1 Department of Protein Engineering, Biomedical Research and Study Centre, University of Latvia, Ratsupites Str., 1, LV-1067 Riga, Latvia
2 Department of Biosciences at Novum, Karolinska Institute, S-141 57 Huddinge, Sweden
Correspondence
Anna Zajakina
Anna{at}biomed.lu.lv
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ABSTRACT |
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INTRODUCTION |
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The main HBV mRNA (3·4 kb) or pregenome RNA (pgRNA), serves as a template for reverse transcription and genomic DNA formation inside the viral nucleocapsid, as well as for translation of the two proteins: HBc and Pol in the bicistronic way, where the Pol protein is synthesized via ribosome leaky scanning mechanism (Lin & Lo, 1992; Fouillot et al., 1993
). Another genomic transcript the precore RNA (pcRNA) differs from that of the pgRNA by the presence of an additional initiation codon of the preC protein at its 5' end (Ou et al., 1986
; Yaginuma et al., 1987
). The three HBs proteins have two 3' co-terminal mRNAs (2·4 and 2·1 kb) (Heermann et al., 1984
). The 2·4 kb mRNA is a template for all three related HBs proteins, which are translated from a single open reading frame (ORF) by the use of three different translation start sites, dividing this ORF into three domains: the amino-terminal pre-S1 domain, which occurs exclusively in the L protein; pre-S2 domain, which is present in both L and M proteins and forms the amino-terminal end of the M protein; and the S domain, which is common to the S, M and L proteins (see Fig. 1a
). The 2·1 kb mRNA is probably a template only for the S, or for both M and S protein synthesis. Efficient simultaneous synthesis of the three HBs variants from the same template was shown by transient expression of the L gene in different cell lines (Bruss & Vieluf, 1995
; Bruss, 1997
; Le Seyec et al., 1999
), where glycosylated and non-glycosylated forms of the L, M and S proteins were detected.
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Three ways of internal initiation might be considered context-dependent leaky scanning, when the first AUG is sometimes ignored and the distantly located start codons became functional; translation reinitiation, when the already initiated protein translation is somehow aborted or terminated and a new protein with distantly located start codon is translated; and direct internal initiation allowing access to AUG codons through IRES elements. As a possible mechanism of internal translation initiation of HBs proteins and Pol from alternative start sites in HBV mRNAs, the context-dependent ribosome leaky scanning of the mRNA was proposed (Ou et al., 1990; Fouillot et al., 1993
; Fouillot & Rossignol, 1996
; Hwang & Su, 1999
). However, this model was not studied in detail, especially for HBs initiation, where such a mechanism was suggested only by the analogy with Pol synthesis. The leaky scanning mechanism of translation provides inefficient synthesis of downstream proteins, which is true for Pol, but not for HBs proteins. The specific secondary structure of mRNA and/or the suboptimal context around the start codon, proposed by Kozak (1999)
, may be important in the case of HBs translation.
Therefore, it is important to investigate the possible leaky scanning model for internal translation of HBs proteins. In this study, we examined whether the other HBV templates, such as pgRNA and pcRNA, could promote an additional synthesis of HBs proteins, and how the length of the RNA's 5' end could interfere with translational efficiency. The expression system on the basis of Semliki Forest virus (SFV), providing effective cytoplasmic synthesis of the corresponding mRNAs (Liljestrom & Garoff, 1991), was used in these experiments.
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METHODS |
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Plasmid construction.
Constructs used in this work are represented in Fig. 1. All HBV sequences were amplified by PCR using pHBT as a template plasmid harbouring a tandem repeat of HBV320 genomes, genotype D and subtype ayw (Bichko et al., 1985
). PCR fragments were cut out with SmaI and ligated into the pSFV1 vector (Liljestrom & Garoff, 1991
), which had also been cleaved with SmaI. Oligonucleotides used for L gene amplification to create the pSFV1/L construct were: 5'-GCCCCGGGATGGGGCAGAATCTTTCCA-3' and 5'-CGCCCGGGTTTAAATGTATACCCAAAG-3' (here and below the SmaI site is underlined). Oligonucleotides used for HBV pgRNA amplification to create the pSFV1/pgRNA construct were: 5'-CCCGGGAACTTTTTCACCTCTGCCTAATCATC-3' and 5'-CCCGGGTAACTCCACAGTAGCTCCAAATTCTTTATAAG-3'. Oligonucleotides used for 3'-truncated HBV pgRNA amplification to create the pSFV1/pgRNA3'
construct were: 5'-CCCGGGAACTTTTTCACCTCTGCCTAATCATC-3' and 5'-CCCGGGTCAAGGTCGGTCG-3'. Oligonucleotides used for preC containing pgRNA amplification to create the pSFV1/pcRNA construct were: 5'-CCCGGGCCATGCAACTTTTTCAC-3' and 5'-CCCGGGTAACTCCACAGTAGCTCCAAATTCTTTATAAG-3'. The preC ATG codon is marked with bold letters. Oligonucleotides used for HBV polymerase gene amplification to create the pSFV1/Pol construct were: 5'-CCCGGGCCACCAAATGCCCCTATC-3' and 5'-CCCGGGTCAAGGTCGGTCG-3'. The Pol ATG codon is marked with bold letters.
RNA transcription and transfection.
RNA transcripts were produced in vitro from 3 µg NruI-linearized pSFV1/L, pSFV1/HBVpgRNA, pSFV1/pgRNA3', pSFV1/pcRNA and pSFV1/Pol plasmids using SP6 RNA polymerase (Fermentas). RNA (35 µg) was transfected into
1x107 BHK cells by electroporation at 850 V (2125 V cm1), 25 µF, pulsed twice, using Bio-Rad Gene Pulser apparatus without the pulse controller unit. Electroporated cells were diluted into 15 ml complete BHK medium, transferred into tissue culture plates and incubated at 37 °C (5 % CO2). About 70 % of the cells survived the transfection step.
Generation of recombinant viruses and cell infection.
For in vivo packaging of recombinant RNA into SFV particles, in vitro-transcribed RNA was electroporated into BHK cells together with SFV helper RNA under above-mentioned conditions. After 20 h, SFV particles in the culture medium were collected and frozen rapidly to be stored as a virus stock. Titres of stocks were determined by infecting cells with serial dilutions of the stocks followed by indirect immunocytochemistry assay for the expressed HBs proteins. The achieved titres were from 1x107 to 5x107 viral particles per ml. The infection of BHK cells was carried out in serum-free medium with appropriate dilution of virus stocks, which infect 100 % of cells.
Analysis of cellular RNA.
At 18 h after infection, total cellular RNA (from cells grown on 3 cm diameter plates) was isolated by Tri Reagent (Sigma) as described by the manufacturer. RNA was dissolved in 30 µl DEPC-treated water and immediately frozen at 70 °C. RNA (5 µl) was analysed by ECL direct nucleic acid labelling and detection system (Amersham Biosciences) using Northern blotting protocol as described by the manufacturer. RNA was immobilized on a Hybond-N+ membrane (Amersham Biosciences). The PCR fragment of pgRNA isolated from the agarose gel (the same as used for construction of pSFV1/pgRNA) was used for the anti-HBV probe preparation as described in ECL direct nucleic acid labelling and detection system.
Metabolic labelling of infected cells.
At 1620 h after infection, cell monolayers (on 3 cm diameter plates) were washed with PBS, overlaid with starvation medium (methionine-free MEM, 2 mM glutamine, 20 mM HEPES) and incubated at 37 °C in 5 % CO2 for 30 min. The starvation media was then replaced with the same media containing 100 µCi ml1 (3·7 MBq) of [35S]methionine (Amersham Biosciences) and incubated at 37 °C (5 % CO2) for 2 h. Cells were lysed with 300 µl lysis buffer [1 % Nonidet P-40 (NP-40), 50 mM Tris/HCl pH 7·6, 150 mM NaCl, 2 mM EDTA, 1 µg PMSF ml1] and left to stand on ice for 10 min. Lysates were transferred to microcentrifuge tubes for cell nuclei centrifugation at 3000 g for 5 min. Supernatants were used for specific protein immunoprecipitation (IP).
IP of proteins from cell lysates.
For IP of HBc and preC proteins, rabbit polyclonal anti-HBc antibodies (gift from I. Sominskaya, Biomedical Research and Study Center, University of Latvia, Riga) were used. IP of HBs proteins was performed with goat polyclonal anti-HBs antibodies (gift from V. V. Tsibinogin, Biomedical Research and Study Center, University of Latvia, Riga). Pol protein IP was done by rabbit anti-Pol Ab (gift from M. Seifer, Idenix Pharmaceuticals, MA). Cell lysates in amounts of 300 µl (pSFV1/L and pSFV1/Pol) or 600 µl (the remaining constructs) were incubated for 2 h with appropriate antibodies (12 µl) on ice. After incubation, 80 µl protein A Sepharose (Amersham) soaked in lysis buffer was added, and incubated with rotation overnight at 4 °C. Protein A Sepharose was washed twice with buffer containing 0·2 % NP-40, 10 mM Tris/HCl pH 7·5, 150 mM NaCl, 2 mM EDTA; twice with buffer containing 0·2 % NP-40, 10 mM Tris/HCl pH 7·5, 500 mM NaCl, 2 mM EDTA; and twice with 10 mM Tris/HCl pH 7·5. Protein A Sepharose pellet was resuspended in 30 µl Laemmli buffer (Laemmli, 1970) and analysed by 12 % SDS-PAGE. Gels were dried and exposed to autoradiography film (Amersham) at 70 °C for overnight or longer.
Immunocytochemical detection of intracellular HBV antigens by mAb.
BHK cells grown on sterile tissue culture chamber slides (NUNC A/S) were infected with recombinant SFV and incubated at 37 °C (5 % CO2) for 20 h. After drying the slides at room temperature, cells were fixed with ethanol/acetic acid (3 : 1) for 20 min and rinsed thoroughly (three times) in distilled water. Slides were immersed in PBS for 10 min, rinsed with PBS supplied with 0·25 % Triton X-100, incubated for 24 h in a humidity chamber at 4 °C with the anti-HBs mAb (gift from I. Sominskaya) and then rinsed in PBS with 0·25 % Triton X-100. Cells were then incubated with anti-mouse IgG conjugated with alkaline phosphatase (Sigma) at room temperature, in the dark for 1 h, then rinsed with PBS, and alkaline phosphatase activity was developed by Sigma FAST reagent, which produces an intense red stain. Finally, cells were rinsed in deionized water; counterstained with haematoxylin and mounted in glycerol gelatin (Sigma). The evaluation was done using a light microscope.
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RESULTS |
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In order to enhance efficiency of the expression system, infection of cells was used instead of transfection. Therefore, recombinant SFV particles were produced for each HBV construct by the simultaneous infection of cells with RNA of an appropriate construct and SFV helper RNA.
Expression of HBV L gene and pgRNA in BHK cells
The concomitant synthesis of HBV M and S proteins during the transient L gene expression have been described previously (Stibbe & Gerlich, 1983; Bruss, 1997
; Le Seyec et al., 1998
). For pgRNA, translation was shown for HBc and Pol proteins, but not for HBs proteins (L, M, and S) that have the start codons located more downstream. We proposed that translation of HBs proteins were also from the HBV pgRNA.
To test this idea, BHK cells infected with pSFV1/L and pSFV1/pgRNA constructs were metabolically labelled with [35S]methionine and lysed with NP-40 containing lysis buffer. The analysis of anti-HBs and -HBc immunoprecipitates in SDS-PAGE is demonstrated in Fig. 2. The L gene mRNA, efficiently synthesized in cytoplasm by SFV replicases (pSFV1/L), served as a template for translation of all three variants of HBs proteins (Fig. 2a
, lane 1), which were found in glycosylated and non-glycosylated forms corresponding to earlier findings (Stibbe & Gerlich, 1983
; Bruss, 1997
). Thus, the S products existed as non-glycosylated p24 and mono-glycosylated gp27 molecules. The M products revealed two bands: mono- and di-glycosylated gp33 and gp36 forms. The non-glycosylated form of the M protein was not identified as p31 protein. However, a protein of lower molecular mass of about 29 kDa was found. Taking into consideration that the electrophoretical mobility of the MW protein marker could vary in different gel systems, we suppose that this protein represents the non-glycosylated form of the M protein (p31). The products of the L protein were found in non-glycosylated form p39 and mono-glycosylated form gp42.
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Beside this, the anti-HBc IP of SFV1/pgRNA-infected cell lysate revealed a clear p21 band, which corresponded to the HBc protein (Fig. 2c, lane 3). An additional upper band of about 26 kDa (p26), the origin of which is still unclear (see Discussion), was also seen.
Unfortunately, we could not detect Pol protein translation in SFV1/pgRNA-infected cell lysate. The IP with anti-Pol Ab demonstrated strong non-specificity (data not shown). Moreover, the very low level of production of this protein caused by (i) translation via the ribosome leaky scanning model (Lin & Lo, 1992; Fouillot et al., 1993
) and (ii) unfavourable sequence context around the Pol start codon (Kozak, 1987
) established additional difficulties for the detection of the HBV Pol protein.
RNA analysis of infected cells
Although proteins with the molecular mass corresponding to the appropriate HBs proteins have been detected in pSFV1/L- and pSFV1/pgRNA-driven expression, the question remained whether such translation is directed only by the full-length L transcript and pgRNA as subgenomic RNAs of SFV constructs or whether other smaller mRNAs appeared during synthesis, which were used by translational machinery for production of HBs proteins.
The SFV subgenomic promoter (26S) recognized by SFV replicases (nsP14) is responsible for the recombinant mRNA production (Liljestrom & Garoff, 1991). The comparative analysis of the sequence of SFV 26S subgenomic promoter with the sequence of the HBV genome did not display any similarity (not shown), allowing us to exclude the SFV-driven synthesis of additional RNAs from HBV genome sequence.
Nevertheless, the potential capability of pgRNA to be transported into the cell nucleus (Kann et al., 1999) and to initiate production of all HBV mRNAs through the replication of the genome, prompted us to analyse HBV-specific mRNAs in infected BHK cells. Total RNA from cells producing pgRNA (pSFV1/pgRNA) and L (pSFV1/L) transcripts was isolated, and HBV-specific sequences were detected by Northern blot technique (Fig. 3
). As expected, the recombinant SFV provided synthesis of two types of mRNAs: genomic and subgenomic. In the pSFV1/L construct, genomic RNA carries 8908 bases, the subgenomic 1534 bases. The length of pSFV1/pgRNA genomic and subgenomic transcripts are: 11 047 and 3673 bases, respectively (Fig. 3
). No other mRNAs, which could be considered as natural HBs transcripts for translation of HBs proteins, were found. These results confirm the hypothesis that all HBV proteins synthesized by our constructs are translated from recombinant SFV subgenomic RNA.
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Pol gene expression provoked efficient synthesis of HBs proteins
For further investigation of internal translation initiation of HBs proteins, we shortened the 5' end of pgRNA. The construct used for this experiment represented the full-length Pol gene (pSFV1/Pol), carrying internally intact sequences of HBs proteins. Our attention was focused on the possible enhancement of the L, M and S synthesis from Pol template in the absence of preferable HBc and preC protein synthesis, which was inevitable for pgRNA-derived translation. As we supposed, the infection of BHK cells with recombinant SFV1/Pol virus resulted in the successful production of all HBs proteins (Fig. 4c), with significantly higher yield than in the case of SFV1/pgRNA, SFV1/pcRNA and SFV1/pgRNA3'
constructs.
However, we were unable to detect the synthesis of the Pol protein, as in the case of the pgRNA expression, probably because of its low production and limitations in the detection method (see above).
Immunocytochemical analysis of BHK cells producing HBs proteins
As was shown above, the level of production of HBs proteins by pgRNA-like templates was low. The detection of anti-HBs immunoprecipitates in SDS-PAGE requires overexposure of the film for an indefinite time. Therefore, it is difficult to compare different gels. Moreover, the analysis of cell lysates did not allow us to evaluate the distribution of the product over the cells. To compare different constructs and estimate the level of HBs translation, we used an immunocytochemical method, allowing us to show the expressed product in infected BHK cells more obviously (Fig. 5).
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The BHK cells, which were used in these experiments, are not natural host cells for HBV. However, they are optimal for infection with and production of recombinant SFV particles, allowing the highest yields of recombinant proteins. Besides the BHK cells, we established similar expression patterns of HBs proteins for all studied constructs in other cell lines (HuH-7, HepG2 and COS-7), only the level of production, even for HBc and preC proteins, were lower (data not shown).
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DISCUSSION |
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It is difficult to evaluate the influence of SFV to such internal translation of HBs proteins. The SFV replication within the cell shuts down the host-cell protein synthesis. The mechanism of this regulation is not understood. Probably, the strong cytopathic effect of SFV is caused by the efficiency of its structural protein production, which is regulated by a specific translational enchancer (Sjoberg et al., 1994). However, our observations suggest that pSFV1-vector-driven expression, where no SFV structural proteins are presented, does not affect the cell protein synthesis dramatically. Specifically, the analysis of the lysates of HBV-producing cells and non-treated cells did not display a significant difference in the level of cellular protein synthesis (results not shown). Nevertheless, the question about the possible affect of the SFV replication on recombinant protein production remains open.
It is not typical for eukaryotic cells or animal viruses to use polycistronic templates, when more than one protein is initiated from the same mRNA. The common way, especially for viruses, is the expression of a polyprotein from the single mRNA with further post-translational cleavage of the polyprotein into separate proteins. HBV is a unique object in this context, which is able to express HBc and Pol in different ORFs, and three HBs variants in the one ORF by initiation of them from the same mRNA. It ensures an evolutionary advantage in terms of restricted genome size, when the virus needs to express sufficient quantity of specific proteins, but in different amounts, which is difficult in the case of the polyprotein model.
Probably, all HBV mRNAs express the maximum number of proteins by individual translation of them. We found that the largest HBV template pcRNA provided translation of preC, HBc and all variants of HBs proteins. The synthesis of Pol protein from pcRNA was described earlier (Lin & Lo, 1992). Moreover, we did not exclude the possible translation of HBx (X gene) protein from the pcRNA, as well as from the pgRNA templates. A weak anti-HBx immunostaining was revealed in BHK cells infected with pcRNA and pgRNA viruses (data not shown).
Besides the proteins mentioned above, we observed the appearance of an additional high-molecular mass protein (p26) of unknown nature in anti-HBc immunoprecipitates of pcRNA and pgRNA-infected cells. There were no additional AUG codons found upstream and located in-frame of the authentic HBc AUG as a possible consequence of cloning. This was confirmed by sequencing of the corresponding region around the SFV 26S subgenomic promoter in both (pSFV1/pcRNA and pSFV1/pgRNA) constructs. Therefore, the p26 protein could represent (i) a host protein co-precipitated together with the HBc or (ii) a post-translationally modified form of HBc. However, this protein was also revealed by the anti-HBc immunoblot in the total SDS cell lysate (not shown). This result excludes the possibility of co-precipitation. There were several studies devoted to the post-translational modification of the core protein, such as phosphorylation (Yeh & Ou, 1991; Duclos-Vallee et al., 1998
; Kann et al., 1999
). However, the appearance of a 26 kDa band as HBc protein post-translational modification product was not described before and is the subject of further investigation.
Does the HBV genome carry IRESs? IRES cannot yet be predicted by the presence of characteristic RNA sequence or structural motifs (Vagner et al., 2001). As a rule, there are no significant similarities among individual IRES elements (unless they are from related viruses). Nevertheless, probably all IRES elements are also functional when they are positioned at the midpoint (intercistronic gap) of dicistronic artificial mRNAs and their activity does not depend on the length and the properties of the first 5' cistron (Sachs, 2000
). In contrast, our results show the extreme correlation between the length of the 5' end of mRNA and the level of the HBs protein production. Therefore, this suggests that specific IRES elements are not involved in the translation of HBV mRNAs. However, taking into consideration the presence of many sites of 916 nt in pgRNA, which are complementary to the 18S rRNA, we cannot completely exclude the possible assistance of internal ribosome assembly for the expression of HBs proteins. Despite the lack of specific experiments unravelling the intimate mechanisms of HBV RNA translation, we propose that the ribosome leaky scanning through pgRNA and pgRNA-like templates results in initiation of HBs proteins synthesis. The translation of HBs proteins decreases from the highest level for the pSFV1/L construct to the pSFV1/Pol and finally to the pSFV1/pgRNA, pSFV1/pgRNA3'
, pSFV1/pcRNA constructs. The latter three constructs supported similar low synthesis of HBs proteins, all having long 5' end preceding the start codon of the L gene. The reason for low HBs expression in pgRNA and pgRNA-like templates may also be because of the extensive translation of HBc or preC proteins. The corresponding coding regions occupy the 5' end of the mRNAs and may therefore reduce the capacity for downstream protein translation. One may speculate that only a small proportion of ribosomes is able to reach the start codons of the remote L, M and S genes in such conditions. HBs proteins synthesized from pgRNA were found in the NP-40 fraction, as when expressed from the L gene transcript. This suggests that the HBs are made by endoplasmic reticulum-associated polysomes in both cases.
We do not know whether this additional synthesis of HBs proteins from pgRNA could play a definite role in HBV biology. Transcription of specific mRNAs in the nucleus is the initial regulation stage of HBV gene expression. This process depends on the activation of promoter/enhancer elements, which are sensitive to the presence of hepatocyte-specific factors (Antonucci & Rutter, 1989; Kosovsky et al., 1996
; Tang & McLachlan, 2001
). In the case when L and S gene promoters are silent, due to accidental mutations or deficiency of the cellular factors, the pgRNA may appear as a unique source of HBs protein translation. Moreover, the capability of HBV to infect cells of other organs, such as kidney, pancreas (Dejean et al., 1984
) or some blood cells (Blum & Vyas, 1983
; Lobbiani et al., 1990
), where the expression is hampered by the absence of hepatocyte-specific transcription factors, may be provided by additional mechanisms for the HBs protein synthesis including such from pgRNA. Therefore, the pgRNA alone may be able to initiate viral production by ensuring synthesis of Pol and all HBV structural proteins.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Bichko, V., Pushko, P., Dreilina, D., Pumpen, P. & Gren, E. (1985). Subtype ayw variant of hepatitis B virus. DNA primary structure analysis. FEBS Lett 185, 208212.[CrossRef][Medline]
Blum, H. E. & Vyas, G. N. (1983). Hepatitis B virus in non-hepatocytes. Lancet 2, 920.[Medline]
Bruss, V. (1997). A short linear sequence in the pre-S domain of the large hepatitis B virus envelope protein required for virion formation. J Virol 71, 93509357.[Abstract]
Bruss, V. & Vieluf, K. (1995). Functions of the internal pre-S domain of the large surface protein in hepatitis B virus particle morphogenesis. J Virol 69, 66526657.[Abstract]
Dejean, A., Lugassy, C., Zafrani, S., Tiollais, P. & Brechot, C. (1984). Detection of hepatitis B virus DNA in pancreas, kidney and skin of two human carriers of the virus. J Gen Virol 65, 651655.[Abstract]
Duclos-Vallee, J. C., Capel, F., Mabit, H. & Petit, M. A. (1998). Phosphorylation of the hepatitis B virus core protein by glyceraldehyde-3-phosphate dehydrogenase protein kinase activity. J Gen Virol 79, 16651670.[Abstract]
Fouillot, N. & Rossignol, J. M. (1996). Translational stop codons in the precore sequence of hepatitis B virus pre-C RNA allow translation reinitiation at downstream AUGs. J Gen Virol 77, 11231127.[Abstract]
Fouillot, N., Tlouzeau, S., Rossignol, J. M. & Jean-Jean, O. (1993). Translation of the hepatitis B virus P gene by ribosomal scanning as an alternative to internal initiation. J Virol 67, 48864895.[Abstract]
Heermann, K. H., Goldmann, U., Schwartz, W., Seyffarth, T., Baumgarten, H. & Gerlich, W. H. (1984). Large surface proteins of hepatitis B virus containing the pre-s sequence. J Virol 52, 396402.[Medline]
Hu, M. C., Tranque, P., Edelman, G. M. & Mauro, V. P. (1999). rRNA-complementarity in the 5' untranslated region of mRNA specifying the Gtx homeodomain protein: evidence that base-pairing to 18S rRNA affects translational efficiency. Proc Natl Acad Sci U S A 96, 13391344.
Hwang, W. L. & Su, T. S. (1999). The encapsidation signal of hepatitis B virus facilitates preC AUG recognition resulting in inefficient translation of the downstream genes. J Gen Virol 80, 17691776.[Abstract]
Kann, M., Sodeik, B., Vlachou, A., Gerlich, W. H. & Helenius, A. (1999). Phosphorylation-dependent binding of hepatitis B virus core particles to the nuclear pore complex. J Cell Biol 145, 4555.
Kosovsky, M. J., Huan, B. & Siddiqui, A. (1996). Purification and properties of rat liver nuclear proteins that interact with the hepatitis B virus enhancer 1. J Biol Chem 271, 2185921869.
Kozak, M. (1987). At least six nucleotides preceding the AUG initiator codon enhance translation in mammalian cells. J Mol Biol 196, 947950.[Medline]
Kozak, M. (1991). An analysis of vertebrate mRNA sequences: intimations of translational control. J Cell Biol 115, 887903.[Abstract]
Kozak, M. (1999). Initiation of translation in prokaryotes and eukaryotes. Gene 234, 187208.[CrossRef][Medline]
Kozak, M. (2001). New ways of initiating translation in eukaryotes? Mol Cell Biol 21, 18991907.
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680685.[Medline]
Le Seyec, J., Chouteau, P., Cannie, I., Guguen-Guillouzo, C. & Gripon, P. (1998). Role of the pre-S2 domain of the large envelope protein in hepatitis B virus assembly and infectivity. J Virol 72, 55735578.
Le Seyec, J., Chouteau, P., Cannie, I., Guguen-Guillouzo, C. & Gripon, P. (1999). Infection process of the hepatitis B virus depends on the presence of a defined sequence in the pre-S1 domain. J Virol 73, 20522057.
Liljestrom, P. & Garoff, H. (1991). A new generation of animal cell expression vectors based on the Semliki Forest virus replicon. Biotechnology (N Y) 9, 13561361.[Medline]
Lin, C. G. & Lo, S. J. (1992). Evidence for involvement of a ribosomal leaky scanning mechanism in the translation of the hepatitis B virus pol gene from the viral pregenome RNA. Virology 188, 342352.[CrossRef][Medline]
Lobbiani, A., Lalatta, F., Lugo, F. & Colucci, G. (1990). Hepatitis B virus transcripts and surface antigen in human peripheral blood lymphocytes. J Med Virol 31, 190194.[Medline]
Mauro, V. P. & Edelman, G. M. (1997). rRNA-like sequences occur in diverse primary transcripts: implications for the control of gene expression. Proc Natl Acad Sci U S A 94, 422427.
Mauro, V. P. & Edelman, G. M. (2002). The ribosome filter hypothesis. Proc Natl Acad Sci U S A 99, 1203112036.
Ou, J. H., Laub, O. & Rutter, W. J. (1986). Hepatitis B virus gene function: the precore region targets the core antigen to cellular membranes and causes the secretion of the e antigen. Proc Natl Acad Sci U S A 83, 15781582.[Abstract]
Ou, J. H., Bao, H., Shih, C. & Tahara, S. M. (1990). Preferred translation of human hepatitis B virus polymerase from core protein but not from precore protein-specific transcript. J Virol 64, 45784581.[Medline]
Sachs, A. B. (2000). Cell cycle-dependent translation initiation: IRES elements prevail. Cell 101, 243245.[Medline]
Sjoberg, E. M., Suomalainen, M. & Garoff, H. (1994). A significantly improved Semliki Forest virus expression system based on translation enhancer segments from the viral capsid gene. Biotechnology (N Y) 12, 11271131.[Medline]
Stibbe, W. & Gerlich, W. H. (1983). Structural relationships between minor and major proteins of hepatitis B surface antigen. J Virol 46, 626628.[Medline]
Summers, J. & Mason, W. S. (1982). Replication of the genome of a hepatitis B-like virus by reverse transcription of an RNA intermediate. Cell 29, 403415.[Medline]
Tang, H. & McLachlan, A. (2001). Transcriptional regulation of hepatitis B virus by nuclear hormone receptors is a critical determinant of viral tropism. Proc Natl Acad Sci U S A 98, 18411846.
Vagner, S., Galy, B. & Pyronnet, S. (2001). Irresistible IRES. Attracting the translation machinery to internal ribosome entry sites. EMBO Rep 2, 893898.
Yaginuma, K., Shirakata, Y., Kobayashi, M. & Koike, K. (1987). Hepatitis B virus (HBV) particles are produced in a cell culture system by transient expression of transfected HBV DNA. Proc Natl Acad Sci U S A 84, 26782682.[Abstract]
Yeh, C. T. & Ou, J. H. (1991). Phosphorylation of hepatitis B virus precore and core proteins. J Virol 65, 23272331.[Medline]
Received 22 June 2004;
accepted 26 July 2004.
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