Oxidative/nitrosative stress has been increasingly implicated in viral infections, including HCV, but the sources of ROS during HCV infection have not been completely characterized. Here, using state-of-the-art HCV cell culture systems and human liver samples, we present evidence that hepatocyte Nox1 and Nox4 are prominent sources of ROS during complete HCV replication. In agreement with a recent report that JFH1 core does not localize to the mitochondria, we did not find a significant elevation of mitochondrial ROS or ATP depletion with JFH1 (3
). However, it is possible that the role of the mitochondria in the HCV-induced oxidative stress is more pronounced with certain viral genotypes or cell types. Previously, HCV core protein was suggested to reduce the cell's ability to upregulate its antioxidant defenses (1
). However, hepatitis C patients have elevated levels of antioxidant genes, and JFH1 increased GSH concentration in our study (Supplement Fig. 2B
); thus, to what extent HCV interferes with the antioxidant defense mechanisms during complete viral replication remains to be further examined.
In this study, our objective was not only to find a source of ROS during complete HCV replication but to find the source of superoxide for peroxynitrite generation which we predicted would occur near the cell nucleus. Consistent with this hypothesis, nitrotyrosine and Nox activity were increased in the JFH1-transfected cell nucleus, and this increase was attenuated with siRNAs to Nox. Also, although the relative amount of nuclear vs. cytoplasmic Nox4 tended to vary from one experiment to another, Nox4 was always at least partly nuclear and colocalized with lamin A/C, particularly in the presence of HCV. Furthermore, HCV elevated intracellular superoxide concentration, and Huh7 cells overexpressing Nox4 showed increased superoxide level. These data do not completely rule out the possibility that Nox4 generates superoxide indirectly through other source(s) of superoxide in the cell, and the significant effect that Nox1 siRNA had on nuclear nitrotyrosine could at least in part be due to the uncoupling of nitric oxide synthase by peroxynitrite. Nevertheless, our data strongly indicate that Nox enzymes can elevate intracellular superoxide concentration either directly or indirectly in the cell, leading to increased generation of peroxynitrite in the hepatocyte nucleus during HCV infection. Indeed, although Nox4 has recently been suggested to generate H2
rather than superoxide, by the virtue of their chemical mechanism involving a terminal electron transfer from the one-electron carrying heme B, Nox family proteins must generate superoxide first, prior to formation of secondary products (6
). Thus, reported inability to detect superoxide with some Nox/Duox enzymes is likely to be due to rapid dismutation of superoxide to form H2
, which under some circumstances occurs more rapidly than the reaction with superoxide detecting probe. Also, if anything can outcompete superoxide dismutase (SOD) for superoxide, it will be nitric oxide reacting with superoxide to generate peroxynitrite (5
). In fact, we were able to detect higher amount of 2-OH-E+ by inhibiting SOD in our cells, suggesting a significant competition between the probe and SOD for reacting with superoxide (unpublished observation). ROS/RNS thus generated would then cooperate with other Nox/Duox enzymes and other potential sources of ROS outside nucleus to induce a chronic state of oxidative/nitrosative stress during HCV infection. In this scheme, ROS generated by nuclear Nox4 and other extra-nuclear sources of ROS would form concentration gradients, the probability of their reacting with target molecules diminishing with increasing distance from their respective origin ().
Our discovery of nuclear Nox4 raises questions as to exactly where in the nucleus Nox4 is located and how HCV changes the location of Nox4 without affecting the location of Nox1. Nox family enzymes have multiple transmembrane domains and are membrane-bound (6
). In this regard, it may be important to note that the ER membrane is contiguous with the nuclear membrane. Also, the nucleoplasm is generally membrane free but intranuclear membrane structures have been reported (19
), and Nox4 might be located within the inner or outer nuclear membrane or intranuclear cisternae of hepatocytes. If Nox4 is responsible for peroxynitrite-dependent DNA damage, it is most likely be located on the inner nuclear membrane or intranuclear membrane, with its active site facing the nucleoplasm. Notice that in our nuclear Nox activity assays, the nuclear pore is likely to allow NADPH, cytochrome c, and SOD to enter the nucleus. Detailed analysis of the subcellular location of Nox4 by electron microscopy is underway. In regards to the mechanism of increased nuclear localization of Nox4, HCV is known to induce severe membrane and nuclear alterations (20
). Thus, increased nuclear location of Nox4 might be a consequence of virus modifying the cell for its replication. In addition, as some Nox4 could be found in the nucleus even without HCV (), nuclear Nox4 is likely to represent a normal cellular process that is enhanced by HCV. In this study, we focused primarily on 50 to 65 kDa Nox1/4 protein bands which corresponded to the expected sizes of Nox1 and Nox4 proteins. The higher molecular weight bands, however, also increased with HCV and could be partly decreased with siRNAs ( and ), and might represent Nox protein complexes or post-translationally modified Nox (9
Nox enzymes are implicated in anti-microbial defense, toll-like receptor signaling, lung fibrosis, and cancers. H2
and Nox enzymes can also increase iron uptake and mediate many biological effects of TGFβ (14
). Tumor necrosis factor alpha and TGFβ, which are pro-inflammatory and fibrogenic cytokines, are elevated during HCV infection, and these cytokines are known inducers of Nox4 (6
). We also found that TGFβ played a major role in the HCV-induced elevation of Nox4 in hepatocytes, although other factors might also be involved (; also, unpublished findings). Some of these findings were corroborated by a recent study by Boudreau et al.
). Importantly, hepatocellular carcinoma, associated with chronic hepatitis C, is typically preceded by cirrhosis. Therefore, hepatocyte Nox protein(s) may provide a link between inflammation, fibrogenesis, and hepatocarcinogenesis during chronic hepatitis C. In particular, nuclear Nox4 is likely to be highly significant in the HCV-induced DNA damage in the initiation of cancer as well as reversible and/or irreversible protein modifications in the modulation of cell signaling and host gene expression. The ability to regulate Nox proteins also makes them potential targets for therapy. Therefore, our study provides new insights into possible mechanism of HCV-induced pathogenesis and points to potential targets for therapy, directed at the source of ROS.