1 Department of Medicine, Fukuoka Dental College, 2-15-1 Tamura, Sawara-ku, Fukuoka 814-0193, Japan
2 Department of Medicine and Biosystemic Science, Graduate School of Medical Sciences, Kyushu University, Higashi-ku, Fukuoka 812-8582, Japan
3 Department of Surgery and Science, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582, Japan
4 Division of Disease Genes, Research Center for Genetic Information, Medical Institute of Bioregulation, Kyushu University, Fukuoka 812-8582, Japan
Correspondence
Kosei Moriyama
moriyama{at}college.fdcnet.ac.jp
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ABSTRACT |
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Published ahead of print on 1 May 2003 as DOI 10.1099/vir.0.19170-0
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INTRODUCTION |
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Standring et al. (1983) demonstrated an RNA polymerase III-dependent antisense transcript (asRNA) of cloned HBV DNA in an in vitro transcription system using a HeLa cell extract. The non-polyadenylated transcript was initiated at position 1631/1632 or 1628 and terminated at a (dT)4 tract in the cloning plasmid. It has not been proven whether these polymerase III transcripts are present in vivo.
Using Northern blotting, Zelent et al. (1987) found several polyadenylated asRNAs in a murine cell line carrying integrated HBV genomes. S1 nuclease analysis revealed that the transcripts initiated at position 1861 and terminated at position 2381. It was speculated that one of the asRNAs was 2·8 kb in length. Chen et al. (1993)
looked for asRNAs of woodchuck hepatitis virus (WHV) and its coding ORF6 protein in the livers of acutely or chronically infected woodchucks, but their study yielded negative results. Using a luciferase assay of HepG2 cells, Velhagen et al. (1995)
observed antisense promoter activity of the HBV X gene at positions 13751575, which was similar to that of the X gene promoter. They also found extremely low antisense promoter activity in the StyI fragment of the precore/core (preC/C) promoter at positions 16451885. This activity was one-tenth that of the sense orientation. Shimoda et al. (1998)
demonstrated bidirectional promoter activity within the X gene of WHV by luciferase assay in human and woodchuck hepatoma cell lines. Initiation sites were mapped near the upstream region of ORF6 and the antisense promoter activity was comparable to that of the S gene promoter in the absence of an enhancer. Since these reporter genes have a polyadenylation signal, these findings suggest RNA polymerase II promoter activity in the antisense orientation. However, in human patients, it has yet to be determined whether asRNAs are expressed and transcribed by RNA polymerase II.
In this study, we examined liver tissue from humans with HBV infection and found a low level of asRNAs expression. We determined both ends of the asRNAs as well as their copy numbers by PCR. The 0·7 kb asRNAs in vivo were not polyadenylated and presumed to be transcribed by RNA polymerase III. We discuss the hypothetical 10·5 kDa protein encoded by the transcript as well as the significance of the asRNA in the negative regulation of transcription and translation of the virus.
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METHODS |
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Liver DNA and RNA preparation.
Tissue DNA was extracted from each 20 mg liver sample. The sample was transferred to 500 µl lysis buffer (Qiagen) with 1 mg proteinase K ml-1 and incubated at 56 °C for 2 h. DNA was purified using silica columns from the QIAamp DNA Mini kit (Qiagen). Total RNA for anchored-5'RACE, RLM-3'RACE, oligo(dT)-primed 3'RACE and quantitative RT-PCR was prepared using an RNAeasy Mini kit (Qiagen). Total RNA (40 µg) was treated with 1 U DNase1 (Promega) in 50 µl appended buffer (Moriyama, 1997) at 37 °C for 1 h. PCR for amplifying positions 15242067 with primers B105 and B108 was done before cDNA synthesis to confirm the complete elimination of undigested HBV DNA.
Nucleotide sequencing of tissue HBV DNA.
The entire 3·2 kb genome was amplified by PCR with KOD polymerase (Toyobo) and 20 pmol of each of the HB-P1 and HB-P2 primers (Gunther et al., 1995). PCR was done at 94 °C for 2 min (for initial denaturation of DNA and inactivation of anti-KOD polymerase antibody), with 30 cycles of 94 °C for 15 s, 60 °C for 15 s and 68 °C for 1 min. PCR products were digested with NotI and cloned into pBluescriptKS+ (Stratagene). The nucleotide sequences of the inserts were determined by a Dideoxynucleotide Sequence kit (ABI) using the primers T7Pro (T7 promoter sequence), T3Pro (T3 promoter sequence), B2500, AS11 and B202 (Table 1
). For each patient, nucleotide sequences of three clones were determined.
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Southern blot of RLM-3'RACE products.
Five microlitres of 500-fold diluted RLM-3'RACE products were run in 1 % agarose gel and electroblotted onto a nylon membrane (Hybond-N+, Amersham). Hybridization was carried out with cloned one-genome-length HBV DNA labelled with alkaline phosphatase in 6x SSC and 0·25 % non-fat dried milk at 55 °C for 8 h. The filter was washed under highly stringent conditions and specific bands were visualized with CDP-Star Chemiluminescence Detection Reagent (Amersham).
Oligo(dT)-primed 3'RACE.
cDNA was synthesized with 1 µg total RNA and 100 pmol oligo(dT) primer (12 dT residues). After incubation at 65 °C for 5 min, reverse transcription was done with Superscript II in 20 µl appended buffer at 42 °C for 40 min. RNaseH (1 U, Invitrogen) was added and incubated at 37 °C for 15 min to eliminate template RNA. Excess primers and dNTPs were removed by passage through an agarose column Chromaspin-100. The first PCR was done with the primers PRIMER-OUTER and AS11. Nested PCR was done with the primers PRIMER-INNER and AS22. PCR, cloning and nucleotide sequencing were done in the same manner as those for anchored-5'RACE.
Quantitative RT-PCR.
cDNA was synthesized with 0·25 µg total RNA in 20 µl of reaction mixture containing a primer mixture of 2·5 pmol of each of B026, B301 and GAPDH-R, and 1 U of Superscript II. The reaction mixture was then diluted with 120 µl 10 mM Tris, pH 8·4, and stored at -20 °C. Each 4 µl was amplified by competitive PCR in the presence of each 10-fold- or 2-fold-diluted competitor DNA Comp-AS (430 bp). For competitor DNA template Comp-AS, the 430 bp region of pBluescriptKS+ was amplified with the primers AS11pBSF and B026pBSR. PCR primers B301pBSR and B302pBSF2 were used for making the competitor DNA Comp-PC. Primers GAPDHpBSF1 and GAPDHpBSR2 were used for making the competitor DNA Comp-GAPDH. Both the 5' and 3' ends of these competitor DNAs have annealing sites for primer sets AS11 and B026, B301 and B302 or GAPDH-F and GAPDH-R. Each PCR product was passed through a Chromaspin-100 column to eliminate residual dNTPs and primers. The purified competitor was assayed for DNA concentration by a UV spectrophotometer and normalized by adding 10 mM Tris, pH 8·0. A 4 µl sample of the cDNA product was mixed with 2 µl of serially diluted competitor DNA Comp-PC, Comp-AS or Comp-GAPDH. Then, the mixture was reacted with 20 pmol of each primer pair using Taq polymerase in 50 µl appended buffer for 30 cycles of 94 °C for 20 s, 60 °C for 30 s and 72 °C for 30 s. The cDNA of asRNA was amplified with the primers AS11 and B026. The cDNAs of preC/C, pregenome, preS/S and X RNA were amplified with the primers B301 and B302, and the cDNA of GAPDH mRNA was amplified with the primers GAPDH-F and GAPDH-R. After agarose gel electrophoresis, the lane showing a density equal to that of the objective band and the competitor product was determined and the concentration of each cDNA was deduced from the copy number of the competitor.
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RESULTS |
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Next, we used primer B036 (positions 742762) to synthesize the cDNA (Fig. 1A). It was considered that 5'RACE would detect the asRNA 5' ends initiating downstream from the former cDNA primer B026. However, under several conditions, we did not detect any 5'RACE products containing the viral nucleotide sequences.
Determination of 3' ends of asRNAs by RLM-3'RACE
To determine the 3' ends of asRNAs, we performed RLM-3'RACE. We ligated the 3' ends of total RNA with ANCHOR-B100 and synthesized the cDNA using the primer RLMP1, which anneals with the anchor (Fig. 1B). We used the first and nested PCR primers AS11 and AS22 to anneal with the region near the 5' ends of the asRNAs. PCR of the joined region produced several fragments (Fig. 3
A). Therefore, we performed a Southern blot of the 500-fold diluted PCR products using HBV DNA as a probe (Fig. 3B
). It was found that only the 0·7 kb products contained the viral sequence. Amplification of the 0·7 kb product from patient 4 was unsuccessful. Cloning and sequencing of the 0·7 kb products revealed that the asRNAs ended at position 844/846, 929/930/931, 951 or 966. All of them, except for one clone from patient 5, were located in the (dT)4 or (dT)5 tracts that can be termination cleavage signals for RNA polymerase III (Fig. 3C
). The procedure did not detect a polyadenylated asRNA.
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Search for spliced forms and quantification of asRNA
Anchored-5'RACE, RLM-3'RACE or oligo(dT)-primed 3'RACE failed to reveal any nucleotide sequences compatible with the spliced forms. Therefore, to find any spliced forms of asRNAs, we performed further RT-PCRs of the region encompassing positions 12821650. However, only non-spliced products were obtained consistently and some smaller products were observed in a limited number of cases. We cloned and sequenced the RT-PCR products smaller than 0·7 kb but their deleted sequences lacked the intron GT or AG at both ends; they were made by homologous recombination or polymerase jumping in PCR (data not shown). Therefore, it is likely that the 0·7 kb non-polyadenylated asRNAs lack introns.
We estimated the amounts of HBV transcripts by competitive PCR. We synthesized the cDNAs using primer B026, which anneals at positions 12621282. To the same reaction mixture, we added primers B301 and GAPDH-R to reverse transcribe the sense RNA of the virus as well as GAPDH mRNA. We then amplified the cDNA by PCR in the presence of either a 10-fold- or a 2-fold-diluted competitor DNA. The amounts of asRNAs and sense RNAs varied among the five liver tissue samples examined and their ratios were estimated to be approximately 1 : 2501 : 2000 (Table 2).
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DISCUSSION |
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Anchored-5'RACE amplified the transcripts initiating at position 1655 in four cases. A database search showed that the co-existence of T1678 and G1679 in patient 2 was a rare event and appeared to have altered the initiation site of the asRNA from 1680/1681. This suggests that this region is, in fact, an antisense promoter region.
Position 1655 lies near the position that was reported previously to be the initiation site of RNA polymerase III at positions 1629, 1631 or 1632 (Fig. 2C) (Standring et al., 1983
). There is no typical TATA, CAAT or GC box around this region and the asRNAs terminated at (dT)4 or (dT)5 tracts (Fig. 3C
). Therefore, it is possible that asRNAs are transcribed by a polymerase III system in vivo (Geiduschek & Tocchini-Valentini, 1988
; Willis 1993
) and the difference between our results and those of Standring et al. (1983)
may be due to the different nucleotide sequences of this region (Fig. 2C
).
asRNAs detected in our patients had ORF6, capable of encoding a protein of 95 amino acids with a predicted molecular mass of 10·5 kDa. It has been demonstrated that an RNA transcribed from a strong polymerase III promoter of a plasmid construct in transfected HeLa cells produces the coding protein (Gunnery & Mathews, 1995). Thus, human cells can utilize polymerase III transcripts as functional mRNAs and neither a cap nor a poly(A) tail is essential for translation. However, there is no known natural case of translation of RNAs made by polymerase III. One asRNA initiating at position 1680/1681 in patient 2 had the initiation and adjacent termination codons in an area 40 nucleotides upstream of ORF6. In addition, the most widely reported HBV subtype adw has an upstream initiation codon at positions 16311629. Moreover, the length and amino acid sequence of the carboxy-terminal region varies among reported strains. Therefore, ORF6 and its product are not required for virus proliferation.
asRNAs can regulate the transcription of sense mRNA by annealing with the complementary sequences of DNA templates (reviewed by Kumar & Carmichael, 1998). asRNAs can also hybridize with complementary sense RNAs. The dsRNA molecules in the nuclear compartment can be a substrate for enzymes that deaminate adenosine to inosine. Extensively modified RNAs are either degraded rapidly or retained within the nucleus. Transcripts with few modifications may be transported to the cytoplasm, where they serve to produce altered proteins. Therefore, asRNAs can cause amino acid alterations of polymerase and X proteins. dsRNA molecules in the cytoplasm can trigger sequence-non-specific interferon synthesis. Minced small molecular dsRNAs in the cytoplasm can also induce the Dicer pathway (Ahlquist, 2002
). Since this cycle does not require a large amount of trigger RNA, the small amount of cytoplasmic asRNA, if any, might be sufficient to inactivate any molar excess of sense RNAs. Therefore, the existence of endogenous asRNAs complementary to the common ends of all sense RNAs of HBV suggests an antisense-mediated self-regulation of hepadnavirus.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 18 February 2003;
accepted 11 April 2003.
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