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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) exhibited no significant effect on nuclear export of the 3.5 kb pgRNA.A) HuH-7 cells were transfected with various plasmids of HBV genomes (based on plasmid pCHT-9/3091), containing wild type (WT) HBc, mutant ΔHBc (a core-deficient HBV genome), and a combination of ΔHBc and an expression vector of full-length HBc 183, respectively. HBV RNAs were extracted from nuclear and cytoplasmic compartments according to the Vendor’s protocol (Materials and Methods). Plasmid ΔHBc resulted in no apparent effect on the relative distribution of HBV pgRNA levels between nucleus and cytoplasm (N/C) by RT-qPCR analysis. The N/C ratio of HBV pgRNA from a WT HBV genome (plasmid pCHT-9/3091) was shown as 1. HBc protein expression was monitored by Western blot analysis (right panel). The graph represents an average from at least three independent experiments. B) Northern blot analysis revealed no significant reduction of the 3.5 kb pgRNA, in the cytoplasm of HuH-7 cells transfected with either a ΔHBc mutant or a combination of plasmids ΔHBc and HBc 183. Ribosomal RNA was included as an internal control for sample loading.
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pone-0106683-g007: HBV core protein (HBc) exhibited no significant effect on nuclear export of the 3.5 kb pgRNA.A) HuH-7 cells were transfected with various plasmids of HBV genomes (based on plasmid pCHT-9/3091), containing wild type (WT) HBc, mutant ΔHBc (a core-deficient HBV genome), and a combination of ΔHBc and an expression vector of full-length HBc 183, respectively. HBV RNAs were extracted from nuclear and cytoplasmic compartments according to the Vendor’s protocol (Materials and Methods). Plasmid ΔHBc resulted in no apparent effect on the relative distribution of HBV pgRNA levels between nucleus and cytoplasm (N/C) by RT-qPCR analysis. The N/C ratio of HBV pgRNA from a WT HBV genome (plasmid pCHT-9/3091) was shown as 1. HBc protein expression was monitored by Western blot analysis (right panel). The graph represents an average from at least three independent experiments. B) Northern blot analysis revealed no significant reduction of the 3.5 kb pgRNA, in the cytoplasm of HuH-7 cells transfected with either a ΔHBc mutant or a combination of plasmids ΔHBc and HBc 183. Ribosomal RNA was included as an internal control for sample loading.

Mentions: Since HBc can associate with RNA [50], as well as NXF1, p15, and ALY proteins (Fig. 2 and Fig. 3), we asked whether HBc could serve as an RNA-binding adaptor for HBV pgRNA nuclear export. We compared the relative distribution of HBV pgRNA between nucleus and cytoplasm by RT-qPCR analysis in the presence or absence of HBV core protein (Fig. 7A). The subcellular distribution of pgRNA appeared to be independent of wild type HBc, as detected by pgRNA specific primer pair 2414–2606 (Fig. 1). In this assay for the N/C ratio by RT-qPCR (Fig. 7A), HBV pgRNA in nucleus vs. cytoplasm was always normalized first with snRNA U1 as an internal control (or GAPDH, data not shown). Finally, by Northern blot analysis, we also detected no significant difference in the cytoplasmic HBV pgRNA levels, with or without HBc (Fig. 7B). In summary, HBV pgRNA nuclear export does not appear to require HBc protein.


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) exhibited no significant effect on nuclear export of the 3.5 kb pgRNA.A) HuH-7 cells were transfected with various plasmids of HBV genomes (based on plasmid pCHT-9/3091), containing wild type (WT) HBc, mutant ΔHBc (a core-deficient HBV genome), and a combination of ΔHBc and an expression vector of full-length HBc 183, respectively. HBV RNAs were extracted from nuclear and cytoplasmic compartments according to the Vendor’s protocol (Materials and Methods). Plasmid ΔHBc resulted in no apparent effect on the relative distribution of HBV pgRNA levels between nucleus and cytoplasm (N/C) by RT-qPCR analysis. The N/C ratio of HBV pgRNA from a WT HBV genome (plasmid pCHT-9/3091) was shown as 1. HBc protein expression was monitored by Western blot analysis (right panel). The graph represents an average from at least three independent experiments. B) Northern blot analysis revealed no significant reduction of the 3.5 kb pgRNA, in the cytoplasm of HuH-7 cells transfected with either a ΔHBc mutant or a combination of plasmids ΔHBc and HBc 183. Ribosomal RNA was included as an internal control for sample loading.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0106683-g007: HBV core protein (HBc) exhibited no significant effect on nuclear export of the 3.5 kb pgRNA.A) HuH-7 cells were transfected with various plasmids of HBV genomes (based on plasmid pCHT-9/3091), containing wild type (WT) HBc, mutant ΔHBc (a core-deficient HBV genome), and a combination of ΔHBc and an expression vector of full-length HBc 183, respectively. HBV RNAs were extracted from nuclear and cytoplasmic compartments according to the Vendor’s protocol (Materials and Methods). Plasmid ΔHBc resulted in no apparent effect on the relative distribution of HBV pgRNA levels between nucleus and cytoplasm (N/C) by RT-qPCR analysis. The N/C ratio of HBV pgRNA from a WT HBV genome (plasmid pCHT-9/3091) was shown as 1. HBc protein expression was monitored by Western blot analysis (right panel). The graph represents an average from at least three independent experiments. B) Northern blot analysis revealed no significant reduction of the 3.5 kb pgRNA, in the cytoplasm of HuH-7 cells transfected with either a ΔHBc mutant or a combination of plasmids ΔHBc and HBc 183. Ribosomal RNA was included as an internal control for sample loading.
Mentions: Since HBc can associate with RNA [50], as well as NXF1, p15, and ALY proteins (Fig. 2 and Fig. 3), we asked whether HBc could serve as an RNA-binding adaptor for HBV pgRNA nuclear export. We compared the relative distribution of HBV pgRNA between nucleus and cytoplasm by RT-qPCR analysis in the presence or absence of HBV core protein (Fig. 7A). The subcellular distribution of pgRNA appeared to be independent of wild type HBc, as detected by pgRNA specific primer pair 2414–2606 (Fig. 1). In this assay for the N/C ratio by RT-qPCR (Fig. 7A), HBV pgRNA in nucleus vs. cytoplasm was always normalized first with snRNA U1 as an internal control (or GAPDH, data not shown). Finally, by Northern blot analysis, we also detected no significant difference in the cytoplasmic HBV pgRNA levels, with or without HBc (Fig. 7B). In summary, HBV pgRNA nuclear export does not appear to require HBc protein.

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