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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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Interference with the NXF1-p15 complex reduced HBV DNA synthesis.HuH-7 cells were transiently co-transfected with an HBV genome (plasmid pCHT-9/3091) and siRNAs, followed by Southern blot analysis using HBV probe (nt 1521–3164) (Fig. 1). Upon treatment with siRNAs specific for p15 or NXF1-p15 complex, HBV replication was significantly inhibited. In contrast, no apparent effect on HBV replication was noted by treatment with various siRNAs specific for TREX components and CRM-1. RC: relaxed circle DNA, and SS: single-strand DNA.
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pone-0106683-g008: Interference with the NXF1-p15 complex reduced HBV DNA synthesis.HuH-7 cells were transiently co-transfected with an HBV genome (plasmid pCHT-9/3091) and siRNAs, followed by Southern blot analysis using HBV probe (nt 1521–3164) (Fig. 1). Upon treatment with siRNAs specific for p15 or NXF1-p15 complex, HBV replication was significantly inhibited. In contrast, no apparent effect on HBV replication was noted by treatment with various siRNAs specific for TREX components and CRM-1. RC: relaxed circle DNA, and SS: single-strand DNA.

Mentions: Next, we asked whether perturbation of the NXF1-p15 pathway could influence HBV DNA replication. As shown in the Southern blot analysis of Fig. 8, treatments with siRNAs specific for NXF1 or p15, but not with siRNAs specific for TREX components and CRM-1, resulted in reproducible reduction of HBV DNA replication (lane 2, left panel; lane 6 and 8, right panel,Fig. 8). Most likely, the reduced HBV DNA synthesis was at least in part due to the reduced nuclear export of pgRNA (Fig. 6), when the efficiency of pgRNA nuclear export was compromised by the siRNA treatment specific for NXF1-p15.


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)

Interference with the NXF1-p15 complex reduced HBV DNA synthesis.HuH-7 cells were transiently co-transfected with an HBV genome (plasmid pCHT-9/3091) and siRNAs, followed by Southern blot analysis using HBV probe (nt 1521–3164) (Fig. 1). Upon treatment with siRNAs specific for p15 or NXF1-p15 complex, HBV replication was significantly inhibited. In contrast, no apparent effect on HBV replication was noted by treatment with various siRNAs specific for TREX components and CRM-1. RC: relaxed circle DNA, and SS: single-strand DNA.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0106683-g008: Interference with the NXF1-p15 complex reduced HBV DNA synthesis.HuH-7 cells were transiently co-transfected with an HBV genome (plasmid pCHT-9/3091) and siRNAs, followed by Southern blot analysis using HBV probe (nt 1521–3164) (Fig. 1). Upon treatment with siRNAs specific for p15 or NXF1-p15 complex, HBV replication was significantly inhibited. In contrast, no apparent effect on HBV replication was noted by treatment with various siRNAs specific for TREX components and CRM-1. RC: relaxed circle DNA, and SS: single-strand DNA.
Mentions: Next, we asked whether perturbation of the NXF1-p15 pathway could influence HBV DNA replication. As shown in the Southern blot analysis of Fig. 8, treatments with siRNAs specific for NXF1 or p15, but not with siRNAs specific for TREX components and CRM-1, resulted in reproducible reduction of HBV DNA replication (lane 2, left panel; lane 6 and 8, right panel,Fig. 8). Most likely, the reduced HBV DNA synthesis was at least in part due to the reduced nuclear export of pgRNA (Fig. 6), when the efficiency of pgRNA nuclear export was compromised by the siRNA treatment specific for NXF1-p15.

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