In the present study, we verified that NO possesses potent mutagenic activity for SeV, which is consistent with our previous in vivo experiment showing that GFP-SeV propagated in iNOS-deficient mice had a much lower level of mutation frequency than GFP-SeV grown in wild-type mice (4
). No appreciable antiviral effect of NO was observed on influenza virus and SeV replicated in cells in culture. This finding again supports our earlier data obtained with mouse models of influenza virus- and SeV-induced pneumonia (4
). Furthermore, it is of great importance that our present work provides the first demonstration that 8-nitroguanosine, which is produced endogenously by NO or its reactive derivatives, as evidenced here and in our earlier report (8
), induced mutagenesis of GFP-SeV in cultured cells.
The mutation frequency of GFP-SeV increased almost 10-fold during multicycle replication of GFP-SeV in iNOS-SW480 cells compared with the GFP-SeV mutation frequency in l
-NMMA-treated iNOS-SW480 cells and in parent SW480 cells that had no appreciable iNOS expression (Fig. ). A similar magnitude of increase in NO-dependent viral mutation was observed with GFP-SeV replicated in vivo in mouse lungs, which was reported previously (4
). The mutation rates of GFP and F protein genes were found to be similar (Table ), which suggests that mutation of viral genes other than the GFP and F genes may also be accelerated by NO to the same extent during viral replication. This NO-induced viral mutagenesis may explain the heterogeneity and increased repertories of variants from which a particular genotype can evolve rapidly under selective pressure. Therefore, GFP-SeV mutation induced by NO is considered biologically relevant and may have important implications for viral pathogenesis and evolution, particularly when a virus is replicating in vivo in the presence of NO.
In our recent work with influenza virus- and SeV-induced pneumonia in mice, formation of 8-nitroguanosine was found to be localized mainly in bronchial and bronchiolar epithelial cells of the lung (8
), where viral replication primarily occurs. The same study found an appreciable amount of 8-nitroguanosine in the total RNA isolated from iNOS-expressing cells in culture. It is therefore highly plausible that NO-dependent viral mutagenesis was brought about at least in part by NO-generated 8-nitroguanosine, which is in turn incorporated into the viral genome during replication and thus accelerates viral mutation (Fig. ).
FIG. 7. Schematic drawing of hypothetical mechanisms for NO-induced viral mutagenesis proposed by the present work. NO may accelerate viral mutation via formation of 8-nitroguanosine (8-nitroGuo), which may be a substantial contributor to erroneous RNA replication (more ...)
Peroxynitrite formed via superoxide and NO generation during infections possesses the potential for potent nitrating and oxidizing effects on many biomolecules, including nucleic acids (14
). Peroxynitrite has mutagenic effects on prokaryotic DNA, possibly via nitration of guanine residues of DNA (35
). Wogan's group documented NO-induced mutation of an endogenous hypoxanthine-guanine phosphoribosyltransferase (hprt
) gene in murine macrophages expressing iNOS (60
). The same group showed that mutagenicity was enhanced by NO overproduction in vivo, as evidenced by mutation of an exogenously expressed lacZ
-containing pUR288 transgenic mice (26
). Also important, Ohshima's group reported that p53 was inactivated by peroxynitrite, which may indirectly increase genetic mutation related to oxidative damage of DNA (15
). Therefore, excess production of NO by iNOS induced by proinflammatory cytokines, possibly through reactive nitrogen intermediates, may cause nucleic acid modifications and thus mutagenesis in various pathogens as well as hosts. This process may occur during infections in biological systems as a result of host defense.
In addition, oxidative stress caused by NO and 8-nitroguanosine may have a great impact in terms of mutagenic potential (2
). Our other studies have revealed that 8-nitroguanosine has strong redox activity, which stimulates superoxide generation from NADPH-cytochrome P450 reductase and various isoforms of NOS (8
). It has been known for a long time that many naturally occurring mutagens and carcinogens may act as generators of free radicals (10
). Moreover, oxygen radicals and reactive oxygen species, as endogenous initiators of DNA damage and mutation, are involved in multiple stages of carcinogenesis (11
). In fact, human leukocytes producing superoxide but not leukocytes lacking superoxide-generating activity from patients with chronic granulomatous disease caused mutation of Salmonella enterica serovar Typhimurium
). It is therefore logical that NO-induced viral mutation may be mediated by NO-generated 8-nitroguanosine through two different mechanisms: direct modification of nucleic acid (e.g., via 8-nitroguanosine formation), and indirect augmentation by 8-nitroguanosine of oxidative stress via superoxide generation (Fig. ).
It is intriguing that the mutation profile of the GFP gene in the GFP-SeV mutants induced by 8-nitroguanosine appeared to resemble that of the mutants occurring in mouse lungs in vivo (with a predominant C-to-U transition), in which NO is produced in excess from iNOS (4
). A base substitution that was relatively characteristic of the GFP mutants induced by 8-nitroguanosine and mutants occurring in vivo was the C-to-U transition (Table ). This finding suggests indirectly that 8-nitroguanosine formed in vivo could indeed contribute to enhanced viral mutation induced by NO.
The mechanism for the C-to-U point mutation may involve incorporation of 8-nitroguanosine into the positive-strand antigenomic RNA, with subsequent G-to-A (positive sense) and C-to-U transitions in the viral genome during RNA replication. However, because a G-to-A substitution did not occur very often in the GFP gene of GFP-SeV mutants, except for mutants produced in vivo, other mechanisms may be involved in the C-to-U mutation. Similarly, the exact mechanism for the frequent A-to-G mutation, which was found in various GFP-SeV mutants, has not yet been identified. In addition, no G-specific alteration was detected in GFP-SeV mutants, which is consistent with our previous analysis (4
). These mutation profiles seem to differ from the DNA mutations induced by NO, in which G-to-T transversion was typical in eukaryotic and prokaryotic DNA treated with peroxynitrite (35
). In fact, transversions occurred much less frequently than transitions did, and the G-to-U transversion in the GFP-SeV mutants isolated in the present study was rare (Table ). This result suggests again that NO may cause RNA mutagenesis by a mechanism different from that of NO-elicited DNA mutagenesis. Further analyses in a cell-free replication system are needed to elucidate the molecular mechanism of RNA mutagenesis involving NO and 8-nitroguanosine.
The most striking feature of a virus is its considerable adaptability to various environmental stresses (21
). For example, RNA viruses exist as highly heterogeneous populations, called quasispecies, primarily because of the error-prone nature of the replicase of the viruses. In general, RNA viruses have a high mutation rate, ranging from 10−3
misincorporations/nucleotide site/replication, which is more than 104
-fold higher than the error rate for DNA viruses (20-23, 30). The low fidelity of RNA replication has been believed to be due to the lack of proofreading and repair functions of RNA polymerase or reverse transcriptase (21
). Our earlier and present studies, however, showed that RNA viral mutation was greatly affected by NO and its reactive derivatives and that guanosine nitration (8-nitroguanosine formation) occurred more in RNA than in DNA. Also, the degree of RNA viral mutation was reportedly increased by chemical mutagens, including nitrous acid (16
). Thus, the higher incidence of erroneous RNA viral replication may be due to greater susceptibility of RNA than of DNA to NO or oxidative stress.
Several reports have showed a possible association between oxidative stress and viral mutation. For example, oxidative stress augmented the integration of duck hepatitis B virus DNA into genomic DNA in cells through DNA damage and deficient DNA repair (44
). Beck et al. documented that the pathogenicity of coxsackievirus B3 is potentiated in vivo in mice fed a selenium-deficient diet, which impairs the antioxidant systems of the host (13
). Similar results that were obtained with animals deficient in vitamin E and glutathione peroxidase suggest that oxidative stress facilitates the selection and generation of virulent mutants (13
). Impaired immunological clearance of virus that is induced by oxidative stress aids the survival of heterogeneous mutants, which would result in selection of highly pathogenic variants of coxsackievirus (12
). In this context, it is of great interest that NO has immunosuppressive and regulatory effects by modulating the T-cell immune response during viral infection (39
In conclusion, NO-induced mutagenesis may result in greater heterogeneity of variants of RNA viruses, which would lead to rapid viral evolution under selective pressure and to the production of drug-resistant, immunologically tolerant, and cell tropism-altered mutants. It is now accepted that NO is generated during infection caused by any type of pathogen. Therefore, further clarification of the mechanism of NO-induced mutation of viruses is quite important, with particular focus on the role of 8-nitroguanosine in NO-dependent mutagenesis, as suggested by our current work.