The HBx protein has been reported to exhibit a variety of different activities in tissue culture cells, including induction of cell death and stimulation of HBV replication. Previous work established the functional importance of an interaction between HBx and the cellular protein DDB1 in HBx-mediated cell death (20
). In the present study, we show that this interaction is also critical for HBx to promote HBV genome replication in human hepatoma cells. This stimulatory function of HBx shows the same requirements with respect to DDB1 binding as its previously reported effect on cell viability; it needs HBx and DDB1 to interact through their natural binding regions and to form a complex in the nuclear compartment of the cell. However, HBx exhibits DDB1-dependent cytotoxic properties in both HepG2 and Huh-7 hepatoma cells, whereas it enhances HBV replication only in HepG2 cells, suggesting that the two activities are distinct. The finding that HBx performs these activities when expressed at more physiological levels from its own viral genome rather than from a recombinant vector suggests that they are potentially relevant to HBV infection and/or associated liver carcinogenesis.
Recent studies (4
) reported that HBx functions in the cytoplasm to stimulate HBV DNA replication by enhancing the reverse transcription activity of the viral polymerase while having little effect on viral mRNA steady-state levels. In marked contrast, we found that HBx acts predominantly, if not exclusively, to increase viral mRNA levels regardless of whether it is expressed from its own viral genome or from a recombinant vector. Furthermore, the use of HBx variants engineered to accumulate in either the cytoplasm or the nucleus suggests that HBx performs this activity by a mechanism that requires its nuclear location. These contrasting results are not easy to explain, since the same HBV genomic constructs and recipient HepG2 cell line were used in both studies. One possibility is that HBx can promote HBV replication by performing distinct activities, depending on the cell culture conditions. It would be interesting to see if the effect of HBx on viral polymerase does also require its interaction with DDB1.
Although we did not specifically address this issue, HBx is likely to increase the steady-state levels of HBV mRNAs by stimulating viral transcription (7
). Indeed, the protein is widely recognized to display transactivation activity (26
), and evidence has been presented that at certain promoters this activity requires an interaction of HBx with DDB1 (30
). Although HBx is believed to function predominantly in the cytoplasm to stimulate various signal transduction pathways through its association with mitochondria (1
), our finding that HBx exerts its effect in the nucleus is more consistent with the HBx-DDB1 complex promoting viral replication by another mechanism. In agreement with such a possibility, previous studies have shown that HBx increases expression of an HBV enhancer I reporter construct through a pathway that involves a nuclear function of HBx (12
). HBx and DDB1 are therefore more likely to function directly at the HBV promoter level. DDB1 has been reported to bind to and function as a transcription factor at the apolipoprotein B (apoB
) and double-stranded RNA-dependent protein kinase (PKR) promoters (10
), whereas HBx does not bind to DNA directly but exhibits transactivation properties when fused to a heterologous DNA-binding protein (28
). It is possible, therefore, that DDB1 binds to some HBV regulatory sequence with HBx acting as a coactivator. Alternatively, HBx may associate with HBV enhancer-bound transcription factors, such as CREB, ATF-2 (23
), or AP-2 (28
), and through its interaction with DDB1 recruits either STAGA (24
) or a DDB1-containing E3 ubiquitin ligase complex (15
) to modify chromatin components or transcription factors.
Intriguingly, although a direct comparison of the levels of viral replication in HepG2 and Huh-7 cells is made difficult due to large differences in transfection efficiencies, HBV appears to replicate at comparable if not higher levels in Huh-7 cells, in which HBx has no stimulatory effect, than wild-type HBV does in HepG2 cells. These results suggest that HBx may act by relieving the effect of an inhibitory activity. It has been reported that p53, which is functional in HepG2 cells and mutated in Huh-7 cells (5
), can repress HBV transcription by binding specifically to the HBV enhancer I and that HBx can prevent p53 from exerting this repressive effect (11
), perhaps by interacting directly with p53 (19
). However, under our experimental conditions, we did not detect any effect on replication of the wild-type or HBx mutant HBV genome upon siRNA-mediated downregulation of endogenous p53 expression in HepG2 cells or cotransfection of a wild-type p53 expression construct in Huh-7 cells (data not shown). Hence, the mechanism through which HBx mediates its activity in association with DDB1 remains to be established.
Whether the DDB1-dependent stimulatory activity of HBx described in the present work is related to the essential function of HBx during natural viral infection remains to be determined. However, evidence has been presented that mutations in the woodchuck hepatitis virus X gene that variably affect binding of WHx to DDB1 in vitro either delay or abolish productive virus infection in woodchucks, consistent with this interaction being functionally important (29
). Surprisingly, in these studies, no correlation was observed when the WHx mutant virus constructs were tested for replication in HepG2 cells (29
), in which HBx-mediated HBV replication involves DDB1. However, we note that HBV replication shows a strong dependence on HBx only under certain experimental conditions, for reasons that are not clear. Since HBx fully substitutes for WHx in activating woodchuck hepatitis virus replication (17
), it is more likely that the WHx mutant constructs were tested under conditions where replication was mostly independent of WHx.
The finding that, when expressed in its most native context from the viral genome, HBx affects the viability of hepatoma cell lines is rather unexpected, since HBV is a noncytopathic virus. An interesting possibility is that HBx exhibits deleterious activities only in actively dividing cells but not in quiescent cells, and experiments aimed at addressing this issue are ongoing. Following this hypothesis, HBx could promote HBV replication without altering cell viability at the time of infection, when most hepatocytes are in a quiescent state. However, HBx might become cytotoxic at later stages of the infection, and thereby possibly contribute to liver cancer development, when hepatocytes divide to replace those destroyed by the host immune response. Since both activities require the binding of HBx to DDB1, this HBV-host protein interaction may represent a promising new target for therapeutic intervention.