Oertzenweg 19 b
+49 30 838 62225
The human hepatitis B virus (HBV) belongs to the family of hepadnaviridae (Schädler und Hildt, 2009). It harbors a partially double-stranded DNA genome which is about 3.2 kb in size (Schädler und Hildt, 2009). HBV is a major cause of liver disease which can cause acute or chronic hepatitis. In addition, HBV is considered to be a major etiological factor in the development of human hepatocellular carcinoma (HCC) (Lupberger und Hildt, 2007, Cougot et al., 2005). Beside the viral particles there are also so called subviral particles (SVP) detectable in the blood of infected patients (Ganem, 1991).
SVPs are composed of SHBs-Ag, do not contain viral DNA and are therefore not infectious.
HBV itself is not cytopathologic. But the risk of HCC in HBV infected patients is much higher than in uninfected patients (Beasley et al., 1981). Therefore HBV plays an important role in the pathogenesis of HCC (Beasley, 1988). Recent studies showed that -taxilin, a newly identified membrane traffic-related molecule, is upregulated in malignant tissues of HCCs (Ohtomo et al., 2010), which can be the consequence of a chronic HBV infection. -taxilin is a ubiquitinously expressed protein of 62 kDa.
There is not much known about the function of this protein. Therefore in this study the interference between HBV and -taxilin should be investigated in more detail.
Western blot analysis and immunofluorescence staining revealed higher -taxilin amounts in stable HBV-expressing and transiently transfected cells. This investigation could be confirmed in in vivo models, such as HBV-transgenic mice and livers of HBV infected patients (see chapter 5.1 fig. 5.1, chapter 5.2.1 fig. 5.4, chapter 5.2.2 fig. 5.9 and 5.10). In contrast to the finding of high -taxilin mRNA levels in HBV-replicating cells (see chapter 5.1 fig. 5.2) and in human liver samples (see chapter 5.2.2 fig. 5.11), the -taxilin mRNA levels in HBV-transgenic mice were lower compared to the control (see chapter 5.2.1 fig. 5.5). A possible explanation to this finding could be a different regulation mechanism of -taxilin in humans and mice. A long half life of the protein -taxilin in mice which causes the lower amounts of mRNA, could be possible.
A different explanation could be a negative feedback mechanism caused by the high -taxilin amounts in order to downregulate the mRNA levels. Based on the higher -taxilin amounts in several HBV infection models, we wanted to know if an overexpression of -taxilin would influence the HBV replication. However, overexpression of -taxilin showed no significant effect on the HBV replication (see chapter 5.3 fig. 5.13).
The -taxilin amounts are so high that a moderate overexpression has presumably no effect. In contrast to the overexpression, inhibition of -taxilin by siRNA abolishes HBV replication almost completely (see chapter 5.4 fig. 5.15).
This supports the hyopthesis that -taxilin plays an important role in HBV replication. But not only the viral replication is massively regulated by -taxilin, but also the release of viral paticles. ELISA measurements of supernatants of -taxilin knockdown cells revealed higher amounts of HBs- and HBe-antigen (see chapter 5.4 fig. 5.16).
As already described, it is speculated that -taxilin acts as a SM-protein by binding free syntaxin and therefore prevents the SNARE complex formation and the vesicle transport (Nogami et al., 2003, Nogami et al., 2003a). When the expression of -taxilin is inhibited, the syntaxins are available for SNARE complex formation and therefore the vesicular transport can take place which explains the elevated levels of HBe- and HBs-antigen in the supernatant. In order to get more information about the subcellular distribution of -taxilin, we performed immunofluorescence staining. It was found that -taxilin colocalizes with core and LHBs-antigen, but not with SHBs-antigen, which is the main constituent of SVPs (see chapter 5.5 fig. 5.17 and 5.18). To verify if this colocalization is due to an interaction or not, we performed co-immunoprecipitation. These data confirm the interaction between -taxilin and LHBs-Ag and PreS1PreS2, respectively (see chapter 5.6 fig. 5.19 and chapter 5.7 fig. 5.20), which confirms our assumption of an essential role of -taxilin for the HBV lifecycle. Recent work provides evidence that viral particles leave the cell by a pathway that is different from the pathway used by SVPs (Lambert et al., 2007). Although HBV is an enveloped DNA virus, there is evidence that maturation and egress of HBV depends on intraluminal vesicles (ILV) of maturing endosomes – the multivesicular bodies (MVB) (Patient et al., 2009, Lambert et al., 2007, Watanabe et al., 2007). It could be shown that HBV assembly and egress were potently blocked by perturbing the MVB machinery. The release of subviral particles was not affected by MVB inhibitors (Lambert et al., 2007). However, efficient release of enveloped viruses using the ESCRT system requires the presence of viral late domains including PXXP motives (Hurley und Hanson, 2010, Lambert et al., 2007). For HIV it could be shown that the structural gag protein binds to tsg101 (a component of the ESCRT-I complex) via its PXXP motives (Garrus et al., 2001, VerPlank et al., 2001). The sequence of -taxilin harbors a variety of such PXXP motives, which are found in the N-terminal domain and clustered in the C-terminal part of the protein. Therefore the ESCRT system could represent a potential secretion pathway for HBV viral particles. In order to analyze the influence of HBV on the ESCRT system, western blot analysis and immunofluorescence stainings were performed.
Western blot (data not shown) and immunofluorescence analysis could show that the expression of the ESCRT-component tsg101 is upregulated in BV-positive cells in parallel to the higher -taxilin amounts (see chapter 5.8 fig. 5.21). In chapter 5.9 fig. 5.23 a partial colocalization of both proteins can be seen. Vice versa, inhibition of -taxilin expression results in decreased levels of intracellular tsg101 (see chapter 5.8 fig. 5.22). In co-immunoprecipitation experiments we could prove an interaction between -taxilin and tsg101 (see chapter 5.10 fig. 5.24). Taken together, these results indicate that -taxilin plays an essential role in HBV replication, as well as an important role in the morphogenesis of HBV. HBV infection has an enhancing effect on the -taxilin expression, which could be shown in several infection models. The important role of -taxilin could be proved by inhibition of -taxilin which abolished HBV replication almost completely. The interaction of -taxilin with the viral structural protein LHBs on one side and the interaction of -taxilin with the ESCRT system on the other side makes it very likely that -taxilin serves as an adaptor protein. Because HBV structural proteins lack the late domains which are essential for the binding to the ESCRT system we conclude that -taxilin serves as an adaptor protein binding LHBs-Ag and provides the PXXP motives which are essential for the binding to the ESCRT system. In future experiments, it could be investigated whether -taxilin also interacts with other components of the ESCRT machinery. If this is not the case, one could inhibit the interaction with tsg101 and thus prevent the virus secretion. If this would succeed, it could be a potential therapeutic approach that would prevent the spread of the virus. Previously, the functional binding sites of -taxilin which are responsible for for the binding to the ESCRT machinery and the virus must be identified. This could be analyzed by means of a genetic mapping of -taxilin. Moreover, it would be interesting to see whether -taxilin harbors a ubiquitin interaction motif which also provides contact to the ESCRT machinery. In further experiments, rescue experiments could shed light on whether the HBV replication can be recovered by the transfection of an -taxilin expression plasmid.