Restrictive influence of SAMHD1 on Hepatitis B Virus life cycle.

Deoxynucleotide triphosphates (dNTPs) are essential for efficient hepatitis B virus (HBV) replication. Here, we investigated the influence of the restriction factor SAMHD1, a dNTP hydrolase (dNTPase) and RNase, on HBV replication. We demonstrated that silencing of SAMHD1 in hepatic cells increased HBV replication, while overexpression had the opposite effect. SAMHD1 significantly affected the levels of extracellular viral DNA as well as intracellular reverse transcription products, without affecting HBV RNAs or cccDNA. SAMHD1 mutations that interfere with the dNTPase activity (D137N) or in the catalytic center of the histidine-aspartate (HD) domain (D311A), and a phospho-mimetic mutation (T592E), abrogated the inhibitory activity. In contrast, a mutation diminishing the potential RNase but not dNTPase activity (Q548A) and a mutation disabling phosphorylation (T592A) did not affect antiviral activity. Moreover, HBV restriction by SAMHD1 was rescued by addition of deoxynucleosides. Although HBV infection did not directly affect protein level or phosphorylation of SAMHD1, the virus upregulated intracellular dATPs. Interestingly, SAMHD1 was dephosphorylated, thus in a potentially antiviral-active state, in primary human hepatocytes. Furthermore, SAMHD1 was upregulated by type I and II interferons in hepatic cells. These results suggest that SAMHD1 is a relevant restriction factor for HBV and restricts reverse transcription through its dNTPase activity.

Characterization of α-taxilin as a novel factor controlling the release of hepatitis C virus.

Although it is well established that the release of HCV (hepatitis C virus) occurs through the secretory pathway, many aspects concerning the control of this process are not yet fully understood. α-Taxilin was identified as a novel binding partner of syntaxin-4 and of other members of the syntaxin family, which are part of SNARE (soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptor) complexes and so are involved in intracellular vesicle traffic. Since α-taxilin prevents t-SNARE (target SNARE) formation by binding exclusively to free syntaxin-4, it exerts an inhibitory effect on the vesicular transport. HCV-replicating Huh7.5 cells and HCV-infected primary human hepatocytes and liver samples of patients suffering from chronic HCV contain significantly less α-taxilin compared with the controls. HCV impairs the expression of α-taxilin via NS5A-dependent interruption of the Raf/MEK [MAPK (mitogen-activated protein kinase)/ERK (extracellular-signal-regulated kinase) kinase] signal transduction cascade. Moreover, the half-life of α-taxilin is significantly reduced in HCV-replicating cells. Whereas modulation of α-taxilin expression does not significantly affect genome replication, the overexpression of α-taxilin prevents the release of HCV. In contrast with this, silencing of α-taxilin expression leads to increased release of infectious viral particles. This is due to the negative effect of α-taxilin on t-SNARE formation that leads to impaired vesicular trafficking. Accordingly, overexpression of the t-SNARE component syntaxin-4 increases release of HCV, whereas silencing leads to an impaired release. These data identify α-taxilin as a novel factor that controls the release of HCV and reveal the mechanism by which HCV controls the activity of α-taxilin.

Identification of α-taxilin as an essential factor for the life cycle of hepatitis B virus.

BACKGROUND & AIMS: α-taxilin was identified as binding partner of syntaxins and is supposed to regulate vesicular trafficking. However, the physiological functions of α-taxilin and its potential relevance for the life cycle of hepatitis B virus (HBV) are still poorly understood. METHODS: Transfected hepatoma cells, infected primary human hepatocytes, and liver tissue of HBV-infected patients were used to study the expression of α-taxilin. Subcellular localization and colocalization were analyzed by confocal laser scanning microscopy (CLSM). Protein-protein interactions were further investigated by co-immunoprecipitations. Silencing of α-taxilin expression was performed by lentiviral gene transfer. RESULTS: HBV producing cells show a significant higher level of α-taxilin. HBV induces α-taxilin expression, by its regulatory proteins HBx and LHBs via c-Raf. This indicates that α-taxilin is essential for the release of HBV particles. CLSM and co-immunoprecipitations demonstrated that the PreS1PreS2 domain of LHBs interacts with α-taxilin. α-taxilin harbors a YXXL motif that represents a classic late domain. In accordance with this, it was found by co-immunoprecipitations that α-taxilin interacts with the ESCRT I component tsg101. CLSM revealed that a fraction of α-taxilin colocalizes with LHBs and tsg101. CONCLUSIONS: α-taxilin plays an essential role for release of HBV-DNA containing particles. It might act as an adapter that binds, on the one hand, to LHBs and, on the other hand, to tsg101 and thereby helps recruit the ESCRT machinery to the viral envelope proteins.