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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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Both NXF1 and p15 can contribute to the efficient nuclear export of HBV pgRNA.A)Upper panel: A cartoon illustrates an HBV pgRNA reporter plasmid containing a Renilla luciferase gene inserted into a major intron of HBV pgRNA [25]. Lower panel: HuH-7 cells were co-transfected with an HBV pgRNA reporter plasmid and siRNAs. Reduction of the luciferase reporter activity was observed by treatment with siRNAs specific for NXF1-p15, but not with siRNAs specific for TREX components. To control for the indirect effect of siRNA knockdown (Material and Methods), Renilla luciferase activity was normalized with a co-transfected internal reference plasmid (firefly luciferase reporter). The relative Renilla luciferase activity of mock transfection was presented as 1. The graph here represents an average from at least three independent experiments. B)Upper panel: The knockdown efficacies of siRNAs specific for p15 and UIF were measured by RT-qPCR analysis, respectively. The copy number of RNA from siNonTarget treatment was presented as 1. Lower panel: Similarly, the efficacies of siRNAs specific for BAT1/DDX39 (lane 2, left panel) and ALY (lane 2, right panel) were measured by Western blot analysis, respectively. Expression vectors pCMV-BAT1, pCMV-DDX39 (lane 3, left panel), and pCMV-ALY (lane 3, right panel), were shown to produce their respective protein products by Western blot analysis. C) Only the siRNAs specific for NXF1 and p15 reduced the cytoplasmic core+ RNA levels by Northern blot (NB) analysis using an HBc specific probe (Fig. 1). Only weak signals of pgRNA were detected in the nuclear fraction. The faint signals of 45S, 32S and 20S ribosomal RNA precursors in the nuclear fraction can be seen after longer exposure [17]. D) The knockdown efficacy of siRNAs specific for CRM-1 was measured by RT-qPCR (left panel) and Western blot analyses (right panel). The copy number of RNA from siNonTarget treatment was shown as 1. *Transfected cells in the Western blot experiment were enriched by puromycin selection. The relative distribution of pgRNA (E) and HBV core+ RNAs (F) between the nuclear and cytoplasmic compartments was measured by RT-qPCR analysis. HBV RNAs were extracted from HuH-7 cells, which were co-transfected with an HBV genome and siRNAs specific for NXF1-p15, ALY and UIF, BAT1 and DDX39. Only the siRNA specific for NXF1 and p15 exhibited a higher N/C ratio of pgRNA, while no apparent effect was observed by siRNA specific for the TREX complex. The snRNA U1 was included as an internal control to normalize the N/C ratio. N/C: relative RNA levels between nucleus (N) and cytoplasm (C). The N/C ratio of HBV pgRNA or core+ RNAs from siNonTarget treatment was shown as 1. The graph represents an average from at least three independent experiments. G) Treatment with siRNA specific for CRM-1 resulted in no effect on the N/C ratio of HBV pgRNA. The snRNA U1, known to be exported by CRM-1 [49], was used as a positive control here. GAPDH was used as an internal control to normalize the N/C ratio. The N/C ratio of HBV pgRNA from siNonTarget treatment was shown as 1. The data here represent an average from at least three independent experiments.
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pone-0106683-g006: Both NXF1 and p15 can contribute to the efficient nuclear export of HBV pgRNA.A)Upper panel: A cartoon illustrates an HBV pgRNA reporter plasmid containing a Renilla luciferase gene inserted into a major intron of HBV pgRNA [25]. Lower panel: HuH-7 cells were co-transfected with an HBV pgRNA reporter plasmid and siRNAs. Reduction of the luciferase reporter activity was observed by treatment with siRNAs specific for NXF1-p15, but not with siRNAs specific for TREX components. To control for the indirect effect of siRNA knockdown (Material and Methods), Renilla luciferase activity was normalized with a co-transfected internal reference plasmid (firefly luciferase reporter). The relative Renilla luciferase activity of mock transfection was presented as 1. The graph here represents an average from at least three independent experiments. B)Upper panel: The knockdown efficacies of siRNAs specific for p15 and UIF were measured by RT-qPCR analysis, respectively. The copy number of RNA from siNonTarget treatment was presented as 1. Lower panel: Similarly, the efficacies of siRNAs specific for BAT1/DDX39 (lane 2, left panel) and ALY (lane 2, right panel) were measured by Western blot analysis, respectively. Expression vectors pCMV-BAT1, pCMV-DDX39 (lane 3, left panel), and pCMV-ALY (lane 3, right panel), were shown to produce their respective protein products by Western blot analysis. C) Only the siRNAs specific for NXF1 and p15 reduced the cytoplasmic core+ RNA levels by Northern blot (NB) analysis using an HBc specific probe (Fig. 1). Only weak signals of pgRNA were detected in the nuclear fraction. The faint signals of 45S, 32S and 20S ribosomal RNA precursors in the nuclear fraction can be seen after longer exposure [17]. D) The knockdown efficacy of siRNAs specific for CRM-1 was measured by RT-qPCR (left panel) and Western blot analyses (right panel). The copy number of RNA from siNonTarget treatment was shown as 1. *Transfected cells in the Western blot experiment were enriched by puromycin selection. The relative distribution of pgRNA (E) and HBV core+ RNAs (F) between the nuclear and cytoplasmic compartments was measured by RT-qPCR analysis. HBV RNAs were extracted from HuH-7 cells, which were co-transfected with an HBV genome and siRNAs specific for NXF1-p15, ALY and UIF, BAT1 and DDX39. Only the siRNA specific for NXF1 and p15 exhibited a higher N/C ratio of pgRNA, while no apparent effect was observed by siRNA specific for the TREX complex. The snRNA U1 was included as an internal control to normalize the N/C ratio. N/C: relative RNA levels between nucleus (N) and cytoplasm (C). The N/C ratio of HBV pgRNA or core+ RNAs from siNonTarget treatment was shown as 1. The graph represents an average from at least three independent experiments. G) Treatment with siRNA specific for CRM-1 resulted in no effect on the N/C ratio of HBV pgRNA. The snRNA U1, known to be exported by CRM-1 [49], was used as a positive control here. GAPDH was used as an internal control to normalize the N/C ratio. The N/C ratio of HBV pgRNA from siNonTarget treatment was shown as 1. The data here represent an average from at least three independent experiments.

Mentions: Next, we performed a reporter assay for the functional study of HBV pgRNA export (Fig. 6A). The renilla luciferase reporter was inserted into the major intron of an HBV genome [25]. Therefore, only the non-spliced RNA in the cytoplasm can express the luciferase activity. As described in the legend of Fig. 6A, cell lysates of the reporter plasmid transfected HuH-7 cells, with or without siRNA treatment, were subject to the luciferase assay (Materials and Methods). The siRNAs specific for NXF1 or p15, but not TREX components (ALY, UIF, BAT1, and DDX39), resulted in significant reduction of the luciferase activity. The result in Fig. 6A suggests that the non-spliced reporter-containing RNA relies on the NXF1-p15 machinery for nuclear export. No apparent cytotoxicity from siRNA treatments was detected by the MTT assay (data not shown). The lack of effect from siRNA treatments specific for TREX components (ALY, UIF, BAT1 and DDX39), could be due to the functional redundancy between these TREX components and other cellular factors involved in the RNA processing and export machinery in the nucleus [46]. The efficacies of these siRNA treatments were monitored by RT-qPCR and Western blot analyses (Fig. 6B). The result of the reporter assay of Fig. 6A was confirmed by Northern blot analysis using an HBc specific probe, which does not overlap with HBs and HBx specific RNAs (Fig. 1). We found that the cytoplasmic levels of both pgRNA and the spliced 2.2 kb RNA were reproducibly reduced upon treatment with siRNA against NXF1 and p15 (compare lane 2 vs. lane 3, Fig. 6C), but not by siRNA treatments specific for TREX components (compare lane 4, 5 vs. lane 2, Fig. 6C). As mentioned in the Introduction, RNA export could also be mediated by another CRM-1 dependent pathway. As shown in Fig. 6C, no apparent change in pgRNA and 2.2 kb spliced RNA was observed by siRNA treatment specific for CRM-1 (compare lane 1 vs. lane 2, Fig. 6C). The efficacy of CRM-1 siRNA treatment was monitored by Western blot and RT-qPCR analyses (Fig. 6D). The reduction of cytoplasmic pgRNA by siRNA treatment specific for NXF1 and p15, suggests that the nuclear level of pgRNA should be elevated. However, we could detect only similar levels of weak HBV RNA signals in the nucleus by Northern blot analysis (Fig. 6C). It remains unclear to us what could be the explanation for this phenomenon. One possibility, which cannot be excluded, is the very rapid degradation of unsuccessfully exported mRNA by “nuclear exosome” of the nuclear RNA surveillance machinery [8], [47]. Other possibilities include the altered transcription of pgRNA and an indirect effect of the siRNA depletions. However, since the weak signals of pgRNA in the nucleus were similar between siRNA treatments specific for NXF1 or control siRNA, it is unlikely that cytoplasmic reduction of pgRNA was due to altered transcription.


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)

Both NXF1 and p15 can contribute to the efficient nuclear export of HBV pgRNA.A)Upper panel: A cartoon illustrates an HBV pgRNA reporter plasmid containing a Renilla luciferase gene inserted into a major intron of HBV pgRNA [25]. Lower panel: HuH-7 cells were co-transfected with an HBV pgRNA reporter plasmid and siRNAs. Reduction of the luciferase reporter activity was observed by treatment with siRNAs specific for NXF1-p15, but not with siRNAs specific for TREX components. To control for the indirect effect of siRNA knockdown (Material and Methods), Renilla luciferase activity was normalized with a co-transfected internal reference plasmid (firefly luciferase reporter). The relative Renilla luciferase activity of mock transfection was presented as 1. The graph here represents an average from at least three independent experiments. B)Upper panel: The knockdown efficacies of siRNAs specific for p15 and UIF were measured by RT-qPCR analysis, respectively. The copy number of RNA from siNonTarget treatment was presented as 1. Lower panel: Similarly, the efficacies of siRNAs specific for BAT1/DDX39 (lane 2, left panel) and ALY (lane 2, right panel) were measured by Western blot analysis, respectively. Expression vectors pCMV-BAT1, pCMV-DDX39 (lane 3, left panel), and pCMV-ALY (lane 3, right panel), were shown to produce their respective protein products by Western blot analysis. C) Only the siRNAs specific for NXF1 and p15 reduced the cytoplasmic core+ RNA levels by Northern blot (NB) analysis using an HBc specific probe (Fig. 1). Only weak signals of pgRNA were detected in the nuclear fraction. The faint signals of 45S, 32S and 20S ribosomal RNA precursors in the nuclear fraction can be seen after longer exposure [17]. D) The knockdown efficacy of siRNAs specific for CRM-1 was measured by RT-qPCR (left panel) and Western blot analyses (right panel). The copy number of RNA from siNonTarget treatment was shown as 1. *Transfected cells in the Western blot experiment were enriched by puromycin selection. The relative distribution of pgRNA (E) and HBV core+ RNAs (F) between the nuclear and cytoplasmic compartments was measured by RT-qPCR analysis. HBV RNAs were extracted from HuH-7 cells, which were co-transfected with an HBV genome and siRNAs specific for NXF1-p15, ALY and UIF, BAT1 and DDX39. Only the siRNA specific for NXF1 and p15 exhibited a higher N/C ratio of pgRNA, while no apparent effect was observed by siRNA specific for the TREX complex. The snRNA U1 was included as an internal control to normalize the N/C ratio. N/C: relative RNA levels between nucleus (N) and cytoplasm (C). The N/C ratio of HBV pgRNA or core+ RNAs from siNonTarget treatment was shown as 1. The graph represents an average from at least three independent experiments. G) Treatment with siRNA specific for CRM-1 resulted in no effect on the N/C ratio of HBV pgRNA. The snRNA U1, known to be exported by CRM-1 [49], was used as a positive control here. GAPDH was used as an internal control to normalize the N/C ratio. The N/C ratio of HBV pgRNA from siNonTarget treatment was shown as 1. The data here represent an average from at least three independent experiments.
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pone-0106683-g006: Both NXF1 and p15 can contribute to the efficient nuclear export of HBV pgRNA.A)Upper panel: A cartoon illustrates an HBV pgRNA reporter plasmid containing a Renilla luciferase gene inserted into a major intron of HBV pgRNA [25]. Lower panel: HuH-7 cells were co-transfected with an HBV pgRNA reporter plasmid and siRNAs. Reduction of the luciferase reporter activity was observed by treatment with siRNAs specific for NXF1-p15, but not with siRNAs specific for TREX components. To control for the indirect effect of siRNA knockdown (Material and Methods), Renilla luciferase activity was normalized with a co-transfected internal reference plasmid (firefly luciferase reporter). The relative Renilla luciferase activity of mock transfection was presented as 1. The graph here represents an average from at least three independent experiments. B)Upper panel: The knockdown efficacies of siRNAs specific for p15 and UIF were measured by RT-qPCR analysis, respectively. The copy number of RNA from siNonTarget treatment was presented as 1. Lower panel: Similarly, the efficacies of siRNAs specific for BAT1/DDX39 (lane 2, left panel) and ALY (lane 2, right panel) were measured by Western blot analysis, respectively. Expression vectors pCMV-BAT1, pCMV-DDX39 (lane 3, left panel), and pCMV-ALY (lane 3, right panel), were shown to produce their respective protein products by Western blot analysis. C) Only the siRNAs specific for NXF1 and p15 reduced the cytoplasmic core+ RNA levels by Northern blot (NB) analysis using an HBc specific probe (Fig. 1). Only weak signals of pgRNA were detected in the nuclear fraction. The faint signals of 45S, 32S and 20S ribosomal RNA precursors in the nuclear fraction can be seen after longer exposure [17]. D) The knockdown efficacy of siRNAs specific for CRM-1 was measured by RT-qPCR (left panel) and Western blot analyses (right panel). The copy number of RNA from siNonTarget treatment was shown as 1. *Transfected cells in the Western blot experiment were enriched by puromycin selection. The relative distribution of pgRNA (E) and HBV core+ RNAs (F) between the nuclear and cytoplasmic compartments was measured by RT-qPCR analysis. HBV RNAs were extracted from HuH-7 cells, which were co-transfected with an HBV genome and siRNAs specific for NXF1-p15, ALY and UIF, BAT1 and DDX39. Only the siRNA specific for NXF1 and p15 exhibited a higher N/C ratio of pgRNA, while no apparent effect was observed by siRNA specific for the TREX complex. The snRNA U1 was included as an internal control to normalize the N/C ratio. N/C: relative RNA levels between nucleus (N) and cytoplasm (C). The N/C ratio of HBV pgRNA or core+ RNAs from siNonTarget treatment was shown as 1. The graph represents an average from at least three independent experiments. G) Treatment with siRNA specific for CRM-1 resulted in no effect on the N/C ratio of HBV pgRNA. The snRNA U1, known to be exported by CRM-1 [49], was used as a positive control here. GAPDH was used as an internal control to normalize the N/C ratio. The N/C ratio of HBV pgRNA from siNonTarget treatment was shown as 1. The data here represent an average from at least three independent experiments.
Mentions: Next, we performed a reporter assay for the functional study of HBV pgRNA export (Fig. 6A). The renilla luciferase reporter was inserted into the major intron of an HBV genome [25]. Therefore, only the non-spliced RNA in the cytoplasm can express the luciferase activity. As described in the legend of Fig. 6A, cell lysates of the reporter plasmid transfected HuH-7 cells, with or without siRNA treatment, were subject to the luciferase assay (Materials and Methods). The siRNAs specific for NXF1 or p15, but not TREX components (ALY, UIF, BAT1, and DDX39), resulted in significant reduction of the luciferase activity. The result in Fig. 6A suggests that the non-spliced reporter-containing RNA relies on the NXF1-p15 machinery for nuclear export. No apparent cytotoxicity from siRNA treatments was detected by the MTT assay (data not shown). The lack of effect from siRNA treatments specific for TREX components (ALY, UIF, BAT1 and DDX39), could be due to the functional redundancy between these TREX components and other cellular factors involved in the RNA processing and export machinery in the nucleus [46]. The efficacies of these siRNA treatments were monitored by RT-qPCR and Western blot analyses (Fig. 6B). The result of the reporter assay of Fig. 6A was confirmed by Northern blot analysis using an HBc specific probe, which does not overlap with HBs and HBx specific RNAs (Fig. 1). We found that the cytoplasmic levels of both pgRNA and the spliced 2.2 kb RNA were reproducibly reduced upon treatment with siRNA against NXF1 and p15 (compare lane 2 vs. lane 3, Fig. 6C), but not by siRNA treatments specific for TREX components (compare lane 4, 5 vs. lane 2, Fig. 6C). As mentioned in the Introduction, RNA export could also be mediated by another CRM-1 dependent pathway. As shown in Fig. 6C, no apparent change in pgRNA and 2.2 kb spliced RNA was observed by siRNA treatment specific for CRM-1 (compare lane 1 vs. lane 2, Fig. 6C). The efficacy of CRM-1 siRNA treatment was monitored by Western blot and RT-qPCR analyses (Fig. 6D). The reduction of cytoplasmic pgRNA by siRNA treatment specific for NXF1 and p15, suggests that the nuclear level of pgRNA should be elevated. However, we could detect only similar levels of weak HBV RNA signals in the nucleus by Northern blot analysis (Fig. 6C). It remains unclear to us what could be the explanation for this phenomenon. One possibility, which cannot be excluded, is the very rapid degradation of unsuccessfully exported mRNA by “nuclear exosome” of the nuclear RNA surveillance machinery [8], [47]. Other possibilities include the altered transcription of pgRNA and an indirect effect of the siRNA depletions. However, since the weak signals of pgRNA in the nucleus were similar between siRNA treatments specific for NXF1 or control siRNA, it is unlikely that cytoplasmic reduction of pgRNA was due to altered transcription.

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