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Nucleoporin 153 arrests the nuclear import of hepatitis B virus capsids in the nuclear basket.

Schmitz A, Schwarz A, Foss M, Zhou L, Rabe B, Hoellenriegel J, Stoeber M, Panté N, Kann M - PLoS Pathog. (2010)

Bottom Line: Investigating the difference between immature and mature capsids, we found that mature capsids had to disintegrate in order to leave the nuclear basket.The binding sites of importin beta and capsids were shown to overlap but capsid binding was 150-fold stronger.In cellulo experiments using digitonin-permeabilized cells confirmed the interference between capsid binding and nuclear import by importin beta.

View Article: PubMed Central - PubMed

Affiliation: Institute of Medical Virology, Justus Liebig University, Giessen, Germany.

ABSTRACT
Virtually all DNA viruses including hepatitis B viruses (HBV) replicate their genome inside the nucleus. In non-dividing cells, the genome has to pass through the nuclear pore complexes (NPCs) by the aid of nuclear transport receptors as e.g. importin beta (karyopherin). Most viruses release their genome in the cytoplasm or at the cytosolic face of the NPC, as the diameter of their capsids exceeds the size of the NPC. The DNA genome of HBV is derived from reverse transcription of an RNA pregenome. Genome maturation occurs in cytosolic capsids and progeny capsids can deliver the genome into the nucleus causing nuclear genome amplification. The karyophilic capsids are small enough to pass the NPC, but nuclear entry of capsids with an immature genome is halted in the nuclear basket on the nuclear side of the NPC, and the genome remains encapsidated. In contrast, capsids with a mature genome enter the basket and consequently liberate the genome. Investigating the difference between immature and mature capsids, we found that mature capsids had to disintegrate in order to leave the nuclear basket. The arrest of a karyophilic cargo at the nuclear pore is a rare phenomenon, which has been described for only very few cellular proteins participating in nuclear entry. We analyzed the interactions causing HBV capsid retention. By pull-down assays and partial siRNA depletion, we showed that HBV capsids directly interact with nucleoporin 153 (Nup153), an essential protein of the nuclear basket which participates in nuclear transport via importin beta. The binding sites of importin beta and capsids were shown to overlap but capsid binding was 150-fold stronger. In cellulo experiments using digitonin-permeabilized cells confirmed the interference between capsid binding and nuclear import by importin beta. Collectively, our findings describe a unique nuclear import strategy not only for viruses but for all karyophilic cargos.

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Analysis of cross-linked core particles.A. Lane 1: 32P-labelled Mat-C-UV and (lane 2) 32P-labelled Mat-C were separated on a 4–12% SDS-PAGE. Phosphoimaging showed that the core proteins of Mat-C-UV did not enter the separating gel indicating successful cross-linking. In contrast the core proteins of Mat-C migrated as a 21.5 kDa band. m: 14C-labelled molecular weight marker. B. Immune blot of Mat-C-UV and non cross-linked P-rC after native agarose gel electrophoresis. Mat-C-UV migrated as the P-rC standard indicating that the core proteins were linked within the individual capsids and that the capsids were not linked to each other. The identical migration indicates that UV irradiation has not changed the surface charge. The numbers on top of the standard dilution series give the amount of the P-rC in pg.
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ppat-1000741-g005: Analysis of cross-linked core particles.A. Lane 1: 32P-labelled Mat-C-UV and (lane 2) 32P-labelled Mat-C were separated on a 4–12% SDS-PAGE. Phosphoimaging showed that the core proteins of Mat-C-UV did not enter the separating gel indicating successful cross-linking. In contrast the core proteins of Mat-C migrated as a 21.5 kDa band. m: 14C-labelled molecular weight marker. B. Immune blot of Mat-C-UV and non cross-linked P-rC after native agarose gel electrophoresis. Mat-C-UV migrated as the P-rC standard indicating that the core proteins were linked within the individual capsids and that the capsids were not linked to each other. The identical migration indicates that UV irradiation has not changed the surface charge. The numbers on top of the standard dilution series give the amount of the P-rC in pg.

Mentions: We next asked how Mat-C proteins could enter the nucleus despite the strong binding to Nup153. We followed the hypothesis that capsids should remain intact while genome maturation continues but should liberate the genome from the basket once rcDNA is formed. To test this hypothesis we cross-linked the Mat-C subunits, which were 32P-labelled by UV irradiation (Mat-C UV) and analyzed their integrity. Figure 5A shows that the cross-linking caused a strong reduced migration of the core protein subunits in SDS PAGE. Most of the protein was retained at the entry site of the SDS PAGE; only traces resulted in a smear >45 kDa. To test whether cross-linking occurred between capsids, which would have caused unsuitable particle aggregates we separated Mat-C UV on native agarose gel and detected them by anti-capsid antibody used before. We observed no difference in migration compared to the P-rC standard (Fig. 5B), demonstrating that UV irradiation only induced bonds between subunits of individual capsids. The result further shows that the UV treatment had not changed the surface charge.


Nucleoporin 153 arrests the nuclear import of hepatitis B virus capsids in the nuclear basket.

Schmitz A, Schwarz A, Foss M, Zhou L, Rabe B, Hoellenriegel J, Stoeber M, Panté N, Kann M - PLoS Pathog. (2010)

Analysis of cross-linked core particles.A. Lane 1: 32P-labelled Mat-C-UV and (lane 2) 32P-labelled Mat-C were separated on a 4–12% SDS-PAGE. Phosphoimaging showed that the core proteins of Mat-C-UV did not enter the separating gel indicating successful cross-linking. In contrast the core proteins of Mat-C migrated as a 21.5 kDa band. m: 14C-labelled molecular weight marker. B. Immune blot of Mat-C-UV and non cross-linked P-rC after native agarose gel electrophoresis. Mat-C-UV migrated as the P-rC standard indicating that the core proteins were linked within the individual capsids and that the capsids were not linked to each other. The identical migration indicates that UV irradiation has not changed the surface charge. The numbers on top of the standard dilution series give the amount of the P-rC in pg.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2813275&req=5

ppat-1000741-g005: Analysis of cross-linked core particles.A. Lane 1: 32P-labelled Mat-C-UV and (lane 2) 32P-labelled Mat-C were separated on a 4–12% SDS-PAGE. Phosphoimaging showed that the core proteins of Mat-C-UV did not enter the separating gel indicating successful cross-linking. In contrast the core proteins of Mat-C migrated as a 21.5 kDa band. m: 14C-labelled molecular weight marker. B. Immune blot of Mat-C-UV and non cross-linked P-rC after native agarose gel electrophoresis. Mat-C-UV migrated as the P-rC standard indicating that the core proteins were linked within the individual capsids and that the capsids were not linked to each other. The identical migration indicates that UV irradiation has not changed the surface charge. The numbers on top of the standard dilution series give the amount of the P-rC in pg.
Mentions: We next asked how Mat-C proteins could enter the nucleus despite the strong binding to Nup153. We followed the hypothesis that capsids should remain intact while genome maturation continues but should liberate the genome from the basket once rcDNA is formed. To test this hypothesis we cross-linked the Mat-C subunits, which were 32P-labelled by UV irradiation (Mat-C UV) and analyzed their integrity. Figure 5A shows that the cross-linking caused a strong reduced migration of the core protein subunits in SDS PAGE. Most of the protein was retained at the entry site of the SDS PAGE; only traces resulted in a smear >45 kDa. To test whether cross-linking occurred between capsids, which would have caused unsuitable particle aggregates we separated Mat-C UV on native agarose gel and detected them by anti-capsid antibody used before. We observed no difference in migration compared to the P-rC standard (Fig. 5B), demonstrating that UV irradiation only induced bonds between subunits of individual capsids. The result further shows that the UV treatment had not changed the surface charge.

Bottom Line: Investigating the difference between immature and mature capsids, we found that mature capsids had to disintegrate in order to leave the nuclear basket.The binding sites of importin beta and capsids were shown to overlap but capsid binding was 150-fold stronger.In cellulo experiments using digitonin-permeabilized cells confirmed the interference between capsid binding and nuclear import by importin beta.

View Article: PubMed Central - PubMed

Affiliation: Institute of Medical Virology, Justus Liebig University, Giessen, Germany.

ABSTRACT
Virtually all DNA viruses including hepatitis B viruses (HBV) replicate their genome inside the nucleus. In non-dividing cells, the genome has to pass through the nuclear pore complexes (NPCs) by the aid of nuclear transport receptors as e.g. importin beta (karyopherin). Most viruses release their genome in the cytoplasm or at the cytosolic face of the NPC, as the diameter of their capsids exceeds the size of the NPC. The DNA genome of HBV is derived from reverse transcription of an RNA pregenome. Genome maturation occurs in cytosolic capsids and progeny capsids can deliver the genome into the nucleus causing nuclear genome amplification. The karyophilic capsids are small enough to pass the NPC, but nuclear entry of capsids with an immature genome is halted in the nuclear basket on the nuclear side of the NPC, and the genome remains encapsidated. In contrast, capsids with a mature genome enter the basket and consequently liberate the genome. Investigating the difference between immature and mature capsids, we found that mature capsids had to disintegrate in order to leave the nuclear basket. The arrest of a karyophilic cargo at the nuclear pore is a rare phenomenon, which has been described for only very few cellular proteins participating in nuclear entry. We analyzed the interactions causing HBV capsid retention. By pull-down assays and partial siRNA depletion, we showed that HBV capsids directly interact with nucleoporin 153 (Nup153), an essential protein of the nuclear basket which participates in nuclear transport via importin beta. The binding sites of importin beta and capsids were shown to overlap but capsid binding was 150-fold stronger. In cellulo experiments using digitonin-permeabilized cells confirmed the interference between capsid binding and nuclear import by importin beta. Collectively, our findings describe a unique nuclear import strategy not only for viruses but for all karyophilic cargos.

Show MeSH
Related in: MedlinePlus