Because of the essential role of the X gene in the life cycle of mammalian hepadnaviruses in vivo, characterization of the structural and functional properties of X protein in an infectious animal model is crucial. While the avian hepadnavirus may encode an X-equivalent gene (29
), the X gene of WHV has been studied in the woodchuck, which probably represents the best small animal model to address the issue. We have demonstrated previously the structural and functional interactions between HBX and the proteasome complex and characterized the domain of HBX that is important for this interaction with a well-defined structural and functional correlation (15
). In this study, we introduced various single amino acid mutations into the domains of HBX that have been shown to be functionally important for X in vitro, including the domain interacting with the proteasome. In adult woodchucks, these X mutants appeared to behave like attenuated viruses with low-level replication and anti-WHV seroconversion in some animals. Interestingly, these animals were protected from high-dose challenge of infectious WHV, despite the absence of anti-WHV Ab’s in some animals. They all exhibited anamnestic Ab response after the challenge. A WHV mutant with mutations inactivating the pol gene was constructed as a control and was replication defective in woodchucks. Surprisingly, one of three developed anti-WHc Ab’s, which was likely a result of DNA immunization, but none of them were protected from the challenge (Figure and 6). Given the small amount of DNA injected, the lack of protection is consistent with previous studies on DNA immunization in woodchucks (30
Previous studies of WHVX mutants have shown that they were noninfectious in vivo (31
). The study by Chen et al. (31
) used a WHV construct that is self-ligated monomeric WHV genome, which is likely a less-efficient transfecting construct in vivo than the CMV-driven terminally redundant WHV plasmid used in our study. The advantage of the latter construct is that it does not require any manipulation such as self-ligation, and it is capable of directing a first-round synthesis of pregenomic RNA independent of the endogenous HBV promoter. This construct has also been used to test the effect of X mutations in woodchucks, and the study also failed to show any infectivity of the mutant (32
). However, in the study of Zoulim et al., only one animal was tested for the X mutant, and in our study we tested four animals. Furthermore, the above two studies depended on serology for infectivity determination and did not look for low-level viremia by PCR. As shown in our study, several animals inoculated with WHVX mutants did not develop anti-WHV Ab’s but had demonstrable viremia. Finally the challenge study, performed only in our study, further supported the interpretation that X-deficient mutant can replicate at low level in vivo.
Analyses of WHV sequences during the acute phase of infection revealed the reversion of X mutants to the wild-type sequence except in the animals inoculated with the Xmd–
mutants. Although Xmd–
mutants had diminished transactivation functions and replicated at a reduced efficiency in cell culture, they seemed to behave similarly to the wild-type in vivo. Therefore, the notion of alternative initiations of X gene may not be biologically relevant (24
). However, there may be subtle phenotypic differences that could not be discerned in this study. In animals inoculated with other X mutants, the genotypic reversion to wild-type supports the interpretation that early replication of the X mutants did occur in the woodchucks; otherwise the wild-type revertant would not have a chance to emerge. The temporal sequence of the reversion of X mutants to wild-type occurred rather quickly during the first 2 weeks (Table ). Most of the X mutant–inoculated animals had predominantly the wild-type sequence in circulation by week 2. Only the animals inoculated with X mutant, XP68A, demonstrated a mixture of mutant and wild-type sequences in the first few weeks, which was then replaced completely by the wild-type sequence in circulation (Figure and Table ). Although a minor population of mutant species might still exist, we could not detect them based on the sensitivity of our assay (< 1 of 20).
Based on the calculation of the rate of replication in vivo and the mutational frequency of the error-prone reverse transcriptase, a high probability of the emergence of mutants could be predicted in vivo (28
). At the peak of hepadnaviral infection, all possible single nucleotide changes could occur in one day. However, the emergence of a viral species is determined by many factors, one of which is the replication fitness of the viral species (28
). Because the wild-type replicates more efficiently than the X mutants, it is expected that the wild-type can quickly replace the mutants, especially during the early phase of infection, in which there is little competition for replication space (34
). It is not clear why only the XP68A persisted longer than the other X mutants in vivo. This X mutation may confer a less deleterious effect on the in vivo replication of WHV, as reflected in its partially active transactivation function in vitro (Figure a). This possible explanation awaits further experimentation.
The X mutants, while replicate with an attenuated phenotype, are capable of priming the immune response to protect the animals from subsequent challenge. All the animals demonstrated an anamnestic response with a rapid rise of anti-WHs and/or anti-WHc titers. Although the responses in general were not as robust as those in animals inoculated with the wild-type, some X mutant animals did exhibit a rather brisk rise to high titers of anti-WHV Ab’s as the wild-type animals (Figure ). On the other hand, one animal inoculated with the pol mutant with an anti-WHc response exhibited weak, if any, anamnestic response (anti-WHc titer rose from 20 to 40 four weeks after challenge). This evidence lends further credence to the interpretation that X-deficient mutant is not completely replication defective and behaves as an attenuated virus in vivo.
A paper published recently has also examined the role of various WHVX mutants in vivo, in correlation with the interaction of X with another cellular factor UVDDB (35
). The mutations were introduced into a region (aa 70–100) that corresponds to the interaction domain between X and UVDDB. Similar to our study, the authors also described a phenotype of attenuated infection with reversion to wild-type associated with some of the mutants. However, it is not clear why viremia and genotypic reversion occurred much later (more than 10 weeks) in those X mutant inoculated animals. No longitudinal analyses were performed on these animals to examine the emergence of wild-type from the mutant species. Furthermore, the lack of a completely X-defective mutant (such as the Xlg–
) in their study does not permit a phenotypic comparison of the various X mutants with a X-null mutant. In our study, the Xlg–
mutant behaved similarly to all the other X mutants (excluding Xmd–
). It is evident from these two studies that several domains of X are functionally important in vivo, but the precise roles of these interacting factors with X and productive viral infection await further experimentation.
Our study suggests that X-deficient virus, as an attenuated virus, could be considered as a vaccine candidate because it is capable of inducing protective immune response in vivo. In addition, the intriguing possibility of HBX as a potential target of antiviral development could greatly expand the armamentarium of anti-HBV therapy, which, to date, only consists of interferon and lamivudine.