NO is a negative feedback inhibitor of iNOS protein expression.     

Effect of irradiation on cytokine secretion and nitric oxide production by inflammatory macrophages

This study explored the effects of low-dose and high-dose irradiation on inflammatory macrophage cells, specifically inflammatory cytokine secretion and nitric oxide (NO) production after irradiation. To elucidate the effect of irradiation on active and inactive macrophages, we exposed LPS-treated or untreated murine monocyte/macrophage RAW 264.7 cell lines to low-dose to high-dose radiation (0.01–10 Gy). We analyzed the effects of irradiation on RAW 264.7 cell proliferation by MTT assays and analyzed cytokine secretion and NO production related to inflammation by ELISA assays. Low-to-high doses of radiation did not significantly affect the proliferation of LPS-treated or untreated RAW 264.7 cells. Pro-inflammatory cytokine IL-1ß was generally increased in RAW 264.7 cells at 3 days after radiation. Especially, IL-1ß was significantly increased in only high dose-irradiation (2 and 10 Gy irradiation) groups in LPS-untreated RAW 264.7 cells but increased in both low and high dose-irradiation groups (0.01–10 Gy) in LPS-treated RAW 264.7 cells at 3 days after irradiation. Whereas, the expression of IL-1ß was prolonged in high-dose irradiation group at 5 days after irradiation. The production of anti-inflammatory cytokine IL-10 did not change significantly at 3 days after radiation but was significantly reduced at 5 days after 10 Gy radiation. The effect of irradiation on the secretion of IL-1ß and IL-10 was not significantly different between RAW 264.7 cells treated or not treated with LPS. The effect of irradiation on NO secretion by RAW 264.7 cells showed a specific pattern. NO was produced after low-dose irradiation but reduced in a high-dose irradiation group at 3 days after irradiation. However, NO production was not changed after low-dose irradiation and reduced at 5 days after high-dose irradiation. These results showed that irradiation affected the inflammatory system and regulated NO production in both activated and inactivated macrophages through different regulation mechanisms, depending on irradiation dose.     

Effect of nitric oxide on gamma-ray-induced micronucleus frequency in RAW264.7 cells

The effect of low-dose nitric oxide (NO) on gamma-ray-induced micronucleus (MN) frequency was investigated in RAW264.7 cells. Treatment of RAW264.7 cells with 0.25 mM sodium nitroprusside (SNP), a chemical NO donor, reduced the frequency of micronuclei induced by 5 Gy gamma rays by 43 to 45% between 3 and 12 h post-treatment. This effect was blocked by carboxy-PTIO, suggesting that NO may play a role in the reduction of radiation-induced MN frequency. To examine possible mechanisms underlying this effect, we first looked at changes in the antioxidant system after SNP treatment. A significant increase in intracellular glutathione (GSH) was seen in SNP-treated cells between 3 and 12 h post-treatment. Depletion of GSH with buthionine sulfoximine (BSO) increased the gamma-ray-induced increase in MN frequency. Detailed studies using various inducers of intracellular GSH suggested that GSH induction has a partial role in the reducing effect of NO on the gamma-ray-induced MN frequency. Next, the effect of NO on DNA repair and replication systems was examined. Wortmannin, an inhibitor of DNA-dependent protein kinase (DNA-PK), dose-dependently inhibited the reducing effect of NO, while caffeine, an inhibitor of ATM kinase and ATR kinase, did not. DNA-PK activity was increased by NO treatment. Etoposide, a topoisomerase II inhibitor, dose-dependently blocked the effect of NO in reducing the gamma-ray-induced MN frequency. These results suggest that the mechanisms of the effect of NO on the gamma-ray-induced MN frequency include elevation of GSH and up-regulation of DNA-PK activity for repairing double-strand breaks. NO may act as a signal for repair systems, e.g. for nonhomologous recombination and for the replication system in S phase, to reduce the MN frequency.     

Generation of nitric oxide by the inducible nitric oxide synthase protects gamma delta T cells from Mycobacterium tuberculosis-induced apoptosis.

Gamma delta T cells are early recruited into mycobacterial lesions. Upon microbial Ag recognition, gamma delta cells secrete cytokines and chemokines and undergo apoptosis via CD95/CD95 ligand (CD95L) interaction, possibly influencing the outcome of infection and the characteristics of the disease. In this paper we show that activated phagocytes acquire, upon challenge with Mycobacterium tuberculosis, the ability to inhibit M. tuberculosis-induced gamma delta cell apoptosis. Apoptosis protection was due to NO because it correlated with NO synthase (NOS)-2 induction and activity in scavenger cells and was abrogated by NOS inhibitors. Furthermore, the NO donor S-nitrosoacetylpenicillamine mimicked the effect of enzyme induction. NO left unaffected the expression of CD95 and CD95L, suggesting interference with an event ensuing CD95/CD95L interaction. NO was found to interfere with the intracellular accumulation of ceramide and the activation of caspases, which were involved in gamma delta T cells apoptosis after M. tuberculosis recognition. We propose that NO generated by infected macrophages determines the life span and therefore the function of lymphocytes at the infection site, thus linking innate and adaptive immunity.     

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) interaction with 3' ends of Japanese encephalitis virus RNA and colocalization with the viral NS5 protein

Replication of the Japanese encephalitis virus (JEV) genome depends on host factors for successfully completing their life cycles; to do this, host factors have been recruited and/or relocated to the site of viral replication. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a cellular metabolic protein, was found to colocalize with viral RNA-dependent RNA polymerase (NS5) in JEV-infected cells. Subcellular fractionation further indicated that GAPDH remained relatively constant in the cytosol, while increasing at 12 to 24 hours postinfection (hpi) and decreasing at 36 hpi in the nuclear fraction of infected cells. In contrast, the redistribution patterns of GAPDH were not observed in the uninfected cells. Co-immunoprecipitation of GAPDH and JEV NS5 protein revealed no direct protein-protein interaction; instead, GAPDH binds to the 3'' termini of plus- and minus-strand RNAs of JEV by electrophoretic mobility shift assays. Accordingly, GAPDH binds to the minus strand more efficiently than to the plus strand of JEV RNAs. This study highlights the findings that infection of JEV changes subcellular localization of GAPDH suggesting that this metabolic enzyme may play a role in JEV replication.     

Osteopontin is induced by nitric oxide in RAW 264.7 cells

Nitric oxide (NO) produced by macrophages is thought to contribute to various pathological conditions. Osteopontin (OPN) is a phosphorylated glycoprotein produced principally by macrophages. OPN inhibits inducible nitric oxide synthase (iNOS), which generates large amounts of NO production. However, the relationship between NO and endogenous OPN in activated macrophages has not yet been elucidated. We therefore examined expression of endogenous iNOS and OPN in a murine macrophage cell line, RAW 264.7 cells, by treating the cells with lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma). Treatment of cells with LPS and IFN-gamma resulted in an increase of iNOS mRNA to maximum at 12 h after stimulation. In contrast, OPN mRNA was induced more slowly than iNOS mRNA. Induction of both iNOS and OPN mRNA in RAW 264.7 cells was markedly suppressed by addition of the specific iNOS inhibitor S-2-aminoethyl isothiourea dihydrobromide. The NOS inhibitor NG-methyl-L-arginine also suppressed induction of OPN mRNA but hardly affected iNOS mRNA expression. The NO-releasing agent spermine-NONOate but not peroxynitrite enhanced induction of OPN mRNA. These results suggest that NO directly up-regulates the endogenous OPN in macrophages stimulated with LPS and IFN-gamma. This up-regulation of endogenous OPN may represent a negative feedback system acting to reduce iNOS expression.     

A recombinant vaccinia virus encoding inducible nitric oxide synthase is attenuated in vivo.

To investigate the role of nitric oxide during vaccinia virus (VV) infection of mice, a recombinant VV encoding the inducible nitric oxide synthase (iNOS) gene (VV-HA-iNOS) was constructed. Following infection of immunocompromised or immunocompetent mice, the virus was highly attenuated compared with a control recombinant VV. Athymic and sublethally irradiated mice survived infection with 10(7) PFU of VV-HA-iNOS, a dose that resulted in uniform mortality in mice infected with the control recombinant VV. Attenuated virus growth was evident as early as 24 h following infection, suggesting that NO had direct antiviral activity. We have previously shown that treatment of mice with the inhibitor of NO production N(G)-methyl-L-arginine did not influence the course of VV infection in mice. The present study has indicated that NO can potentially exert an antiviral effect during murine VV infection. We propose that during VV infection, nitric oxide production contributes to the control of virus growth, but that in its absence, other antiviral mechanisms are sufficient to mediate fully effective virus clearance.     

Differential response of three cell types to dual stress of nitric oxide and radiation

The perception of toxicity to nitric oxide (NO) and irradiation (IR) by three different cell types has been studied. The three cell types are the macrophage like RAW264.7 cells, EL4 lymphoma cells, and splenocytes, which represent the different components of a tumor. These three cell types respond differently to NO donors (SNP and SNAP) and radiation treatment. The macrophages were found to be most radio-resistant and insensitive to NO donors. The innate resistance of the macrophages was not due to its antioxidant defense system since there was no significant activation of the enzymes (superoxide dismutases, catalase, and glutathione peroxidase) in RAW264.7 cells after NO donor and irradiation. But the cell cycle arrest of the three cell types was different from each other. The EL4 cells were found to arrest in the G2/M phase while the macrophages were found arrested in the G1 phase of the cell cycle. Such specific killing of the tumor cell in response to NO donor while sparing the macrophages can be of immense importance to radiotherapy.