| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
J. Biol. Chem., Vol. 279, Issue 42, 43437-43447, October 15, 2004
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
¶**
From the
Heinrich-Pette Institut für Experimentelle Virologie und Immunologie an der Universität Hamburg, Martinistrasse 52, D-20251 Hamburg and ||Institut für Molekulare Biotechnologie, Abteilung Biochemie, Beutenbergstrasse 11, D-07745 Jena, Germany
Received for publication, February 27, 2004 , and in revised form, August 6, 2004.
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
HBV is a noncytopathic, hepatotropic virus with a 3.2-kb circular DNA genome. After conversion into a covalently closed circular DNA, this genome serves in the nucleus as a template for transcription of all viral RNAs. Synthesis of these transcripts is driven by at least four promotors, leading to a large size heterogeneity with many different 5'-ends, whereas they all have very similar 3'-ends because of processing at the same polyadenylylation site (6). The so-called pregenomic RNA (slightly longer than genome length) is encapsidated into nucleocapsids where it is reverse-transcribed into viral DNA. This RNA serves also as messenger for synthesis of the viral P protein as well as for the core protein. The viral surface proteins as well as a regulatory protein with a role in hepatocarcinogenesis, designated HBx, are translated from subgenomic mRNAs at 2.4, 2.1, and 0.7 kb in length.
Recently, several factors and cis-acting elements involved in the post-transcriptional control of the HBV gene expression were described. A number of publications highlighted the potential of cytokines to induce the post-transcriptional down-regulation of HBV RNA (for review see Ref. 7) in an HBV transgenic mouse model (8), in human hepatoma cells (9, 10), as well as the inhibition of duck HBV replication (11–13). In the HBV transgenic mouse model, the cytotoxic T lymphocyte (CTL) response to HBV antigens was shown to inhibit HBV replication by a post-transcriptional, noncytotoxic mechanism, leading to effective viral RNA degradation as induced by interferon-
and tumor necrosis factor-
(8, 14, 15). While trying to evaluate the intracellular mechanism(s) responsible for the cytokine-mediated post-transcriptional destabilization of HBV RNA, the mouse La autoantigen (homologue to human La autoantigen) has been identified. The La protein interacts with a small cis-acting element located within the viral RNA between nt 1275 and 1291 (16, 17). The tight temporal correlation between the cytokine-mediated down-regulation of HBV RNA and the cytokine-induced fragmentation of full-length La led to the assumption that full-length La stabilizes HBV RNA by interacting with the cis-acting element (18). Recently, it has been shown that HBV RNA is cleaved close to the La-binding site by an endoribonucleolytic activity present in nuclear extracts prepared from HBV transgenic mice (19). Up-regulation of this activity coincided with the cytokine-induced fragmentation of La and the degradation of HBV RNA, supporting the assumption that HBV RNA is more accessible to endoribonucleolytic cleavage after the disappearance of full-length La protein. More specifically, we hypothesize that La, in concert with additional trans-acting factors, forms an HBV RNA ribonucleoprotein complex stabilizing HBV RNA.
The La protein has been described as important cellular factor involved in the RNA metabolism of a variety of viruses. Most often a function of La was attributed to the translational regulation of viruses like polio and hepatitis C (20–23). In cultured cells relocalization of the predominantly nuclear La protein into the cytoplasm during infection was frequently observed (24, 25). Moreover, La protein was reported to stabilize not only cellular histone mRNA (26) but also RNA of hepatitis C virus (27).
We studied whether hLa is associated with full-length HBV RNA, and we evaluated the importance of the La-binding site for the half-life of HBV RNA. We show that HBV RNA is co-precipitated with hLa, a strong indication for a physical interaction between hLa and HBV RNA in living cells. In addition, we demonstrate in transient transfection experiments that disruption of the proposed La-binding site diminishes the HBV RNA half-life. Furthermore, we show that recombinant hLa binds WT and mutant HBV RNA with the same affinity but with lower specificity, thus arguing for the requirement of accessory factors for a specific interaction. Collectively, these results support the concept that hLa contributes to HBV RNA stability.
|
EXPERIMENTAL PROCEDURES |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
Cell Culture and Transfection Procedure—1.3 x 106 Huh-7 human hepatoma cells were grown as monolayers in 4 ml of Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum per 60-mm dish. All plasmids used for transfection were purified by using the Maxiprep kit (Qiagen). Transfections were performed using FuGENE 6 Transfection Reagent (Roche Applied Science) according to the manufacturer's protocol. HBV expression plasmid (1.5 µg) was co-transfected with 0.5 µg of pCMV/SEAP (kindly provided by J. Taylor, Philadelphia), which codes for a secreted alkaline phosphatase, or, if indicated, with 0.07 µg of pEGFP-N1. To monitor transfection efficiency, Northern blot membranes were hybridized with a GFP mRNA-specific probe, or the amount of secreted alkaline phosphatase activity secreted into the medium of transfected cells was determined as described previously (9).
In Vitro Transcription for Northern Blot Analysis—HBV-specific antisense RNA probe (referred to as HBV probe 1, spanning nucleotides 1403–2907) was generated by in vitro transcription with T3 RNA polymerase (Promega) by using the XhoI-linearized plasmid pBSK c/x (kindly provided by G. Sommer, Hamburg, Germany). GAPDH mRNA antisense RNA probes were generated by in vitro transcription of the SacI-linearized plasmid pBSK-GAPDH (kindly provided by K. Harbers, Hamburg, Germany) using T7 RNA polymerase (Promega). DNA templates used for the in vitro transcription to generate antisense RNA probes specific for HBV pregenomic RNA, histone 2A mRNA, and GFP mRNA were raised by PCR. pWA 175 (containing the histone H2A/a gene, kindly provided by W. Albig, Göttingen, Germany) was used for the production of DNA templates for the generation of histone 2A antisense transcripts. Two primers were used for PCR as follows: the antisense primer (5'-gga tcc taa tac gac tca cta tag gGA ACA TTG AGA TTT CAG GC-3') contained a restriction site for ClaI (shown with underlines), the T7 RNA polymerase promotor sequence (shown in boldface), and the histone 2A sequence spanning nt 829–847 (shown by capitol letters); and the sense primer contained histone 2A sequences from nt 19 to 37 (5'-CAG CAG TGA GAA TGA ACG C-3'). Plasmid pEGFP-N1 was used for the production of DNA templates for the generation of GFP antisense transcripts. Two primers were used as follows: the antisense primer (5'-gga tcc taa tac gac tca cta tag gGT CCA TGC CGA GAG TGA TCC C-3') contained a restriction site for ClaI (shown with underlines), the T7 RNA polymerase promotor sequence (shown in boldface), and the GFP sequence (shown by capitol letters); and the sense primer contained GFP sequences (5'-CCT GGT CGA GCT GGA CGG C-3'). PCRs for the templates were performed with 1 ng of plasmid, and the mixture contained 80 pmol of each primer in 1x PCR buffer, 0.2 mM GTP, ATP, TTP, and CTP, and 2.5 units of TaqDNA polymerase (Roche Applied Science). PCR was performed as follows: 5 min at 95 °C, followed by 35 cycles of 1 min at 95 °C, 1 min at 56 °C, and 1 min at 72 °C and finally once for 5 min at 72 °C. The PCR products were purified by size exclusion using Microspin G-25 columns (Amersham Biosciences), according to the manufacturer's protocol, ethanol-precipitated, and used as templates for in vitro transcription. Transcription reactions were carried out with 0.5–1.0 µg of linearized plasmid or PCR product in a final volume of 20 µl in transcription buffer (Promega) containing 0.31 mM ATP, CTP, and GTP, 0.25 µM UTP, and 5.0 µM [-32P]UTP (800 Ci/mmol) (Hartmann Analytic, Braunschweig, Germany), 5 mM DTT, and 20 units of RNasin (Promega). The reaction was started by the addition of 20 units of T7 RNA polymerase (Promega). After incubation for 45 min at 37 °C, another 20 units of T7 RNA polymerase were added, and the reaction was continued for 45 min at 37 °C. The reaction was terminated by adding 10 µg of yeast tRNA and 1 unit of DNase I (Promega) and incubated for 15 min at 37 °C. Unin-corporated nucleotides were removed by using Microspin G-25 columns.
Northern Blot Analysis—Huh-7 cells were harvested for 48 h or as indicated after transfection. Total RNA was prepared by using TriPure Isolation Reagent (Roche Applied Science) according to the manufacturer's protocol. 10 µg of total RNA was separated on a 1.2% agarose-formaldehyde gel. RNA was blotted onto a nylon membrane (Osmonics, Westborough, MA) and hybridized with 32P-labeled in vitro transcribed RNA probes overnight at 68 °C. To standardize transfection efficiency and RNA loading, Northern blots were hybridized with 32P-labeled in vitro transcribed antisense GFP probe and antisense glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or antisense histone 2A probes, respectively. Blots were exposed to Fuji imaging screens, and signals were quantified by Fujix BAS 2000 bio-imaging analyzer (Fuji, Japan) and by TINA software (Raytest, Germany).
Determination of Pregenomic HBV-RNA Half-life by Using the Tet-Off System—Huh-7 cells were transfected with 1.5 µg of ptetHBV-WT or ptetHBV-M2 and 0.5 µg of pUHD-TA, expressing tTA, the tetracycline-controlled transactivator. Synthesis of pregenomic RNA was blocked by addition of 500 ng/ml doxycycline to the culture medium 24 or 40 h after transfection. Cells were harvested directly upon addition of doxycycline or 3, 6, and 8 h thereafter. Total RNA was prepared and analyzed by Northern blot analysis.
Co-immunoprecipitation and RT-PCR—For immunoprecipitation of endogenous hLa-RNA complexes, about 107 HepG2 2.15 cells were harvested, and cell lysates were prepared as described previously (29). Briefly, cells were washed twice with ice-cold PBS, collected in 500 µlof ice-cold polysomal lysis buffer containing 10 mM HEPES, pH 7.0, 100 mM KCl, 5 mM MgCl2, 1 mM DTT, and 0.5% Nonidet P-40. 120 units of RNasin (Promega), 4 µl of ribonucleoside vanadyl complex (200 µg/µl, Sigma), and protease inhibitor mixture (Roche Applied Science) were freshly added. Cells were pipetted up and down and incubated for 10 min on ice. The lysate was centrifuged at 14,000 x g for 10 min at 4 °C, and the supernatant was removed and centrifuged again. The final supernatant was stored on ice until use. Protein A-Sepharose 4 fast flow beads (Amersham Biosciences) were blocked with bovine serum albumin and pre-coated with specific antibodies as described previously (29). Mouse anti-La antibody (about 7 µg of SW5, kindly provided by M. Bachmann) or a purified mouse IgG (7.5 µg per reaction, Sigma) were used. The antibody-coated beads were washed six times with 900 µl of NT2 buffer containing 50 mM Tris, pH 7.4, 150 mM NaCl, 1 mM MgCl2, 0.05% Nonidet P-40. Finally, the pellets were resuspended in 850 µl of NT2 buffer, supplemented with 4 µl of vanadyl ribonucleoside complex, 160 units of RNasin, and 20 mM EDTA, and 100 µl of cell lysate. The immunoprecipitation reactions were rotated (head over head) at 4 °C overnight. Beads were collected by centrifugation at 800 x g for 2 min, and pellets were washed six times with ice-cold NT2 buffer. Pellets for Western blot analysis were resolved in 100 µl of SDS denaturing buffer, and pellets for RNA analysis were resuspended in 100 µl of NT2 buffer supplemented with 0.01% SDS, 40 µg of proteinase K (Roche Applied Science) and incubated at 50 °C for 30 min in a thermomixer (1000 rpm); subsequently, RNA was extracted by using the TriPure isolation reagent (Roche Applied Science) and precipitated in the presence of 20 µg of glycogen. For Northern blot analysis, the complete RNA pellet was solved in 40 µl of loading buffer, loaded onto a 1.2% agarose-formaldehyde gel, and processed as described above. Western blot analysis was performed by standard methods, using 12.5% polyacrylamide gels and nitrocellulose membranes.
For RT-PCR analysis, the RNA pellets (starting material, pellets, and supernatants) were solved in 43 µl of diethyl pyrocarbonate-treated water; subsequently, 5 µl of RNase-free DNase reaction buffer (Promega) and 2 units of RNase-free DNase (Promega) were added and incubated for 30 min at 37 °C. DNase was inactivated by heating the sample for 10 min at 70 °C. 2 µl of DNase-treated RNA was used as template for RT-PCR. RT-PCR was conducted by using the Titan One-Tube RT-PCR kit (Roche Applied Science), and the following primer pairs were used: HBV sense, 5'-GGACCCCTTCTCGTGTTACAG-3', and antisense, 5'-GCTGGTTGTTGAGGATCCTGG-3'; L-37 sense, 5'-ACGAAGGGAACGTCATCGT-3', and antisense, 5'-CATTCGACCAGTTCCGGT-3'; hnRNP E2 sense, 5'-GACACCGGTGTGATTGAAGG-3', and antisense, 5'-CTCAGGACAATTCCCTTCTGAG-3'.
Expression, Purification, Phosphorylation, Dephosphorylation of Recombinant hLa Protein, and Preparation of Nuclear Extracts—Recombinant hLa protein was prepared as described previously (17). Phosphorylation of recombinant hLa was performed for 3 h at 37 °Cin30-µl reactions containing 1 µg of hLa, 0.2 µl of casein kinase II (New England Biolabs), 3 µl of 10x reaction buffer, and 6 µl of 1 mM ATP. Dephosphorylation occurred with shrimp-alkaline phosphatase (Roche Applied Science) or 
-protein phosphatase (New England Biolabs) according to the manufacturer's instructions. All reactions were controlled by two-dimensional SDS-PAGE, and efficient hyperphosphorylation or dephosphorylation was verified (data not shown).
For preparation of nuclear extracts, Huh-7 cells were plated in 15-cm dishes, grown until confluence, washed twice with 1x PBS, harvested in 1 ml of 1x PBS, and collected by centrifugation at 500 x g for 5 min. Cells were resuspended in 1 ml of 1x PBS and pelleted by centrifugation for 10 s at 10,000 x g. Subsequently, the cell pellet was suspended in 600 µl of buffer A containing 20 mM HEPES, pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.2 mM EDTA, 0.1% Nonidet P-40, and at the time of use 0.5 mM DTT and 40 µl/ml protease inhibitor mix (Roche Applied Science) were added. Samples were placed on ice for 10 min. Cell lysis was controlled by light microscopy. Nuclei were collected by centrifugation for 1 min at 8000 x g and resuspended in 150 µl of buffer B containing 20 mM HEPES, pH 7.9, 1,5 mM MgCl2, 10mM KCl, 420 mM NaCl, 0.2 mM EDTA, 25% glycerol, and freshly added 0.5 mM DTT and 40 µl/ml protease inhibitor mix. Samples were placed on ice for 20 min, and cell lysate was cleared by centrifugation at 10,000 x g for 10 min and stored at –70 °C.
Gel Retardation Assay—The standard binding reaction was carried out in a final volume of 40 µl with 200 ng of purified recombinant hLa protein as indicated and about 200,000 cpm or molar concentrations, as indicated, of 32P-radiolabeled HBV RNA.B in binding buffer containing 10 mM Tris-HCl, pH 7.4, 3 mM MgCl2, 200 mM NaCl, 0.5 mM EDTA, and 0.5% Nonidet P-40 (unless otherwise stated). The in vitro transcribed 32P-labeled RNA.B was denatured at 75 °C for 10 min and renatured by cooling slowly to room temperature prior to addition to the reaction mixture. Samples were incubated for 30 min at room temperature. After addition of 5 µl of electrophoresis buffer containing 10% glycerol and 0.01% bromphenol blue, reaction mixtures were separated on an 8% native polyacrylamide gel (18 x 18 cm) for 3–4 h at 200 V at room temperature. The gels were pre-run at 240 V for 1 h in 1x TBE containing 45 mM Tris, 45 mM boric acid, 1 mM EDTA. Gels were dried (for 1.5 h at 80 °C) on Whatman paper (Whatman) using a Bio-Rad Slab Dryer model 483, and signals were evaluated using a FUJIX BAS 2000 PhosphorImager System (Fuji, Germany) or documented via exposure to x-ray films.
For supershift analysis, 100 ng of NiNTA-purified hLa was preincubated with 5 µl of antibody (as indicated in the figures) and 20 units of RNasin (Promega) in a 20-µl volume of 10 mM Tris-HCl, pH 7.4, 4 °C for 1 h, before the nuclear extracts and the other components of the standard reaction and the labeled RNA were added.
Analysis of RNA-Protein Interactions with Surface Plasmon Resonance Technology—Real time affinity determination of recombinant human La protein with different RNAs was achieved by using a BIA-core-2000 device (BIAcore, Germany) producing the "sensorgram." All experiments were performed at 25 °C with a nitrilotriacetic acid (NTA) sensor chip (Biacore) and the histidine-tagged recombinant hLa proteins and gel-purified in in vitro transcribed HBV RNA.B-WT, -M2, and -M7. The chip contains four flow cells that were treated and detected simultaneously. All four flow cells were loaded with 25 µl of loading buffer (500 µM NiSO4 in running buffer (see below)) at 10 µl min–1, which resulted in a stable average increase of 100–250 response units (RUs). After washing flow cells for 150 s at 10 µl min–1 with running buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 50 µM EDTA, 0.005% Tween 20), proteins were injected from 0.2 µM solutions with 5 µl min–1 into flow cells 1–3. Protein attachment to the chip surface was manually restricted to yield a mean difference of 1000–1300 RUs, where 1000 RUs corresponds to 
1.0 ng/mm2 (30). Later, RNA was injected into all four flow cells, thereby obtaining triple values (flow cells 1–3) each with one negative control (flow cell 4) reflecting potential unspecific binding of RNA to the nickel-coated chip surface. Application of RNAs resulted in RU differences of 30–500, depending on the amount loaded. To exclude that any RNA potentially still bound to the fixed protein after a study could interfere with subsequent analysis, the chip surface was regenerated after measurement by applying 30 µl of regeneration buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 100 mM EDTA, 0.005% Tween 20) with 10 µl min–1 before the chip was loaded and prepared for successive measurements. RNAs were supplied in at least four different concentrations, which resulted in consistent data where the level of response units after RNA loading increased with the amount of RNA loaded. Data were evaluated with the BIAevaluation 3.0.2 software (Biacore); curves of flow cell 4 without protein were subtracted from the corresponding protein containing flow cells 1–3, and results were fitted into Langmuir's model of 1:1 binding. As for the fit, software internal 
2 standards never exceeded values above 0.8, and Rmax values were in the expected ranges, and the residuals had a mean deviation from fitted graphs of less than 1.0 (not shown). Estimated half-life of a given RNA-protein complex was calculated as t
= –ln(0.5)/kd.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
|
|
In an attempt to investigate in greater detail whether the La-binding site in the HBV RNA mediates stabilization of the viral transcripts, we designed cell culture experiments using the HBV expression plasmid pHBV-WT. pHBV-WT encodes the pregenomic RNA under the control of a CMV promotor, whereas the 2.4- and 2.1-kb mRNAs encoding the surface proteins were controlled by endogenous promotors. This plasmid was used to introduce several mutations into the La-binding site referred to as pHBV-M2, pHBV-M7, pHBV-M12, and pHBV-M13 (Fig. 2C). To study the influence of these mutations on HBV RNA levels, wild type and mutant HBV expression plasmids were transiently transfected into HuH-7 cells, and RNA was prepared at the indicated time points thereafter and analyzed by Northern blotting. Quantified HBV mRNA levels were normalized against transfection efficiency controlled by co-expression of GFP or serum alkaline phosphatase, and RNA loading was standardized by detection of GAPDH or histone 2A mRNAs.
First we monitored HBV RNA levels transcribed from pHBV-WT, pHBV-M2, pHBV-M7, pHBV-M12, and pHBV-M13 at 16 and 24 h post-transfection. Differences in the levels of the HBV RNA in cells transfected with pHBV-M13, pHBV-M7, pHBV-M12, or pHBV-WT were not detectable (data not shown), indicating that the respective mutations did not affect mRNA levels. In contrast, lower HBV RNA levels compared with pHBV-WT-transfected cells were observed at both time points when pHBV-M2 was transfected (data not shown). Very similar results were obtained when the RNA levels transcribed from pHBV-WT, pHBV-M2, pHBV-M7, pHBV-M12, and pHBV-M13 were prepared 48 h post-transfection and analyzed by Northern blotting, again only RNA levels transcribed from pHBV-M2 were lower compared with HBV RNA levels transcribed from pHBV-WT (Fig. 3A, lanes 1 and 2 versus 3 and 4). Quantification of six unrelated experiments performed in duplicate and with different plasmid preparations revealed an average decrease both of the pregenomic RNA and of the 2.4- and 2.1-kb subgenomic RNAs by 60% when comparing transcript levels in pHBV-WT- and pHBV-M2-transfected cells (Fig. 3B), indicating a substantial importance of this cis-acting element for HBV RNA expression.
|
|
|
1.0 x 10–9 (16)) and recombinant hLa (KD 0.8 x 10–9 (17)) for HBV RNA.B-WT as acquired by UV-cross-links or gel retardation experiments, respectively. In conclusion, mouse La in nuclear extracts binds HBV RNA.B both with high affinity and high specificity, whereas highly purified recombinant hLa binds HBV RNA.B with high affinity but with low specificity, emphasizing our previous results suggesting that accessory factors specify the binding of La to HBV RNA. Modulation of the hLa-HBV RNA.B Interaction by Nuclear Factors and Phosphorylation—To test whether additional cellular factors modulate the interaction between highly purified recombinant hLa and HBV RNA.B, we analyzed the binding in the presence of nuclear extracts prepared from Huh-7 cells. Different amounts of recombinant hLa or nuclear extracts were incubated with labeled HBV RNA.B-WT and analyzed by electrophoretic mobility shift assay. As shown in Fig. 6, 50 and 100 ng of recombinant hLa without nuclear extract form two major complexes with HBV RNA.B indicated as monomers and multimers (Fig. 6A, lanes 2–7). Next, different amounts of nuclear extracts were tested revealing the formation of a major complex of low mobility (indicated by an arrowhead, Fig. 6B, lanes 2–7) and a minor complex of higher mobility (indicated by an asterisk, Fig. 6B, lanes 2–7). The nature of these complexes is unknown, but it might be possible that the complex indicated by an asterisk represents a complex between endogenous La and HBV RNA.B because this band disappeared in supershift experiments (Fig. 6C, lanes 4 and 5 versus 10 and 11). Addition of increasing amounts of nuclear extract to 50 ng of recombinant hLa led to reduced signals of the multimeric and to increased signals of the monomeric hLa-HBV RNA.B complex (Fig. 6A, lanes 4 and 5 versus 8–13). The predominant formation of the monomeric La-HBV RNA.B complex in presence of nuclear extracts was confirmed if increasing amounts of recombinant hLa were added to 2.0 µg of nuclear extract (Fig. 6B, compare lanes 8–13 with 14 and 15). To verify that hLa-specific RNPs were formed, supershift experiments were performed. As shown in Fig. 6C, La-specific antibodies shifted the multimeric/monomeric hLa-HBV RNA.B complexes (lanes 2 and 3 versus 8 and 9) as well as the dominant monomeric hLa-HBV RNA.B complex formed in presence of nuclear extracts (lanes 6 and 7 versus 12 and 13). In contrast an unrelated antibody was unable to shift the hLa-HBV RNA.B complex (Fig. 6C, lanes 14 and 15). These data demonstrate that components present in nuclear extracts modulate the binding behavior by preferentially supporting the formation of monomeric hLa HBV RNA.B RNPs. These findings indicate that additional factors are involved in specific formation of hLa/HBV RNA RNPs.
|
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
Here, for the first time, we provide experimental data suggesting an association between hLa and HBV RNA in human hepatoma cells. The co-precipitation of full-length HBV RNA with hLa as shown in this report strongly suggests that La physically interacts with HBV RNA in living cells. Although we cannot rule out the formal possibility that the interaction between hLa and HBV RNA is mediated by a bridging factor, the co-precipitation of hLa and HBV RNA would still mean that hLa is part of HBV RNPs, presumably stabilizing the viral RNA. The association of hLa with HBV RNA or HBV stabilizing RNPs combined with previous results showing that HBV RNA degradation coincides with mouse La fragmentation and that mouse La interacts specifically with the predicted stem loop structure (16, 18) strongly support the hypothesis that hLa is involved in the post-transcriptional regulation of HBV RNA. Notably, the putative regulatory element bound by mouse and human La is located in the 3'-region of all viral RNAs targeted by the cytokine-induced post-transcriptional mechanism. To prove the significance of the La-binding site for viral RNA stability, several mutations were introduced, and the half-life of the pregenomic RNAs was determined. These experiments revealed that only mutations disrupting the 4 bp in the stem diminished HBV RNA half-life. Conversely, changing the nucleotides in the stem but maintaining base pairing does not affect HBV RNA levels. These findings indicate that probably structural rather than sequential features of the RNA element are important to uphold HBV RNA half-life. In this context, it is important to note that the sequence of the stem loop is highly conserved between the diversity of HBV genotypes and isolates (Fig. 1B). Our study raises the interesting question whether the nucleotide substitutions within the predicted structure involved in La protein binding as present in various HBV genotypes and isolates have positive or negative consequences for the stability of the viral RNAs and efficiency of virus propagation.
It was shown that mutation M2 diminishes pregenomic RNA half-life by 2 h, indicating that HBV RNA stability depends in part on the structural features of this element. Note, however, with the current data we cannot exclude the possibility that the half-life of pregenomic RNA M2 was reduced by La-independent factors. Therefore, it is reasonable to consider that additional regulatory elements within the viral RNA and cellular factors interacting with HBV RNA and/or the La protein have to be integrated to fully understand the decay of HBV RNA under steady state conditions like in our experiments. Furthermore, the variety and remarkable changes in gene expression profiles observed after CTL injection into HBV transgenic mice (34, 35), probably inducing the inhibition of viral replication and inducing HBV RNA decay, suggest that additional cellular factors and cellular events have to be identified in order to understand the induced decay mechanism specific for the HBV RNA. It is reasonable to speculate that structural features of the La-binding site determine the interaction with trans-acting factors, and it might therefore be possible that mutation M2 leads to disruption of hLa containing RNPs normally protecting HBV RNA against endoribonucleolytic cleavage. However, changing the structure of this RNA element and therefore the HBV RNP composition might be not enough to induce efficient degradation as observed after CTL injection in the mouse system in which HBV RNA degradation was accompanied by the processing of mouse La and the augmentation of an endoribonuclease activity (19). Hence, the identification of factors involved in La-HBV RNP formation is of great importance and might shed light on the mechanism regulating HBV RNA decay under different cellular conditions.
This view concurs with the current accepted pathways of mRNA degradation. In general it is believed that the fate of an mRNA is regulated by trans-acting factors interacting with cis-acting elements within an RNA molecule, thereby protecting or destabilizing the RNA (for review see Ref. 36). Among the best characterized RNA-destabilizing elements are the AU-rich regions first described to regulate the mRNA stability of short lived RNAs (37). The AU-rich regions are recognized by a group of proteins called AU-rich binding proteins (38). In most cases these proteins destabilize RNAs by interacting with the AU-rich regions, but stabilizing effects have also been reported (39–41). To our knowledge, it is not known how exactly this interaction destabilizes/stabilizes RNA. The stable -globin mRNA has been described to interact with several proteins forming the so-called -complex associated with the 3'-untranslated region. Changes in the composition of this complex lead to endoribonucleolytic cleavage of this RNA (42, 43), demonstrating that a variety of trans-acting factors are involved. The requirement of several trans-acting factors was also described for interleukin-2 mRNA stability (44). The transferrin receptor mRNA is stabilized by the iron-regulatory protein, which interacts with the 3'-end of the RNA if cellular iron levels are low, thereby protecting the RNA against endoribonucleolytic cleavage (45). An increase in cellular iron concentration leads to reduced affinity of the iron-regulatory protein to the transferrin receptor mRNA and subsequently to the degradation of the RNA (reviewed in Ref. 46). Furthermore, intensive studies on the post-transcriptional control of albumin and vitellogenin mRNA have shown that albumin mRNA is destabilized after estrogen treatment in parallel with the activation of the polysomal ribonuclease 1 (PMR-1 (47, 48)), whereas vitellogenin mRNA is stabilized by binding of vigilin, protecting the mRNA against PMR-1 cleavage (49). These reports clearly demonstrate and support our assumption that a complex regulatory mechanism determines HBV RNA stability by integrating the action of regulatory trans-acting factors interacting with mRNA stability determinants. Notably, in competitive UV cross-linking experiments, the binding of mouse La in nuclear extracts was highly specific for HBV RNA.B-WT but not for the mutant M2 (16), indicating that under such experimental settings mouse La was able to discriminate between WT and mutant HBV RNA-M2. In contrast highly purified, recombinant hLa binds HBV RNA.B WT and M2 in an unspecific manner, suggesting that additional components, which were present in mouse nuclear extracts, are required for a specific interaction. Until those factors are characterized, it is probably not feasible to explore a difference in binding specificity of recombinant hLa to HBV RNA.B-WT versus -M2 in vitro and to show more directly that the half-life of pregenomic RNA depends on the association with hLa. We recently identified an hLa-interacting protein, and preliminary data show that this RNA-binding protein interacts specifically with HBV RNA.B,2 suggesting that this protein might modulate the association between HBV RNA.B and hLa.
Previously, it has been shown that binding of mouse La to HBV RNA.B in vitro was abolished after dephosphorylation of the La-containing nuclear extract (16). In this previous study, it was not possible to distinguish whether impaired RNA binding activity was because of dephosphorylation of the phosphoprotein La or because of other factors present in the nuclear extracts. Our observation that phosphorylation or dephosphorylation of recombinant hLa had no effect on binding HBV RNA.B, whereas dephosphorylation of nuclear extracts partially inactivated the activity-promoting formation of the monomeric La complex, showed that a phosphorylation-dependent activity modulates hLa binding to HBV RNA.B. We believe that the initial data support our view that the binding specificity of hLa to HBV RNA.B is mediated by phosphorylation-dependent host factors. In the absence of those factors recombinant hLa binds HBV RNA.B with low specificity. In this context it is important to note that the newly identified hLa-interacting protein is also a phosphoprotein and that it will of interest to study whether the interaction between hLa and this protein might be regulated by phosphorylation. Furthermore, it will be interesting to find out which cellular signaling pathway affects the proposed phosphorylation-dependent composition of HBV RNPs thereby potentially regulating the turnover of viral RNAs. Most importantly, it has been shown recently that Janus kinase activity is required for the antiviral effect of interferons on HBV replication (35), indicating that cell signaling pathways and thereby phosphorylation/dephosphorylation events are required. This might also apply for cytokine-induced degradation of HBV RNA decay as supported by our finding that binding of mouse and human La to HBV RNA is modulated by accessory factors in a phosphorylation-dependent manner. Previously, it has been shown that a variety of cellular factors involved in the regulation of mRNA stability, like RNA-binding proteins, was regulated by phosphorylation/dephosphorylation (50, 51) and that ribonuclease activities were elevated by hormones (48, 52, 53), indicating that posttranslational modification and signal transduction pathways are important cellular mechanisms determining mRNA levels.
As a model, we assume that cytokine-induced HBV RNA degradation is initiated at the La-binding site, and this concept is supported by the following findings. First, the cytokine-induced HBV RNA degradation occurring in the mouse system in parallel with La processing is associated with increased endoribonuclease activity that leads to cleavage of the HBV RNA close to the La-binding site (18, 19). Second, mutagenesis of the La-binding site reduces HBV RNA half-life from 5 to 3 h in cell culture experiments. Third, binding of the mouse protein as well as of the human La protein to HBV RNA.B can be modulated by phosphorylation/dephosphorylation of accessory factors. The cytokine-induced HBV RNA decay might be initiated by the loss of protective factors (e.g. La) inducing structural changes and/or exposing the cleavage site located close to the predicted stem loop to endoribonuclease (19), thereby initiating HBV RNA degradation.
We conclude that the La-binding site together with the cleavage sites compose a destabilizing element leading to HBV RNA degradation under specific cellular conditions. It will be of major importance to identify host factors interacting with hLa and/or HBV RNA in a phosphorylation-dependent manner to understand in more detail the initiation of HBV RNA degradation.
|
FOOTNOTES |
|---|
Present address: Developmental Biology Program, Sloan-Kettering Institute, New York, NY 10021.
¶ Present address: Institute of Virology, University Hospital Charité, Schumannstr. 20/21, D-10098 Berlin, Germany.
** To whom correspondence should be addressed: Heinrich-Pette-Institut für Experimentelle Virologie und Immunologie, Universität Hamburg, Postfach 201652, 20206 Hamburg, Germany. Tel.: 49-40-48051-225; Fax: 49-40-48051-222; E-mail: heise{at}hpi.uni-hamburg.de.
1 The abbreviations used are: RNPs, ribonucleoprotein particles; hLa, human La autoantigen; hnRNP, heterogeneous nuclear ribonucleoprotein; WT, wild type; HBV, hepatitis B virus; CTL, cytotoxic T lymphocytes; RT, reverse transcriptase; NiNTA, nickel-nitrilotriacetic acid; DTT, dithiothreitol; RUs, response units; GFP, green fluorescent protein; PBS, phosphate-buffered saline; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; nt, nucleotides; CMV, cytomegalovirus.
2 G. Tettweiler and T. Heise, unpublished data.
|
ACKNOWLEDGMENTS |
|---|
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
