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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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Perturbation of the TREX complex and NXF1-p15 nuclear export machinery by siRNA treatments can result in accumulation of HBc protein in the nucleus by IFA.A) An HBV genome and siRNAs were co-transfected into HuH-7 cells. Transfected culture was fixed 40 hrs post-transfection before IFA. A negative control by siNonTarget treatment exhibited a cytoplasmic predominant pattern (C>N) of HBc (upper left). In contrast, siRNA treatments against NXF1 and p15 (upper right), ALY and UIF (lower left), as well as BAT1 and DDX39 (lower right), induced nuclear accumulation of HBc protein. B) Three different subcellular distribution patterns of HBc are cartoon illustrated. N>C: nucleus predominant pattern; C>N: cytoplasm predominant pattern; N+C: present in both nucleus and cytoplasm, ratio N>C/C>N: tendency of nuclear accumulation. C) Quantitative results of the IFA assay in A) were summarized in the Table. Approximately 100–150 HBc-positive cells were scored in each transfection experiment. The data shown here represent an average from at least three independent transfections.
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pone-0106683-g004: Perturbation of the TREX complex and NXF1-p15 nuclear export machinery by siRNA treatments can result in accumulation of HBc protein in the nucleus by IFA.A) An HBV genome and siRNAs were co-transfected into HuH-7 cells. Transfected culture was fixed 40 hrs post-transfection before IFA. A negative control by siNonTarget treatment exhibited a cytoplasmic predominant pattern (C>N) of HBc (upper left). In contrast, siRNA treatments against NXF1 and p15 (upper right), ALY and UIF (lower left), as well as BAT1 and DDX39 (lower right), induced nuclear accumulation of HBc protein. B) Three different subcellular distribution patterns of HBc are cartoon illustrated. N>C: nucleus predominant pattern; C>N: cytoplasm predominant pattern; N+C: present in both nucleus and cytoplasm, ratio N>C/C>N: tendency of nuclear accumulation. C) Quantitative results of the IFA assay in A) were summarized in the Table. Approximately 100–150 HBc-positive cells were scored in each transfection experiment. The data shown here represent an average from at least three independent transfections.

Mentions: Next, we asked whether the physical associations between HBc, TREX components, and NXF1-p15 proteins (Fig. 2 and 3), have any functional significance in nuclear export of HBc protein. We perturbed the RNA export pathway by siRNAs against TREX components, such as ALY, UIF, BAT1/DDX39, and NXF1-p15 complex. As shown in the IFA (Fig. 4A), HBc protein was preferentially accumulated in the nucleus, when HBV-transfected HuH-7 cells were treated with siRNAs specific for NXF1 and p15 (upper right panel), ALY and UIF (lower left panel), or BAT1 and DDX39 (lower right panel). In contrast, HBc protein was localized predominantly in the cytoplasm, when HBV-transfected cells were treated with the non-target siRNA control (upper left panel, Fig. 4A). The results in Fig. 4A strongly suggest that HBc protein could utilize the TREX and NXF1-p15 RNA export pathway for nuclear exit. To analyze the IFA data in a more quantitative manner, we divided the HBc-positive cells into three different groups (N>C, C>N, and N+C), according to their patterns of subcellular distribution of HBc (Materials and Methods). We defined the “tendency of nuclear accumulation” as the ratio between numbers of cells with a nucleus-predominant pattern over those with a cytoplasm-predominant pattern (N>C/C>N) (Fig. 4B). The scored IFA data are summarized in the Table of Fig. 4C. Interestingly, despite the lack of detectable physical association between HBc and BAT1/DDX39 RNA helicase (Fig. 3C), reduction of BAT1/DDX39 by siRNA treatment did significantly arrest HBc protein in the nucleus (Fig. 4A and Fig. 4C).


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)

Perturbation of the TREX complex and NXF1-p15 nuclear export machinery by siRNA treatments can result in accumulation of HBc protein in the nucleus by IFA.A) An HBV genome and siRNAs were co-transfected into HuH-7 cells. Transfected culture was fixed 40 hrs post-transfection before IFA. A negative control by siNonTarget treatment exhibited a cytoplasmic predominant pattern (C>N) of HBc (upper left). In contrast, siRNA treatments against NXF1 and p15 (upper right), ALY and UIF (lower left), as well as BAT1 and DDX39 (lower right), induced nuclear accumulation of HBc protein. B) Three different subcellular distribution patterns of HBc are cartoon illustrated. N>C: nucleus predominant pattern; C>N: cytoplasm predominant pattern; N+C: present in both nucleus and cytoplasm, ratio N>C/C>N: tendency of nuclear accumulation. C) Quantitative results of the IFA assay in A) were summarized in the Table. Approximately 100–150 HBc-positive cells were scored in each transfection experiment. The data shown here represent an average from at least three independent transfections.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0106683-g004: Perturbation of the TREX complex and NXF1-p15 nuclear export machinery by siRNA treatments can result in accumulation of HBc protein in the nucleus by IFA.A) An HBV genome and siRNAs were co-transfected into HuH-7 cells. Transfected culture was fixed 40 hrs post-transfection before IFA. A negative control by siNonTarget treatment exhibited a cytoplasmic predominant pattern (C>N) of HBc (upper left). In contrast, siRNA treatments against NXF1 and p15 (upper right), ALY and UIF (lower left), as well as BAT1 and DDX39 (lower right), induced nuclear accumulation of HBc protein. B) Three different subcellular distribution patterns of HBc are cartoon illustrated. N>C: nucleus predominant pattern; C>N: cytoplasm predominant pattern; N+C: present in both nucleus and cytoplasm, ratio N>C/C>N: tendency of nuclear accumulation. C) Quantitative results of the IFA assay in A) were summarized in the Table. Approximately 100–150 HBc-positive cells were scored in each transfection experiment. The data shown here represent an average from at least three independent transfections.
Mentions: Next, we asked whether the physical associations between HBc, TREX components, and NXF1-p15 proteins (Fig. 2 and 3), have any functional significance in nuclear export of HBc protein. We perturbed the RNA export pathway by siRNAs against TREX components, such as ALY, UIF, BAT1/DDX39, and NXF1-p15 complex. As shown in the IFA (Fig. 4A), HBc protein was preferentially accumulated in the nucleus, when HBV-transfected HuH-7 cells were treated with siRNAs specific for NXF1 and p15 (upper right panel), ALY and UIF (lower left panel), or BAT1 and DDX39 (lower right panel). In contrast, HBc protein was localized predominantly in the cytoplasm, when HBV-transfected cells were treated with the non-target siRNA control (upper left panel, Fig. 4A). The results in Fig. 4A strongly suggest that HBc protein could utilize the TREX and NXF1-p15 RNA export pathway for nuclear exit. To analyze the IFA data in a more quantitative manner, we divided the HBc-positive cells into three different groups (N>C, C>N, and N+C), according to their patterns of subcellular distribution of HBc (Materials and Methods). We defined the “tendency of nuclear accumulation” as the ratio between numbers of cells with a nucleus-predominant pattern over those with a cytoplasm-predominant pattern (N>C/C>N) (Fig. 4B). The scored IFA data are summarized in the Table of Fig. 4C. Interestingly, despite the lack of detectable physical association between HBc and BAT1/DDX39 RNA helicase (Fig. 3C), reduction of BAT1/DDX39 by siRNA treatment did significantly arrest HBc protein in the nucleus (Fig. 4A and Fig. 4C).

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