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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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Association between HBc ARD and TREX complex.A) The association between GST-HBc ARD and a TREX component ALY was ribonuclease (RNase)-sensitive and DNase-resistant in a GST pull down assay. This assay was performed by using E. coli -expressed GST or GST-HBc ARD protein and untransfected HuH-7 cell lysates (Materials and Methods). B) Full-length HBc 183 protein in HuH-7 cells transiently transfected with an HBV genome can also co-immunoprecipitate the endogenous ALY in an RNase-sensitive manner. C) HuH-7 cells were co-transfected with an HBV genome and a pCMV-DDX39 expression vector. Full-length HBc 183 protein cannot associate with another TREX component BAT1/DDX39 in the co-IP assay. D) A cartoon summarizes the putative associations among HBc, RNA, ALY, TREX components, and others (other known or unknown cellular factors). Such associations are postulated to be involved in nuclear RNA processing and export. NPC: nuclear pore complex.
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pone-0106683-g003: Association between HBc ARD and TREX complex.A) The association between GST-HBc ARD and a TREX component ALY was ribonuclease (RNase)-sensitive and DNase-resistant in a GST pull down assay. This assay was performed by using E. coli -expressed GST or GST-HBc ARD protein and untransfected HuH-7 cell lysates (Materials and Methods). B) Full-length HBc 183 protein in HuH-7 cells transiently transfected with an HBV genome can also co-immunoprecipitate the endogenous ALY in an RNase-sensitive manner. C) HuH-7 cells were co-transfected with an HBV genome and a pCMV-DDX39 expression vector. Full-length HBc 183 protein cannot associate with another TREX component BAT1/DDX39 in the co-IP assay. D) A cartoon summarizes the putative associations among HBc, RNA, ALY, TREX components, and others (other known or unknown cellular factors). Such associations are postulated to be involved in nuclear RNA processing and export. NPC: nuclear pore complex.

Mentions: As mentioned in the Introduction, a so-called TREX complex can couple the pre-mRNA processing and nuclear export events [14]. Having demonstrated that HBc ARD could associate with the NXF1-p15 complex (Fig. 2), we next asked whether HBc ARD can bind to any known TREX components. Using a GST pull down assay (Materials and Methods; Fig. 3A), we demonstrated that the recombinant chimera protein of GST-HBc ARD can pull down a cellular RNA binding adaptor ALY, which is a TREX component known to be involved in the NXF1-p15 RNA export pathway [38]. Furthermore, the association between ALY and HBc ARD was RNase-sensitive and DNase-resistant (Fig. 3A). By co-IP experiment, we also demonstrated that full-length HBc protein can associate with the endogenous ALY in an RNase-sensitive manner (Fig. 3B). However, unlike ALY, HBc cannot associate with a cellular RNA helicase BAT1 (UAP56)/DDX39 (URH49) (Fig. 3C), which is also known to be involved in the NXF1-p15 mediated RNA export pathway [39]. The cartoon of Fig. 3D summarizes a hypothetical RNP complex of HBc, RNA, and various host factors, such as TREX components in the nucleus, which presumably can be involved in RNA splicing, trafficking and export.


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)

Association between HBc ARD and TREX complex.A) The association between GST-HBc ARD and a TREX component ALY was ribonuclease (RNase)-sensitive and DNase-resistant in a GST pull down assay. This assay was performed by using E. coli -expressed GST or GST-HBc ARD protein and untransfected HuH-7 cell lysates (Materials and Methods). B) Full-length HBc 183 protein in HuH-7 cells transiently transfected with an HBV genome can also co-immunoprecipitate the endogenous ALY in an RNase-sensitive manner. C) HuH-7 cells were co-transfected with an HBV genome and a pCMV-DDX39 expression vector. Full-length HBc 183 protein cannot associate with another TREX component BAT1/DDX39 in the co-IP assay. D) A cartoon summarizes the putative associations among HBc, RNA, ALY, TREX components, and others (other known or unknown cellular factors). Such associations are postulated to be involved in nuclear RNA processing and export. NPC: nuclear pore complex.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0106683-g003: Association between HBc ARD and TREX complex.A) The association between GST-HBc ARD and a TREX component ALY was ribonuclease (RNase)-sensitive and DNase-resistant in a GST pull down assay. This assay was performed by using E. coli -expressed GST or GST-HBc ARD protein and untransfected HuH-7 cell lysates (Materials and Methods). B) Full-length HBc 183 protein in HuH-7 cells transiently transfected with an HBV genome can also co-immunoprecipitate the endogenous ALY in an RNase-sensitive manner. C) HuH-7 cells were co-transfected with an HBV genome and a pCMV-DDX39 expression vector. Full-length HBc 183 protein cannot associate with another TREX component BAT1/DDX39 in the co-IP assay. D) A cartoon summarizes the putative associations among HBc, RNA, ALY, TREX components, and others (other known or unknown cellular factors). Such associations are postulated to be involved in nuclear RNA processing and export. NPC: nuclear pore complex.
Mentions: As mentioned in the Introduction, a so-called TREX complex can couple the pre-mRNA processing and nuclear export events [14]. Having demonstrated that HBc ARD could associate with the NXF1-p15 complex (Fig. 2), we next asked whether HBc ARD can bind to any known TREX components. Using a GST pull down assay (Materials and Methods; Fig. 3A), we demonstrated that the recombinant chimera protein of GST-HBc ARD can pull down a cellular RNA binding adaptor ALY, which is a TREX component known to be involved in the NXF1-p15 RNA export pathway [38]. Furthermore, the association between ALY and HBc ARD was RNase-sensitive and DNase-resistant (Fig. 3A). By co-IP experiment, we also demonstrated that full-length HBc protein can associate with the endogenous ALY in an RNase-sensitive manner (Fig. 3B). However, unlike ALY, HBc cannot associate with a cellular RNA helicase BAT1 (UAP56)/DDX39 (URH49) (Fig. 3C), which is also known to be involved in the NXF1-p15 mediated RNA export pathway [39]. The cartoon of Fig. 3D summarizes a hypothetical RNP complex of HBc, RNA, and various host factors, such as TREX components in the nucleus, which presumably can be involved in RNA splicing, trafficking and export.

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