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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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Physical association between HBV pgRNA, TREX and NXF1-p15 complex was revealed by the RNA-immunoprecipitation assay (RNA-IP).A) The NXF1-p15 complex can associate with HBV specific RNAs by the RNA-IP assay. HuH-7 cells were co-transfected with plasmid DNAs of an HBV genome, NXF1-Flag, and p15-Flag. HBV RNAs were extracted from immunoprecipitates using anti-Flag antibody, followed by RT-PCR analysis. PCR primers 1444 and 1702 can detect all species of HBV RNAs (Fig. 1). The most abundant pre-miR-122 in HuH-7 cells was used as a negative control RNA for its lack of physical association with NXF1-p15 complex. Anti-IgG antibody (α-IgG Ab) was included as a control for the specificity of immunoprecipitation. RT: reverse transcriptase. B–D) By the RNA-IP assay, HBV 3.5 kb non-spliced pgRNA was shown to be associated with several known protein components of the NXF1-p15 mediated RNA export machinery: B) NXF1, C) ALY, and D) BAT1/DDX39. PCR primers 2301 and 2598 can detect only the 3.5 kb non-spliced pgRNA (Fig. 1).
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pone-0106683-g005: Physical association between HBV pgRNA, TREX and NXF1-p15 complex was revealed by the RNA-immunoprecipitation assay (RNA-IP).A) The NXF1-p15 complex can associate with HBV specific RNAs by the RNA-IP assay. HuH-7 cells were co-transfected with plasmid DNAs of an HBV genome, NXF1-Flag, and p15-Flag. HBV RNAs were extracted from immunoprecipitates using anti-Flag antibody, followed by RT-PCR analysis. PCR primers 1444 and 1702 can detect all species of HBV RNAs (Fig. 1). The most abundant pre-miR-122 in HuH-7 cells was used as a negative control RNA for its lack of physical association with NXF1-p15 complex. Anti-IgG antibody (α-IgG Ab) was included as a control for the specificity of immunoprecipitation. RT: reverse transcriptase. B–D) By the RNA-IP assay, HBV 3.5 kb non-spliced pgRNA was shown to be associated with several known protein components of the NXF1-p15 mediated RNA export machinery: B) NXF1, C) ALY, and D) BAT1/DDX39. PCR primers 2301 and 2598 can detect only the 3.5 kb non-spliced pgRNA (Fig. 1).

Mentions: In a similar vein to our study of the HBc protein (Fig. 2–4), we asked whether HBV RNA can also utilize the NXF1-p15 machinery for nuclear export. To address this issue, we focused our study here on the 3.5 kb pgRNA, which serves as a template for HBV reverse transcription [4] as well as for translation of HBc and polymerase [3]. We examined whether HBV pgRNA can be physically associated with NXF1 protein by RNA immunoprecipitation (RNA-IP) assay (detailed in Materials and Methods). Briefly, HuH-7 cells were co-transfected with an HBV genome, NXF1-Flag, and p15-Flag plasmid DNAs. Transfected cell lysates were then incubated with anti-Flag antibody. Immunoprecipitated RNAs were then assayed by RT-PCR. As shown in Fig. 5A, the blue color-coded primer pair HBV 1444–1702 is included here as a positive control, since it can detect all the HBV specific RNA transcripts (Fig. 1). Nuclear export of microRNA precursors is known to be mediated by exportin-5 [43]. We therefore used a highly abundant pre-miR-122 as a negative control RNA for the lack of physical association with NXF1-p15 complex (Fig. 5A). Anti-Flag antibody can capture HBV RNAs, which can then be detected in a reverse transcriptase (RT)-dependent manner. On the other hand, control antibodies, such as anti-IgG antibody (Fig. 5A), as well as anti-tubulin antibody and anti-Rev antibody (data not shown), produced no significant HBV-specific RNA signals. Using this RNA-IP assay system established in Fig. 5A, we demonstrated that HBV pgRNA can be preferentially associated with NXF1 (Fig. 5B), ALY (Fig. 5C), and BAT1/DDX39 (Fig. 5D). One caveat here is that the interactions between RNA and RNA-binding proteins are known to be promiscuous in nature. Therefore, the RNA-IP assay may be limited in its specificity. As a side note, the green color-coded primer pair HBV 2301–2598 used in Fig. 5B–5D is relatively specific for the non-spliced pgRNA, since 1) it does not overlap with the HBx and HBsAg specific RNAs (Fig. 1); 2) the reverse primer 2598 is located within the intron of the major 2.2 kb spliced RNA (Fig. 1).


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)

Physical association between HBV pgRNA, TREX and NXF1-p15 complex was revealed by the RNA-immunoprecipitation assay (RNA-IP).A) The NXF1-p15 complex can associate with HBV specific RNAs by the RNA-IP assay. HuH-7 cells were co-transfected with plasmid DNAs of an HBV genome, NXF1-Flag, and p15-Flag. HBV RNAs were extracted from immunoprecipitates using anti-Flag antibody, followed by RT-PCR analysis. PCR primers 1444 and 1702 can detect all species of HBV RNAs (Fig. 1). The most abundant pre-miR-122 in HuH-7 cells was used as a negative control RNA for its lack of physical association with NXF1-p15 complex. Anti-IgG antibody (α-IgG Ab) was included as a control for the specificity of immunoprecipitation. RT: reverse transcriptase. B–D) By the RNA-IP assay, HBV 3.5 kb non-spliced pgRNA was shown to be associated with several known protein components of the NXF1-p15 mediated RNA export machinery: B) NXF1, C) ALY, and D) BAT1/DDX39. PCR primers 2301 and 2598 can detect only the 3.5 kb non-spliced pgRNA (Fig. 1).
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Related In: Results  -  Collection

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

pone-0106683-g005: Physical association between HBV pgRNA, TREX and NXF1-p15 complex was revealed by the RNA-immunoprecipitation assay (RNA-IP).A) The NXF1-p15 complex can associate with HBV specific RNAs by the RNA-IP assay. HuH-7 cells were co-transfected with plasmid DNAs of an HBV genome, NXF1-Flag, and p15-Flag. HBV RNAs were extracted from immunoprecipitates using anti-Flag antibody, followed by RT-PCR analysis. PCR primers 1444 and 1702 can detect all species of HBV RNAs (Fig. 1). The most abundant pre-miR-122 in HuH-7 cells was used as a negative control RNA for its lack of physical association with NXF1-p15 complex. Anti-IgG antibody (α-IgG Ab) was included as a control for the specificity of immunoprecipitation. RT: reverse transcriptase. B–D) By the RNA-IP assay, HBV 3.5 kb non-spliced pgRNA was shown to be associated with several known protein components of the NXF1-p15 mediated RNA export machinery: B) NXF1, C) ALY, and D) BAT1/DDX39. PCR primers 2301 and 2598 can detect only the 3.5 kb non-spliced pgRNA (Fig. 1).
Mentions: In a similar vein to our study of the HBc protein (Fig. 2–4), we asked whether HBV RNA can also utilize the NXF1-p15 machinery for nuclear export. To address this issue, we focused our study here on the 3.5 kb pgRNA, which serves as a template for HBV reverse transcription [4] as well as for translation of HBc and polymerase [3]. We examined whether HBV pgRNA can be physically associated with NXF1 protein by RNA immunoprecipitation (RNA-IP) assay (detailed in Materials and Methods). Briefly, HuH-7 cells were co-transfected with an HBV genome, NXF1-Flag, and p15-Flag plasmid DNAs. Transfected cell lysates were then incubated with anti-Flag antibody. Immunoprecipitated RNAs were then assayed by RT-PCR. As shown in Fig. 5A, the blue color-coded primer pair HBV 1444–1702 is included here as a positive control, since it can detect all the HBV specific RNA transcripts (Fig. 1). Nuclear export of microRNA precursors is known to be mediated by exportin-5 [43]. We therefore used a highly abundant pre-miR-122 as a negative control RNA for the lack of physical association with NXF1-p15 complex (Fig. 5A). Anti-Flag antibody can capture HBV RNAs, which can then be detected in a reverse transcriptase (RT)-dependent manner. On the other hand, control antibodies, such as anti-IgG antibody (Fig. 5A), as well as anti-tubulin antibody and anti-Rev antibody (data not shown), produced no significant HBV-specific RNA signals. Using this RNA-IP assay system established in Fig. 5A, we demonstrated that HBV pgRNA can be preferentially associated with NXF1 (Fig. 5B), ALY (Fig. 5C), and BAT1/DDX39 (Fig. 5D). One caveat here is that the interactions between RNA and RNA-binding proteins are known to be promiscuous in nature. Therefore, the RNA-IP assay may be limited in its specificity. As a side note, the green color-coded primer pair HBV 2301–2598 used in Fig. 5B–5D is relatively specific for the non-spliced pgRNA, since 1) it does not overlap with the HBx and HBsAg specific RNAs (Fig. 1); 2) the reverse primer 2598 is located within the intron of the major 2.2 kb spliced RNA (Fig. 1).

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