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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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Nuclear transport of Mat-C and Mat-C-UV.A. Electron microscopy of the capsids at the NPCs after microinjection into the cytoplasm of Xenopus laevis oocytes. The white arrows indicate capsids. Black scale bar: 100 nm. B. Frequency of NPC-attached capsids and the capsid distribution at the NPCs. Both capsid species showed a similar frequency at the NPCs and similar importation into the nuclear basket. C. In vitro transport assays of the capsids in digitonin-permeabilized HuH-7 cells. NPCs (red) and capsids (green) are visualized by indirect immune stain. The merges are depicted on the right panels. The panels show overviews and strongly magnified images. While Mat-C (upper two panels) caused intranuclear fluorescence cross-linked capsids (lower two panels) failed to enter the karyoplasm.
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ppat-1000741-g006: Nuclear transport of Mat-C and Mat-C-UV.A. Electron microscopy of the capsids at the NPCs after microinjection into the cytoplasm of Xenopus laevis oocytes. The white arrows indicate capsids. Black scale bar: 100 nm. B. Frequency of NPC-attached capsids and the capsid distribution at the NPCs. Both capsid species showed a similar frequency at the NPCs and similar importation into the nuclear basket. C. In vitro transport assays of the capsids in digitonin-permeabilized HuH-7 cells. NPCs (red) and capsids (green) are visualized by indirect immune stain. The merges are depicted on the right panels. The panels show overviews and strongly magnified images. While Mat-C (upper two panels) caused intranuclear fluorescence cross-linked capsids (lower two panels) failed to enter the karyoplasm.

Mentions: As control, six Xenopus laevis oocytes were injected with Mat-C. We restricted the incubation time after injection to 1 h, the earliest time point at which significant numbers of capsids arrive at the nuclear pore (not shown). Such a short time was chosen in order to detect possible differences in nuclear entry of the capsids. As shown in Figure 6A both types of capsids entered the nuclear basket as intact particles. We determined the number of capsids that arrived at 68 NPCs (Mat-C) and 74 NPCs (Mat-C UV), and determined their location at the NPCs. Figure 6B showed that a similar number of capsids arrived at the nuclear pore indicating that the cross-link neither affected the intracytoplasmic transport capacity nor the interaction with the NPCs. We next analyzed the distribution of the capsids, showing that both capsid species exhibited the same distribution at the NPCs with a majority on the cytoplasmic face. To our experience this dominantly cytoplasmic localization is related to the short incubation time. However, the same proportion of Mat-C and Mat-C UV entered the pore and were found in the nuclear basket, implying that their transport competence was the same.


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)

Nuclear transport of Mat-C and Mat-C-UV.A. Electron microscopy of the capsids at the NPCs after microinjection into the cytoplasm of Xenopus laevis oocytes. The white arrows indicate capsids. Black scale bar: 100 nm. B. Frequency of NPC-attached capsids and the capsid distribution at the NPCs. Both capsid species showed a similar frequency at the NPCs and similar importation into the nuclear basket. C. In vitro transport assays of the capsids in digitonin-permeabilized HuH-7 cells. NPCs (red) and capsids (green) are visualized by indirect immune stain. The merges are depicted on the right panels. The panels show overviews and strongly magnified images. While Mat-C (upper two panels) caused intranuclear fluorescence cross-linked capsids (lower two panels) failed to enter the karyoplasm.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1000741-g006: Nuclear transport of Mat-C and Mat-C-UV.A. Electron microscopy of the capsids at the NPCs after microinjection into the cytoplasm of Xenopus laevis oocytes. The white arrows indicate capsids. Black scale bar: 100 nm. B. Frequency of NPC-attached capsids and the capsid distribution at the NPCs. Both capsid species showed a similar frequency at the NPCs and similar importation into the nuclear basket. C. In vitro transport assays of the capsids in digitonin-permeabilized HuH-7 cells. NPCs (red) and capsids (green) are visualized by indirect immune stain. The merges are depicted on the right panels. The panels show overviews and strongly magnified images. While Mat-C (upper two panels) caused intranuclear fluorescence cross-linked capsids (lower two panels) failed to enter the karyoplasm.
Mentions: As control, six Xenopus laevis oocytes were injected with Mat-C. We restricted the incubation time after injection to 1 h, the earliest time point at which significant numbers of capsids arrive at the nuclear pore (not shown). Such a short time was chosen in order to detect possible differences in nuclear entry of the capsids. As shown in Figure 6A both types of capsids entered the nuclear basket as intact particles. We determined the number of capsids that arrived at 68 NPCs (Mat-C) and 74 NPCs (Mat-C UV), and determined their location at the NPCs. Figure 6B showed that a similar number of capsids arrived at the nuclear pore indicating that the cross-link neither affected the intracytoplasmic transport capacity nor the interaction with the NPCs. We next analyzed the distribution of the capsids, showing that both capsid species exhibited the same distribution at the NPCs with a majority on the cytoplasmic face. To our experience this dominantly cytoplasmic localization is related to the short incubation time. However, the same proportion of Mat-C and Mat-C UV entered the pore and were found in the nuclear basket, implying that their transport competence was the same.

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