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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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An HBV transcription map is illustrated.All HBV (ayw) transcripts are shown in solid lines, and a major intron of HBV pgRNA is shown in a dashed line (HBV: 2447–489). PCR primers used for detection of all species of HBV RNAs are shown in blue (HBV: 1444–1702), and primers used for non-spliced pgRNA are shown in red and green (HBV: 2414–2606, HBV: 2301–2598). PCR primers used for detection of HBV core+ RNAs are shown in brown (HBV: 2279–2392). An HBV genome is known to encode a 3.5 kb precore specific RNA which is responsible for the production of precore and HBeAg proteins. This 3.5 kb precore transcript is longer than the 3.5 kb pgRNA by 25–30 nt at the 5′ terminus [45]. The primer pairs HBV 2301–2598 and 2414–2606 cannot distinguish between the precore RNA and pgRNA. However, our HBV replicon system (plasmid pCHT-9/3091) does not produce the 3.5 kb precore RNA [30]. Core+ RNAs refer to the 3.5 kb pgRNA, 3.5 kb precore RNA, and the 2.2 kb major spliced RNA. HBc probe (nt 1903–2447) in Northern blot analysis was specific for HBV core+ RNAs. HBV probe (nt 1521–3164) was prepared for Southern blot analysis of viral replication. C: HBV core, preC: HBV precore, P: polymerase, preS1, preS2, and S: HBV envelope, X: HBx. PRE: posttranscriptional regulatory element [19].
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pone-0106683-g001: An HBV transcription map is illustrated.All HBV (ayw) transcripts are shown in solid lines, and a major intron of HBV pgRNA is shown in a dashed line (HBV: 2447–489). PCR primers used for detection of all species of HBV RNAs are shown in blue (HBV: 1444–1702), and primers used for non-spliced pgRNA are shown in red and green (HBV: 2414–2606, HBV: 2301–2598). PCR primers used for detection of HBV core+ RNAs are shown in brown (HBV: 2279–2392). An HBV genome is known to encode a 3.5 kb precore specific RNA which is responsible for the production of precore and HBeAg proteins. This 3.5 kb precore transcript is longer than the 3.5 kb pgRNA by 25–30 nt at the 5′ terminus [45]. The primer pairs HBV 2301–2598 and 2414–2606 cannot distinguish between the precore RNA and pgRNA. However, our HBV replicon system (plasmid pCHT-9/3091) does not produce the 3.5 kb precore RNA [30]. Core+ RNAs refer to the 3.5 kb pgRNA, 3.5 kb precore RNA, and the 2.2 kb major spliced RNA. HBc probe (nt 1903–2447) in Northern blot analysis was specific for HBV core+ RNAs. HBV probe (nt 1521–3164) was prepared for Southern blot analysis of viral replication. C: HBV core, preC: HBV precore, P: polymerase, preS1, preS2, and S: HBV envelope, X: HBx. PRE: posttranscriptional regulatory element [19].

Mentions: Unlike the aforementioned large DNA viruses, HBV is the smallest DNA animal virus with a genome size near 3.2 kb [3], [18]. As shown in Fig. 1, major non-spliced HBV RNA transcripts include the 3.5 kb pgRNA, 3.5 kb precore RNA, 2.3 kb/2.1 kb HBV surface antigen (HBsAg) RNAs, and 0.7 kb HBx specific RNA. There are two important functions of the 3.5 kb pgRNA. One is to serve as a template of reverse transcription for an HBV genome, and the other is as an mRNA template for translation of polymerase and core protein. Previously, nuclear export of non-spliced HBsAg specific RNAs had been actively investigated. A RNA cis-element, so-called post-transcriptional regulatory element (PRE), was proposed to be important for the nuclear export of HBsAg specific RNAs [19]–[24]. In contrast to HBsAg specific RNAs, very little has been studied regarding how pgRNA and spliced RNAs are exported. Among the multiple HBV spliced transcripts, the most predominant HBV spliced RNA species has a size around 2.2 kb (Fig. 1) [25].


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)

An HBV transcription map is illustrated.All HBV (ayw) transcripts are shown in solid lines, and a major intron of HBV pgRNA is shown in a dashed line (HBV: 2447–489). PCR primers used for detection of all species of HBV RNAs are shown in blue (HBV: 1444–1702), and primers used for non-spliced pgRNA are shown in red and green (HBV: 2414–2606, HBV: 2301–2598). PCR primers used for detection of HBV core+ RNAs are shown in brown (HBV: 2279–2392). An HBV genome is known to encode a 3.5 kb precore specific RNA which is responsible for the production of precore and HBeAg proteins. This 3.5 kb precore transcript is longer than the 3.5 kb pgRNA by 25–30 nt at the 5′ terminus [45]. The primer pairs HBV 2301–2598 and 2414–2606 cannot distinguish between the precore RNA and pgRNA. However, our HBV replicon system (plasmid pCHT-9/3091) does not produce the 3.5 kb precore RNA [30]. Core+ RNAs refer to the 3.5 kb pgRNA, 3.5 kb precore RNA, and the 2.2 kb major spliced RNA. HBc probe (nt 1903–2447) in Northern blot analysis was specific for HBV core+ RNAs. HBV probe (nt 1521–3164) was prepared for Southern blot analysis of viral replication. C: HBV core, preC: HBV precore, P: polymerase, preS1, preS2, and S: HBV envelope, X: HBx. PRE: posttranscriptional regulatory element [19].
© Copyright Policy
Related In: Results  -  Collection

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

pone-0106683-g001: An HBV transcription map is illustrated.All HBV (ayw) transcripts are shown in solid lines, and a major intron of HBV pgRNA is shown in a dashed line (HBV: 2447–489). PCR primers used for detection of all species of HBV RNAs are shown in blue (HBV: 1444–1702), and primers used for non-spliced pgRNA are shown in red and green (HBV: 2414–2606, HBV: 2301–2598). PCR primers used for detection of HBV core+ RNAs are shown in brown (HBV: 2279–2392). An HBV genome is known to encode a 3.5 kb precore specific RNA which is responsible for the production of precore and HBeAg proteins. This 3.5 kb precore transcript is longer than the 3.5 kb pgRNA by 25–30 nt at the 5′ terminus [45]. The primer pairs HBV 2301–2598 and 2414–2606 cannot distinguish between the precore RNA and pgRNA. However, our HBV replicon system (plasmid pCHT-9/3091) does not produce the 3.5 kb precore RNA [30]. Core+ RNAs refer to the 3.5 kb pgRNA, 3.5 kb precore RNA, and the 2.2 kb major spliced RNA. HBc probe (nt 1903–2447) in Northern blot analysis was specific for HBV core+ RNAs. HBV probe (nt 1521–3164) was prepared for Southern blot analysis of viral replication. C: HBV core, preC: HBV precore, P: polymerase, preS1, preS2, and S: HBV envelope, X: HBx. PRE: posttranscriptional regulatory element [19].
Mentions: Unlike the aforementioned large DNA viruses, HBV is the smallest DNA animal virus with a genome size near 3.2 kb [3], [18]. As shown in Fig. 1, major non-spliced HBV RNA transcripts include the 3.5 kb pgRNA, 3.5 kb precore RNA, 2.3 kb/2.1 kb HBV surface antigen (HBsAg) RNAs, and 0.7 kb HBx specific RNA. There are two important functions of the 3.5 kb pgRNA. One is to serve as a template of reverse transcription for an HBV genome, and the other is as an mRNA template for translation of polymerase and core protein. Previously, nuclear export of non-spliced HBsAg specific RNAs had been actively investigated. A RNA cis-element, so-called post-transcriptional regulatory element (PRE), was proposed to be important for the nuclear export of HBsAg specific RNAs [19]–[24]. In contrast to HBsAg specific RNAs, very little has been studied regarding how pgRNA and spliced RNAs are exported. Among the multiple HBV spliced transcripts, the most predominant HBV spliced RNA species has a size around 2.2 kb (Fig. 1) [25].

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