Phosphorylation-dependent Binding of Hepatitis B Virus Core Particles to the Nuclear Pore Complex

Kann, Michael; Sodeik, Beate; Vlachou, Angelika; Gerlich, Wolfram H.; Helenius, Ari

doi:10.1083/jcb.145.1.45

Cited by 230 publications

(313 citation statements)

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“…Core proteins expose a nuclear localization signal (NLS), which causes nuclear import by the nuclear transport receptors of the importin family (Rabe et al, 2003). For assembled capsids at least, nuclear import requires phosphorylation (Kann et al, 1999). While the nuclear transport capacity of core is well established, a recent publication also reported nuclear export of core protein, which was thought to be implicated in export of HBV specific transcripts by the TAP/NxF1 pathway (Li et al, 2010).…”

Section: Introductionmentioning

confidence: 99%

Expression of viral polymerase and phosphorylation of core protein determine core and capsid localization of the human hepatitis B virus

Deroubaix

Osseman

Cassany

et al. 2015

Journal of General Virology

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Biopsies from patients show that hepadnaviral core proteins and capsids -collectively called core -are found in the nucleus and cytoplasm of infected hepatocytes. In the majority of studies, cytoplasmic core localization is related to low viraemia while nuclear core localization is associated with high viral loads. In order to better understand the molecular interactions leading to core localization, we analysed transfected hepatoma cells using immune fluorescence microscopy. We observed that expression of core protein in the absence of other viral proteins led to nuclear localization of core protein and capsids, while expression of core in the context of the other viral proteins resulted in a predominantly cytoplasmic localization. Analysis of which viral partner was responsible for cytoplasmic retention indicated that the HBx, surface proteins and HBeAg had no impact but that the viral polymerase was the major determinant. Further analysis revealed that e, an RNA structure to which the viral polymerase binds, was essential for cytoplasmic retention. Furthermore, we showed that core protein phosphorylation at Ser 164 was essential for the cytoplasmic core localization phenotype, which is likely to explain differences observed between individual cells.

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Section: Introductionmentioning

confidence: 99%

Expression of viral polymerase and phosphorylation of core protein determine core and capsid localization of the human hepatitis B virus

Deroubaix

Osseman

Cassany

et al. 2015

Journal of General Virology

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“…The supernatants were analyzed for the presence of capsids using a native agarose gel and subsequent Western blot analysis as previously described. 26 Capsids were detected via immunostaining using an antibody that binds to HBV capsids but not to unassembled capsid proteins (DAKO 26 ) with an enhanced chemoluminescence kit (Roche) according to the manufacturer's instructions. Migration of the capsids and their quantification was performed via comparison with a geometrical standard dilution series of Escherichia coli-expressed capsids of genotype D. Western Blot Analysis.…”

Section: Methodsmentioning

confidence: 99%

APOBEC‐mediated interference with hepadnavirus production†

et al. 2005

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APOBEC3G is a cellular cytidine deaminase displaying broad antiretroviral activity. Recently, it was shown that APOBEC3G can also suppress hepatitis B virus (HBV) production in human hepatoma cells. In the present study, we characterized the mechanisms of APO-BEC-mediated antiviral activity against HBV and related hepadnaviruses. We show that human APOBEC3G blocks HBV production in mammalian and nonmammalian cells and is active against duck HBV as well. Early steps of viral morphogenesis, including RNA and protein synthesis, binding of pregenomic RNA to core protein, and self-assembly of viral core protein, were unaffected. However, APOBEC3G rendered HBV core protein-associated full-length pregenomic RNA nuclease-sensitive. Ongoing reverse-transcription in capsids that had escaped the block in morphogenesis was not significantly inhibited. The antiviral effect was not modulated by abrogating or enhancing expression of the accessory HBV X protein, suggesting that HBV X protein does not represent a functional homologue to the HIV vif protein. Furthermore, human APOBEC3F but not rat APOBEC1 inhibited HBV DNA production. Viral RNA and low-level DNA produced in the presence of APOBEC3F or rat APOBEC1 occasionally displayed mutations, but the majority of clones were wild-type. In conclusion, APOBEC3G and APOBEC3F but not rat APOBEC1 can downregulate the production of replication-competent hepadnaviral nucleocapsids. In contrast to HIV and other retroviruses, however, APOBEC3G/3F-mediated editing of nucleic acids does not seem to represent an effective innate defense mechanism for hepadnaviruses. H epadnaviruses are a group of small enveloped DNA viruses with a narrow host range and a relative tropism for the liver. Hepatitis B virus (HBV), the prototypic member of the hepadnavirus family, is a major cause of liver disease worldwide, ranging from acute and chronic hepatitis to cirrhosis and hepatocellular carcinoma. 1,2 Other members of the family include the duck hepatitis B virus (DHBV) and the woodchuck hepatitis virus. 3,4 Hepadnaviral replication involves reverse-transcription of a pregenomic RNA (pgRNA) intermediate inside nucleocapsids, which are formed by 180 or 240 core protein subunits. Inside the capsid, the viral polymerase converts pgRNA into minus-strand DNA, which in turn is completed to a double-stranded, relaxed circular DNA molecule. 5,6 This life cycle places HBV into the large family of retroelements, which all require reverse-transcription of an RNA intermediate.Recently, a cellular defense mechanism targeting a wide range of retroviruses was identified. It was shown that the propagation of HIV-1 strains lacking the accessory protein Vif is suppressed in a number of nonpermissive cells and that this block was due to expression of the cytidine deaminase APOBEC3G (A3G). 7,8 Further studies revealed that A3G induces massive C3 U deamination of single stranded retroviral DNA, resulting in DNA degradation or lethal G3 A hypermutation. [9][10][11] Interestingly, A3G can also interfere with the HBV life c...

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“…Furthermore, binding of in vitro generated HBV capsids to the nucleus of digitonin-permeabilized hepatoma cells was previously found to depend on the phosphorylation status of the core protein (15). Hepadnaviral capsid phosphorylation, therefore, represents a candidate mechanism for the regulated nuclear targeting and release of viral rcDNA during natural infection.…”

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confidence: 99%

Central Role of a Serine Phosphorylation Site within Duck Hepatitis B Virus Core Protein for Capsid Trafficking and Genome Release

Köck

Kann

Pütz

et al. 2003

Journal of Biological Chemistry

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Viral nucleocapsids compartmentalize and protect viral genomes during assembly while they mediate targeted genome release during viral infection. This dual role of the capsid in the viral life cycle must be tightly regulated to ensure efficient virus spread. Here, we used the duck hepatitis B virus (DHBV) infection model to analyze the effects of capsid phosphorylation and hydrogen bond formation. The potential key phosphorylation site at serine 245 within the core protein, the building block of DHBV capsids, was substituted by alanine (S245A), aspartic acid (S245D) and asparagine (S245N), respectively. Mutant capsids were analyzed for replication competence, stability, nuclear transport, and infectivity. All mutants formed DHBV DNA-containing nucleocapsids. Wild-type and S245N but not S245A and S245D fully protected capsid-associated mature viral DNA from nuclease action. A negative ionic charge as contributed by phosphorylated serine or aspartic acidsupported nuclear localization of the viral capsid and generation of nuclear superhelical DNA. Finally, wildtype and S245D but not S245N virions were infectious in primary duck hepatocytes. These results suggest that hydrogen bonds formed by non-phosphorylated serine 245 stabilize the quarterny structure of DHBV nucleocapsids during viral assembly, while serine phosphorylation plays an important role in nuclear targeting and DNA release from capsids during viral infection.Hepadnaviruses are a group of small enveloped DNA viruses with a narrow host range and a relative tropism for the liver. Hepatitis B virus (HBV), 1 the prototypic member of the hepadnavirus family, is a major cause of liver disease worldwide, ranging from acute and chronic hepatitis to liver cirrhosis and hepatocellular carcinoma (1, 2). Other members of the family include the duck hepatitis B virus (DHBV) and the woodchuck hepatitis virus (WHV) (3, 4). Similar to other viruses the hepadnaviral genome is protected by a nucleocapsid, which is formed by icosahedrally arranged core proteins. The C-terminal domain of the core protein is rich in basic residues and bears three conserved serine phosphorylation sites (5, 6). While dispensable for capsid assembly, this region is required for packaging of pregenomic RNA (7-11). Inside the capsid the viral polymerase converts pregenomic RNA into minus-strand DNA, which in turn is completed to a double-stranded, relaxed circular (rc) DNA molecule (12). Newly assembled, mature hepadnaviral capsids containing rcDNA have two possible fates: they can either be enveloped by surface proteins and enter the secretory pathway or be redirected to the nucleus where repair of the rcDNA molecule results in the formation of covalently closed circular (ccc) DNA, the template for viral RNA synthesis. Nuclear translocation of viral rcDNA is also mediated by incoming capsids in newly infected cells. However, the precise mechanisms regulating capsid trafficking and uncoating in both settings are unknown.Earlier studies in the DHBV model have shown that capsids from infected liv...

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Phosphorylation-dependent Binding of Hepatitis B Virus Core Particles to the Nuclear Pore Complex

Cited by 230 publications

References 59 publications

Expression of viral polymerase and phosphorylation of core protein determine core and capsid localization of the human hepatitis B virus

Expression of viral polymerase and phosphorylation of core protein determine core and capsid localization of the human hepatitis B virus

APOBEC‐mediated interference with hepadnavirus production†

Central Role of a Serine Phosphorylation Site within Duck Hepatitis B Virus Core Protein for Capsid Trafficking and Genome Release

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