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Nuclear export of human hepatitis B virus core protein and pregenomic RNA depends on the cellular NXF1-p15 machinery.

Yang CC, Huang EY, Li HC, Su PY, Shih C - PLoS ONE (2014)

Bottom Line: Cytoplasm-predominant HBc is clinically associated with severe liver inflammation.Cytoplasmic HBV pgRNA appeared to be reduced by siRNA treatment specific for the NXF1-p15 complex by quantitative RT-qPCR and Northern blot analyses.This result suggests that the pgRNA was also exported via the NXF1-p15 machinery.

View Article: PubMed Central - PubMed

Affiliation: Taiwan International Graduate Program (TIGP) in Molecular Medicine, National Yang-Ming University and Academia Sinica, Taipei, Taiwan; Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan.

ABSTRACT
Hepatitis B virus (HBV) core protein (HBc) can shuttle between nucleus and cytoplasm. Cytoplasm-predominant HBc is clinically associated with severe liver inflammation. Previously, we found that HBc arginine-rich domain (ARD) can associate with a host factor NXF1 (TAP) by coimmunoprecipitation. It is well known that NXF1-p15 heterodimer can serve as a major export receptor of nuclear mRNA as a ribonucleoprotein complex (RNP). In the NXF1-p15 pathway, TREX (transcription/export) complex plays an important role in coupling nuclear pre-mRNA processing with mRNA export in mammalian cells. Here, we tested the hypothesis whether HBc and HBV specific RNA can be exported via the TREX and NXF1-p15 mediated pathway. We demonstrated here that HBc can physically and specifically associate with TREX components, and the NXF1-p15 export receptor by coimmunoprecipitation. Accumulation of HBc protein in the nucleus can be induced by the interference with TREX and NXF1-p15 mediated RNA export machinery. HBV transcripts encodes a non-spliced 3.5 kb pregenomic RNA (pgRNA) which can serve as a template for reverse transcription. Cytoplasmic HBV pgRNA appeared to be reduced by siRNA treatment specific for the NXF1-p15 complex by quantitative RT-qPCR and Northern blot analyses. This result suggests that the pgRNA was also exported via the NXF1-p15 machinery. We entertain the hypothesis that HBc protein can be exported as an RNP cargo via the mRNA export pathway by hijacking the TREX and NXF1-p15 complex. In our current and previous studies, HBc is not required for pgRNA accumulation in the cytoplasm. Furthermore, HBc ARD can mediate nuclear export of a chimeric protein containing HBc ARD in a pgRNA-independent manner. Taken together, it suggests that while both pgRNA and HBc protein exports are dependent on NXF1-p15, they are using the same export machinery in a manner independent of each other.

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HBV core protein (HBc) can physically and specifically associate with the cellular NXF1-p15 complex via the NES (nuclear export signal) motif of HBc arginine rich domain (ARD).A)Upper panel: Full-length HBc (HBc 183) consists of a capsid assembly domain and an arginine rich domain (amino acid 147–183). Lower panel: HuH-7 cells were co-transfected with an HBV genome and a p15-Flag expression vector. Full-length HBc 183 protein can physically and specifically associate with a p15-Flag protein in a ribonuclease (RNase)-sensitive manner by coimmunoprecipitation assay (co-IP) and Western blot analysis (WB). B) Full-length HBc 183 protein was shown to associate with the native exogenous NXF1 protein by the same co-IP assay as described above. C) The upper panel shows a schematic outline of a K128T-HBc ARD chimera construct. The rationale of the experimental design is as detailed in the text. The co-IP result here demonstrated that only the chimera protein K128T-HBc ARD, but not K128T, can physically associate with the p15-Flag protein. D) By the same co-IP assay, only the chimera protein K128T-HBc ARD can physically associate with the native exogenous NXF1 in an RNase sensitive manner. E) Biotin-HBc ARD synthetic polypeptide can invitro pull down purified recombinant proteins of GST-NXF1 and GST-p15 by using streptavidin T1 beads (Materials and Methods). F) A cartoon illustrates that either NXF1 or p15 can bind directly to HBc ARD. Possibility 3 refers to a situation when NXF1 and p15 do not form heterodimer even at high concentrations. The results in Fig. 2E support for possibility 4. G)Upper panel: A schematic outline of the mapped NLS and NES of HBc ARD [26]. Lower panel: HuH-7 cells were co-transfected (CoTf) with plasmid DNAs of NXF1 and p15-Flag expression vectors, and a mutant HBV genome containing R-to-A mutations at HBc NES or NLS subdomains. This NES mutant HBc exhibited significantly decreased physical association with the p15 protein (compare lane 3 with lanes 2 and 4). Ablation of the two NLS motifs appeared to increase slightly the intensity of p15-Flag (compare lane 2 and 4), suggesting that the wild type NLS could have a moderate occlusion effect on the association between p15-Flag and their neighboring NES.
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pone-0106683-g002: HBV core protein (HBc) can physically and specifically associate with the cellular NXF1-p15 complex via the NES (nuclear export signal) motif of HBc arginine rich domain (ARD).A)Upper panel: Full-length HBc (HBc 183) consists of a capsid assembly domain and an arginine rich domain (amino acid 147–183). Lower panel: HuH-7 cells were co-transfected with an HBV genome and a p15-Flag expression vector. Full-length HBc 183 protein can physically and specifically associate with a p15-Flag protein in a ribonuclease (RNase)-sensitive manner by coimmunoprecipitation assay (co-IP) and Western blot analysis (WB). B) Full-length HBc 183 protein was shown to associate with the native exogenous NXF1 protein by the same co-IP assay as described above. C) The upper panel shows a schematic outline of a K128T-HBc ARD chimera construct. The rationale of the experimental design is as detailed in the text. The co-IP result here demonstrated that only the chimera protein K128T-HBc ARD, but not K128T, can physically associate with the p15-Flag protein. D) By the same co-IP assay, only the chimera protein K128T-HBc ARD can physically associate with the native exogenous NXF1 in an RNase sensitive manner. E) Biotin-HBc ARD synthetic polypeptide can invitro pull down purified recombinant proteins of GST-NXF1 and GST-p15 by using streptavidin T1 beads (Materials and Methods). F) A cartoon illustrates that either NXF1 or p15 can bind directly to HBc ARD. Possibility 3 refers to a situation when NXF1 and p15 do not form heterodimer even at high concentrations. The results in Fig. 2E support for possibility 4. G)Upper panel: A schematic outline of the mapped NLS and NES of HBc ARD [26]. Lower panel: HuH-7 cells were co-transfected (CoTf) with plasmid DNAs of NXF1 and p15-Flag expression vectors, and a mutant HBV genome containing R-to-A mutations at HBc NES or NLS subdomains. This NES mutant HBc exhibited significantly decreased physical association with the p15 protein (compare lane 3 with lanes 2 and 4). Ablation of the two NLS motifs appeared to increase slightly the intensity of p15-Flag (compare lane 2 and 4), suggesting that the wild type NLS could have a moderate occlusion effect on the association between p15-Flag and their neighboring NES.

Mentions: It is known that NXF1 and p15 can form a heterodimer [34], [35]. When HuH-7 cells were co-transfected with an HBV genome and p15-Flag or native NXF1 expression vector, full-length HBc can specifically associate with p15 (Fig. 2A) or NXF1 (Fig. 2B) protein by co-IP assay. In our co-IP experiments, both p15 and NXF1 were provided from exogenous source by transfection, since the endogenous levels of these two proteins in HuH-7 cells were not always high enough for the co-IP assay. We used p15-Flag as a substitute to the native p15, because our anti-Flag antibody provided us a very robust detection of the p15-Flag protein in the co-IP experiments. To further map the interaction domain of full-length HBc with p15 or NXF1, we constructed a chimera protein by fusing only the HBc ARD in-frame with a reporter protein of mutant SV40 large T antigen (SVLT-K128T) (Fig. 2C, upper panel). Wild type SVLT is known to contain a potent NLS [29], but without any NES. Mutation K128T inactivated this NLS of SVLT by substitution from lysine to threonine at amino acid 128. Wild type HBc can self-assemble into a 240-mer icosahedral particle with most ARD domains buried in the capsid interior [36]. In the context of this chimera protein of K128T-HBc ARD, both NLS and NES of the HBc ARD domain will no longer be buried in the capsid interior due to the lack of the capsid assembly domain [37] (Upper panel, Fig. 2A, 2C). We demonstrated here that this chimera protein K128T-HBc ARD can also specifically associate with both p15-Flag (Fig. 2C) and NXF1 (Fig. 2D) by co-IP. In contrast, the control protein K128T, without HBc ARD, can bring down only a marginal amount of p15-Flag (Fig. 2C) and NXF1 (Fig. 2D). The physical associations between HBc ARD and NXF1 or p15-Flag were significantly diminished upon treatment with ribonuclease (RNase) in the co-IP assay (lane 3 of Fig. 2A, 2C, 2D; lane 4, Fig. 2B), suggesting that RNA can contribute to the binding between HBc ARD and the NXF1-p15 complex.


Nuclear export of human hepatitis B virus core protein and pregenomic RNA depends on the cellular NXF1-p15 machinery.

Yang CC, Huang EY, Li HC, Su PY, Shih C - PLoS ONE (2014)

HBV core protein (HBc) can physically and specifically associate with the cellular NXF1-p15 complex via the NES (nuclear export signal) motif of HBc arginine rich domain (ARD).A)Upper panel: Full-length HBc (HBc 183) consists of a capsid assembly domain and an arginine rich domain (amino acid 147–183). Lower panel: HuH-7 cells were co-transfected with an HBV genome and a p15-Flag expression vector. Full-length HBc 183 protein can physically and specifically associate with a p15-Flag protein in a ribonuclease (RNase)-sensitive manner by coimmunoprecipitation assay (co-IP) and Western blot analysis (WB). B) Full-length HBc 183 protein was shown to associate with the native exogenous NXF1 protein by the same co-IP assay as described above. C) The upper panel shows a schematic outline of a K128T-HBc ARD chimera construct. The rationale of the experimental design is as detailed in the text. The co-IP result here demonstrated that only the chimera protein K128T-HBc ARD, but not K128T, can physically associate with the p15-Flag protein. D) By the same co-IP assay, only the chimera protein K128T-HBc ARD can physically associate with the native exogenous NXF1 in an RNase sensitive manner. E) Biotin-HBc ARD synthetic polypeptide can invitro pull down purified recombinant proteins of GST-NXF1 and GST-p15 by using streptavidin T1 beads (Materials and Methods). F) A cartoon illustrates that either NXF1 or p15 can bind directly to HBc ARD. Possibility 3 refers to a situation when NXF1 and p15 do not form heterodimer even at high concentrations. The results in Fig. 2E support for possibility 4. G)Upper panel: A schematic outline of the mapped NLS and NES of HBc ARD [26]. Lower panel: HuH-7 cells were co-transfected (CoTf) with plasmid DNAs of NXF1 and p15-Flag expression vectors, and a mutant HBV genome containing R-to-A mutations at HBc NES or NLS subdomains. This NES mutant HBc exhibited significantly decreased physical association with the p15 protein (compare lane 3 with lanes 2 and 4). Ablation of the two NLS motifs appeared to increase slightly the intensity of p15-Flag (compare lane 2 and 4), suggesting that the wild type NLS could have a moderate occlusion effect on the association between p15-Flag and their neighboring NES.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4215830&req=5

pone-0106683-g002: HBV core protein (HBc) can physically and specifically associate with the cellular NXF1-p15 complex via the NES (nuclear export signal) motif of HBc arginine rich domain (ARD).A)Upper panel: Full-length HBc (HBc 183) consists of a capsid assembly domain and an arginine rich domain (amino acid 147–183). Lower panel: HuH-7 cells were co-transfected with an HBV genome and a p15-Flag expression vector. Full-length HBc 183 protein can physically and specifically associate with a p15-Flag protein in a ribonuclease (RNase)-sensitive manner by coimmunoprecipitation assay (co-IP) and Western blot analysis (WB). B) Full-length HBc 183 protein was shown to associate with the native exogenous NXF1 protein by the same co-IP assay as described above. C) The upper panel shows a schematic outline of a K128T-HBc ARD chimera construct. The rationale of the experimental design is as detailed in the text. The co-IP result here demonstrated that only the chimera protein K128T-HBc ARD, but not K128T, can physically associate with the p15-Flag protein. D) By the same co-IP assay, only the chimera protein K128T-HBc ARD can physically associate with the native exogenous NXF1 in an RNase sensitive manner. E) Biotin-HBc ARD synthetic polypeptide can invitro pull down purified recombinant proteins of GST-NXF1 and GST-p15 by using streptavidin T1 beads (Materials and Methods). F) A cartoon illustrates that either NXF1 or p15 can bind directly to HBc ARD. Possibility 3 refers to a situation when NXF1 and p15 do not form heterodimer even at high concentrations. The results in Fig. 2E support for possibility 4. G)Upper panel: A schematic outline of the mapped NLS and NES of HBc ARD [26]. Lower panel: HuH-7 cells were co-transfected (CoTf) with plasmid DNAs of NXF1 and p15-Flag expression vectors, and a mutant HBV genome containing R-to-A mutations at HBc NES or NLS subdomains. This NES mutant HBc exhibited significantly decreased physical association with the p15 protein (compare lane 3 with lanes 2 and 4). Ablation of the two NLS motifs appeared to increase slightly the intensity of p15-Flag (compare lane 2 and 4), suggesting that the wild type NLS could have a moderate occlusion effect on the association between p15-Flag and their neighboring NES.
Mentions: It is known that NXF1 and p15 can form a heterodimer [34], [35]. When HuH-7 cells were co-transfected with an HBV genome and p15-Flag or native NXF1 expression vector, full-length HBc can specifically associate with p15 (Fig. 2A) or NXF1 (Fig. 2B) protein by co-IP assay. In our co-IP experiments, both p15 and NXF1 were provided from exogenous source by transfection, since the endogenous levels of these two proteins in HuH-7 cells were not always high enough for the co-IP assay. We used p15-Flag as a substitute to the native p15, because our anti-Flag antibody provided us a very robust detection of the p15-Flag protein in the co-IP experiments. To further map the interaction domain of full-length HBc with p15 or NXF1, we constructed a chimera protein by fusing only the HBc ARD in-frame with a reporter protein of mutant SV40 large T antigen (SVLT-K128T) (Fig. 2C, upper panel). Wild type SVLT is known to contain a potent NLS [29], but without any NES. Mutation K128T inactivated this NLS of SVLT by substitution from lysine to threonine at amino acid 128. Wild type HBc can self-assemble into a 240-mer icosahedral particle with most ARD domains buried in the capsid interior [36]. In the context of this chimera protein of K128T-HBc ARD, both NLS and NES of the HBc ARD domain will no longer be buried in the capsid interior due to the lack of the capsid assembly domain [37] (Upper panel, Fig. 2A, 2C). We demonstrated here that this chimera protein K128T-HBc ARD can also specifically associate with both p15-Flag (Fig. 2C) and NXF1 (Fig. 2D) by co-IP. In contrast, the control protein K128T, without HBc ARD, can bring down only a marginal amount of p15-Flag (Fig. 2C) and NXF1 (Fig. 2D). The physical associations between HBc ARD and NXF1 or p15-Flag were significantly diminished upon treatment with ribonuclease (RNase) in the co-IP assay (lane 3 of Fig. 2A, 2C, 2D; lane 4, Fig. 2B), suggesting that RNA can contribute to the binding between HBc ARD and the NXF1-p15 complex.

Bottom Line: Cytoplasm-predominant HBc is clinically associated with severe liver inflammation.Cytoplasmic HBV pgRNA appeared to be reduced by siRNA treatment specific for the NXF1-p15 complex by quantitative RT-qPCR and Northern blot analyses.This result suggests that the pgRNA was also exported via the NXF1-p15 machinery.

View Article: PubMed Central - PubMed

Affiliation: Taiwan International Graduate Program (TIGP) in Molecular Medicine, National Yang-Ming University and Academia Sinica, Taipei, Taiwan; Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan.

ABSTRACT
Hepatitis B virus (HBV) core protein (HBc) can shuttle between nucleus and cytoplasm. Cytoplasm-predominant HBc is clinically associated with severe liver inflammation. Previously, we found that HBc arginine-rich domain (ARD) can associate with a host factor NXF1 (TAP) by coimmunoprecipitation. It is well known that NXF1-p15 heterodimer can serve as a major export receptor of nuclear mRNA as a ribonucleoprotein complex (RNP). In the NXF1-p15 pathway, TREX (transcription/export) complex plays an important role in coupling nuclear pre-mRNA processing with mRNA export in mammalian cells. Here, we tested the hypothesis whether HBc and HBV specific RNA can be exported via the TREX and NXF1-p15 mediated pathway. We demonstrated here that HBc can physically and specifically associate with TREX components, and the NXF1-p15 export receptor by coimmunoprecipitation. Accumulation of HBc protein in the nucleus can be induced by the interference with TREX and NXF1-p15 mediated RNA export machinery. HBV transcripts encodes a non-spliced 3.5 kb pregenomic RNA (pgRNA) which can serve as a template for reverse transcription. Cytoplasmic HBV pgRNA appeared to be reduced by siRNA treatment specific for the NXF1-p15 complex by quantitative RT-qPCR and Northern blot analyses. This result suggests that the pgRNA was also exported via the NXF1-p15 machinery. We entertain the hypothesis that HBc protein can be exported as an RNP cargo via the mRNA export pathway by hijacking the TREX and NXF1-p15 complex. In our current and previous studies, HBc is not required for pgRNA accumulation in the cytoplasm. Furthermore, HBc ARD can mediate nuclear export of a chimeric protein containing HBc ARD in a pgRNA-independent manner. Taken together, it suggests that while both pgRNA and HBc protein exports are dependent on NXF1-p15, they are using the same export machinery in a manner independent of each other.

Show MeSH
Related in: MedlinePlus