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Journal of General Virology (1994), 75, 917-923. Printed in Great Brita#l 917 Effect of frameshift mutation in the pre-C region of hepatitis B virus on the X and C genes Seong Kee Kim, 1 Sung Key Jang 2 and H y u n e M o Rho ~* 1Department o f Molecular Biology and Research Center for Cell Differentiation, Seoul National University, Seoul 151-742 and 2Department o f Life Science, Pohang htstitute o f Science and Technology, Pohang 790-784, Korea We have previously cloned a mutant hepatitis B virus (HBV) genome which had one thymidine addition in the pre-C region resulting in a frameshift mutation in the pre-C region and fusion of the X and C genes. We constructed plasmids containing serially deleted and/or back-mutated (authentic) pre-C regions to study the effect of the frameshift mutation. COS cells transfected with plasmids containing the frameshifted pre-C region produced a 21K C protein (P21c) but not a 22K partially processed pre-C protein (P22). On the other hand, COS cells transfected with plasmids containing the back- mutated pre-C region produced P22. This result was also observed in HepG2-K8 cells producing the mutant HBV particles, Therefore, the pre-C region of HBV is likely to be non-essential for virus replication, COS cells transfected with the plasmid containing a fused X-C open reading frame (ORF) produced a 40K X-C fusion protein. This X-C fusion protein exerted transcriptional trans-activation. These results suggest that the mutant HBV has a C gene with a defective pre-C region and a fused X-C ORF, and hence cannot synthesize 16K HBeAg (P16e). Genomic DNA of hepatitis B virus (HBV) has four open reading frames (ORFs) which are designated pre-C/C, P, pre-S/S and X (Ganem & Varmus, 1987; Tiollais et al., 1985), In HBV the coding region for the C protein (P21c) is preceded by an in-phase ORF termed the pre-C region (Ou et al., 1986). Translation initiation from the pre-C initiation codon produces a pre-C protein (P25) that contains the entire C protein sequence and a 29-residue amino-terminal extension (pre-C region). It has been demonstrated that the first 19 amino acids of the pre-C region form a signal sequence to direct the pre-C protein to the endoplasmic reticulum, where it is cleaved, forming the partially processed pre-C protein (P22) (Bruss & Gerlich, 1988; Garcia et al,, 1988, Junker et al., 1987; Ou et al., 1986, 1988; Roossinck et al., 1986; Weimer et al., 1987). P22 is further processed by a protease(s) at its arginine-rich carboxy-terminal domain and secreted as HBV e antigen (HBeAg) (Pl6e) (Ou et al., 1986, 1988; Salfeld et al., 1989). In the course of HBV infection, the presence of HBeAg is generally correlated with active virus replication and often liver disease. On the other hand, the clearance of HBeAg and subsequent rise of the homologous anti-HBe antibody indicate termination of HBV replication and remission of liver disease (Hoofnagle et al., 1981 ; Realdi et al., 1980). The smallest ORF, X, is conserved among all mammalian hepadnaviruses and has been shown to be expressed during virus infection (Kay et al., 1985~ Meyers et al., 1986; Moriarty et al., 1985). It has been demonstrated that the X protein is a transcriptional activator of various enhancer promoter combinations, including the HBV enhancer and C gene promoter, the simian virus 40 (SV40) enhancer and early promoter, and the /q-interferon gene (Spandau & Lee, 1988; Twu & Schloemer, 1987; Zahm et al., 1988). A novel HBV protein of 35K to 40K was discovered in HBV-transfected HepG2 cells (Bchini et al., 1990) and in core particles isolated from HBV-infected livers (Farza et al., 1988; Feitelson, 1986). It was characterized by Western blotting and proposed to be an X-C fusion protein. However, HBV does not have a fused X-C ORF. The biological function and the mechanism of production of such an X-C fusion protein are as yet unknown. The possibility of ribosomal frameshifting causing X C fusion protein production has been excluded (Lo et al., 1990). Previously, we have cloned a mutant HBV (adr) genome from a Korean hepatitis B patient (Choi et al., 1984) and determined the complete sequence of 3213 nucleotides (nt) (Rho et al., 1989). The DNA sequence analysis revealed the addition of one thymidine at nt 1821 of the pre-C region, which resulted in a frameshift mutation in the pre-C region and fusion of the X and C genes. In this paper, we characterized the proteins expressed from the pre-C/C and fused X-C ORFs of the mutant HBV. As the fused X-C ORF contained a region 0001-1967 © 1994SGM Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sat, 17 Jun 2017 12:57:22 918 Short communication (a) 1685 CAATGTCAACGTCCGAccTrG AGGCATACTTC^AAGACTGCTTGGTTAAAGACTGGGAGGACTTGC~GGAGGAGATTAGGTr Hincl I I~ pU$CP1 pUSCP 10 - ~ Frameshi f t e d -.-Elongated M 1757 1 F Precore V L G M G C A H K L V C S P A P Q C N L F F F Precore P S II P C 1 L ] /~TGGATui-i-IGTACTAGGAGGCTGTAGGCATAAATrGffr CTffrTcACCAGC^CCAT66CAAuI-rril-rCAO2T~AATC I I~ pUSCP3 2 A direct repeat 1 pI~CP30 '1" addition --~ Core D I D H G H ~k" M H L 1 1839 L L M S F C M S S C Y P C T S V R H A L Q S k K V L P C | L V G A I L L G W G M D 1 D P Y K P Y K ATCrCATGTTCATGTCCTACTGTTCACGCCTCCAAGCTGTGCCrTGGGTGGCITrGGGGCAT~ACATrGA~ATA~G t,. pLLSC 3 4 4 (b) J S V40 ATG'~T~ 2, ,,,~ I A~G3 I l s J.o,.(.) ] I I I I I I | f I I I I i I I pro-C I C ,: , ,uscP e l[ I T.iddll iOn I I i I I -180 ~ i J--/ I 1 I I ..... [ [ ] ..... [ I I ......... I I I I -129 i I L_ • i i • t~" : pUSCP1 .tJ~ I pUSCPIO y..l I pUSCP3 s~ I pUSCP30 Y.~ t pUSC -32 I Fig. 1. The nucleotide sequence of a pre-C region and mapping of the 5" deletion of the pre-C region in plasmlds. (a) Nucleotlde sequences of the pre-C region. Deleted 5' ends are shown by arrowheads. Three initiation codons are underlined and numbered: 1, ATG codon of possible elongated pre-C protein; 2, ATG codon of frameshifted (authentic) pre-C protein: 3, ATG codon of C protein; 4, two termination codons of frameshifted pre-C protein. The site of one thymidine addition is indicated as a vertical arrowhead. The encoded amino acid sequence is shown above the nucleotide sequence. Termination of the frameshifted pre-C protein is marked by an asterisk. (b) Schematic diagram of recombinant plasmids containing the serially deleted and/or back-mutated pre-C region with the C gene. Vector pUC19 sequences are shown as a thin line. Three ATG codons and endpoints of 5' deletion mutants are the same as in (a). Horizontal dashed lines indicate the region deleted with Bal 31 nuclease. The filled circles indicate the T deletion mutation. Abbreviations: B, BamHI: H, HindlII ; Hc, HinclI ; S, SalI. encoding part o f the X protein, we also tested whether the fused X - C O R F products possess trans-activating activity. In the m u t a n t HBV, the putative initiation c o d o n o f the elongated pre-C region at nt 1758 was located 53 nt upstream o f the initiation c o d o n o f the authentic pre-C region at nt 1811 (Fig. 1 a). Therefore we tested whether the m u t a n t H B V contained an elongated pre-C region instead o f the authentic pre-C region which m a y be involved in generating HBeAg. To investigate the utilization o f the A U G c o d o n s o f the pre-C region for the H B V core antigen (HBcAg) and H B e A g synthesis, we constructed r e c o m b i n a n t plasmids containing serially deleted a n d / o r b a c k - m u t a t e d (authentic) pre-C regions (Fig. 1). To construct 5' deletion m u t a n t plasmids, p U S C P containing the C gene and pre-C region was opened with B a m H I and serially deleted with Bal 31 nuclease. After filling o f the staggered ends with the K l e n o w fragment, B a m H I linkers were attached. The resulting series o f 5' deletion m u t a n t s was selected by D N A sequencing (data not shown). To recover the p r o m o t e r region, the SV40 late p r o m o t e r region o f p U S C P was cloned between B a m H I and H i n d I I I sites o f the deletion m u t a n t plasmids. M u t a n t s with endpoints at - 180 (pUSCP1), - 129 ( p U S C P 3 ) and - 3 2 (pUSC) were selected (numbering system starts with the A o f the C gene initiation codon). F o r the construction o f plasmids p U S C P 1 0 and p U S C P 3 0 containing the backm u t a t e d pre-C region, site-directed mutagenesis was performed using P C R ( K a m m a n n et al., 1989; K i m et al., 1992b; Saiki et al., 1988) with a mutagenic primer. Plasmids p U S C P 1 0 and p U S C P 3 0 were selected by D N A sequencing (data not shown). Each of the vectors was introduced into C O S cells Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sat, 17 Jun 2017 12:57:22 Short communication HBV cannot synthesize pre-C region-derived HBeAg. This observation was further confirmed by Western blotting experiments. Characterization of core antigens by Western blotting (Sambrook et al., 1989) was performed to estimate the relative size of the processed antigens. We expressed the C gene in Escherichia coli (Choi et al., 1991). The recombinant core particles produced by E. coIi cells were purified (Kim & Rho, 1992) and used as an Mr marker. A rabbit polyclonal anti-HBc antiserum against recombinant HBcAg was prepared using techniques reported previously (Sambrook et al., 1989). As shown in Fig. 2(a), COS cells transfected with pUSCP1, pUSCP3 and pUSC produced only a 21K protein (P21c), whereas COS cells transfected with pUSCP10 produced a minor 22K protein (P22) as well as a 21K protein (P21c). After deletion of the ATG codon region of the elongated preC (pUSCP30), the 22K protein became the major species. The 22K protein (P22) then becomes cleaved by a protease(s) and secreted into the medium (Ou et at., 1986, 1988; Salfeld et al., 1989). The production of P22 from pUSCP10 was much lower compared with that from pUSCP30 (Table 1 and Fig. 2). This result suggests that the A U G codon of the elongated pre-C may interfere with translational initiation at the A U G codon of the pre-C, but not with the A U G codon of the C protein. We have also established a mutant-HBVproducing cell line, HepG2-KS, by transfection of mutant HBV DNA into HepG2 cells (Kim et al., 1992a). In HepG2-K8 cells, only the 21K protein was detected (Fig. 2a). These results confirm that our mutant HBV has a defective pre-C region, and hence cannot synthesize Table 1. Relative levels of expression of HBcAg and HBeAg in recombinant plasmid-transfected COS cells Radioactivity (c.p.m.)t Plasmid* Cell extract Culture medium pUC19 pUSCPI pUSCP10 pUSCP3 pUSCP30 pUSC pSVXC 607 17262 19184 24 624 25423 46012 11786 532 1163 3234 1089 14622 1426 1125 919 * Plasmlds are described in Fig. 1 (b) and 4(a). "~ Cell extracts and culture media were one-fifth of the cell lysate, and one-fifth of the supernatant, respectively, from a 100 mm dish. The values are the mean of three determinations. using the calcium phosphate method (Chen & Okayama, 1987). At 72 h post-transfection, cells and culture media were collected separately. Antigens from COS cells transfected with the recombinant plasmids were assayed with the Abbott HBe radioimmunoassay kit (Abbott Laboratories) (Table 1). Note that this assay cannot differentiate between HBcAg and HBeAg. In the case of plasmids pUSCP10 and pUSCP30 (Fig. 1) which contained the C gene with the back-mutated (wild-type) pre-C region, HBcAg/HBeAg was detected in both cell extracts and culture media. These results agreed with the reports of previous studies (Bruss & Gerlich, 1988 ; Ou et al., 1986; Roossinck et al., 1986). In the case of plasmids pUSCP 1, pUSCP3 and pUSC, HBcAg/HBeAg was detected only in cell extracts but not in the culture media (Table 1). These results suggest that our mutant (b) (a) / ~'~, ~ - - 36K I ~:,,,, ' ,.~;,:"j.,~¢ 45K -,~ID - - 36K - ,~£ " --29K - - 24K X-CD,,- P22 P21c I~- ~ aw~,..e - --29K .... ~ - - 20K - - 24K - i - -~IM 20K - - 14K Fig. 2. Western blotting analysis of HBV antigens. Extracts of COS cells transfected with recombinant plasmids and HepG2 cells producing the mutant HBV particles were separated by S D S - P A G E and transferred to a nitrocellulose filter. Core proteins (P21c and P22) (a) and X - C fusion protein (b) were detected by an anti-HBcAg antiserum and 125I-labelled Protein A. The COS control refers to pUC19-transfected COS cells. M r values are shown on the right. M and D indicate the positions of the monomer (21K) and dimer (42K) forms of P21c purified from E. coli. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sat, 17 Jun 2017 12:57:22 920 Short communication HBV (adw2) ! 1837 v 1903 I V I m -c I X 1376 2557 V c I it 1816 1837 1903 HBV-ctl I 1376 Mutant HBV (adr) DefecUve pre-C l 1371 DHBV I x Defectwe pre-C ler~-Cl A 2518 A 2647 2557 * I c x-cl c it 1899 i ] A 2447 c A 3021 I A 411 Fig. 3. Genetic organizationof X and C ORFs in various hepadnaviruses. Numbers indicatemap positions. Sequenceswere taken from the followingreferences:HBV(adw2) (Valenzuelaet al., 1980);mutant HBV (adO (Rho et al., 1989); HBV-71 (Tong et al., 1990); DHBV (Mandart et aL, 1984). the 16K HBeAg, in agreement with the radioimmunoassay results (Table 1). This implies that the mutant HBV did not utilize the AUG codon of the elongated pre-C region for HBcAg and HBeAg synthesis. Kozak (1986) proposed that the optimal context for initiation is CCPuCCAUGG (Pu, purine). However, if the sequence surrounding the first AUG triplet is suboptimal, some 40S subunits bypass that site and initiate translation further downstream. Translation initiation from the AUG codon of the elongated pre-C (in the context GGUUAAUGA) may therefore be suboptimal. Several laboratories have found HBV variants mutated in the pre-C region (Brunetto et aI., 1989, 1990; Carman et al., 1989; Fiordalisi et al., 1990; Li et al., 1990; Okamoto et al., 1990; Riamondo et al., 1990; Tong et al., 1990). Most of these HBV variants contained a TAG stop codon formed by a point mutation in the distal pre-C region. We have previously established a cell line that produces such mutants (Kim et al., 1992a) and therefore the pre-C region of the HBV is likely to be nonessential for virus replication. Introduction of a frameshift and nonsense mutations in the duck hepatitis B virus (DHBV) pre-C region demonstrated that expression of the region is not essential for viral genome replication (Chang et al., 1987; Schlicht et al., 1987). The variant HBV-~I with a nonsense mutation in the distal pre-C region had an in vitro replication capacity (Tong et al., 1991). These results suggest that HBV may not require HBeAg for its replication. The HBV variant contained a TAG stop codon in the distal pre-C region due to a G to A transition. However, our HBV with one thymidine addition at nt 1821 has not only a C gene with a defective pre-C region but also a fused X-C ORF. Our mutant HBV is different from HBV-~I (Tong et al., 1990) and DHBV (Mandart et al., 1984) in its genetic organization (Fig. 3). The HBV-e 1 has a C gene with a defective pre-C region and wild-type X gene. The DHBV has only a pre-C/C ORF. Furthermore, the 5' end of the DHBV C gene shows no homology with the X gene of mammalian hepadnaviruses (Feitelson & Miller, 1988). Therefore, the DHBV C gene cannot be considered as a fusion between the C and X genes. To determine whether an X-C fusion protein is synthesized, we constructed the plasmid pSVXC which contained a fused X-C ORF under the control of the SV40 promoter. Extracts of COS cells transfected with pSVXC contained HBcAg/HBeAg activity (Table 1). In RNA analysis experiments, a 1.9 kb transcript which had the fused X-C ORF coding sequences was detected (data not shown). However, a transcript of approx. 1 kb which encodes the C protein, transcribed from the initiation site of C mRNA, was not detected. Approximately 70 % of the transcripts from a region near the initiation site of the X mRNA did not stop at the HBV transcription termination signal (data not shown). This value was higher than that in a previous report (Guo et al., 1991) which showed that 50 % of X gene transcripts could read through the transcription termination signal. As shown in Fig. 2 (b), COS cells transfected by pSVXC produced a 40K X-C fusion protein. Bands above the 40K band appear to be due to proteins interacting non-specifically with the anti-HBcAg antiserum. The fused X-C ORF could encode an X-C fusion protein of 359 amino acids, consisting of 151 amino acids encoded by the wild-type X ORF and 208 amino acids encoded by the pre-C/C ORF region. Previous results have shown that the carboxyl-terminal amino acids of X protein are dispensable for its function as a transcriptional trans-activator (Levrero et al., 1990; Ritter et al., 1991); we therefore examined the transactivating activity of the X-C fusion protein. The reporter plasmid pSV2CAT containing the SV40 enhancer and early promoter driving the chloramphenicol acetyltransferase (CAT) gene was cotransfected with test plasmids into HepG2 cells, and a CAT assay was then performed (Gorman et al., 1982). Plasmid pHX contained the wild-type X gene (Won et al., 1989) under the control of the HBV enhancer/X promoter and the poly(A) signal of the herpes simplex virus thymidine kinase gene (HSV TK). Plasmid pHXC, on the other hand, was similar to pHX but contained the fused X-C ORF instead of the X gene (Fig. 4). Plasmid pHXCM is similar to pHXC except that the poly(A) signal sequence UAUAAA was mutated to UACAAG. In the case of plasmid pHXC, the poly(A) signal is located inside the C gene. To exclude the effect of the TATAAA sequence when it encounters the transcription termination signal, Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sat, 17 Jun 2017 12:57:22 Short communication (a) HBV ! ~I x iI 0 (1069) C pUC19 pHX I I x pHXCM I I x-c * I ~. _ I I x c @ |__ 1-AC 3-AC pHXC I A v (b) CM pUC19 g pHX ~ pHXCM Q pHXC Q ll} l~ 1-0 6.9 + 1.5 O ~ Activation (fold) 81 + 1 9 Q 70+16 Fig. 4 Transcriptional trans-actlvation of SV40 enhancer/promoter by the fused X-C ORF product. (a) Schematic maps of test plasmids pHX, pHXC and pHXCM, the correct constructions of which were confirmed by DNA sequence analysis. Numbers in parentheses indicate map positions on the HBV genome. E/P, HBV enhancer/X promoter; open boxes, ORFs; open circles, HBV poly(A) signal: sohd circles, herpes simplex virus TK poly(A) signal; star, mutated HBV poly(A) signal. (b) CAT assay for the trans-activation function of the X C fusion protein. HepG2 cells were cotransfected with 5 lag of pSV2CAT and 15 lag of each of the plasmids pUC19, pHX, pHXCM or pHXC. Activation (fold) represents the ratio of the percentage of chloramphenlcol acetylation in cotransfection with the X and X-C gene expression vectors to that with control plasmid pUC19. All values are given as the mean+s.D, obtained from experiments carried out in triplicate. this sequence was mutated to TACAAG. These mutations do not change any amino acids of the X-C fusion protein. In a SV40 mutant, deletion of the entire AAUAAA sequence shifted polyadenylation to just downstream from the next most proximal AAUAAA (Fitzgerald & Shenk, 1981). A single mutation, a U to G transversion in the AAUAAA hexanucleotide of the E1A RNAs of adenovirus, decreases the efficiency of RNA cleavage of the primary transcript by over 95 % (Montell et al., 1983). Therefore, in the case of plasmid pHXCM, over 70 % of the transcripts transcribed from a region near the initiation site of the X mRNA may stop at the HSV TK transcription termination signal. As shown in Fig. 4, the CAT activities derived from cells cotransfected with pHX, pHXC or pHXCM were about sevenfold higher than those from cells cotrans- 921 fected with the control vector pUC19. CAT activity in pHXCM-transfected cells was slightly higher than in pHXC-transfected cells. Therefore, it is evident that the X-C fusion protein expressed from the fused X C ORF using the X promoter is active in trans-activation. These results suggest that the mutant HBV contains a fused X-C ORF. As the fused X-C ORF contains the entire X and pre-C/C genes, the X-C fusion protein might have the functions of both the X and pre-C proteins. In HepG2-K8 cells, six HBV-specific transcripts of 4.0, 3-5, 2.2, 2.1, 1.2 and 0"9 kb were detected (Kim et at., 1992a) and the 4-0 kb species may encode the X-C fusion protein during the life cycle of the mutant HBV, The cDNA cloning and sequence analysis of X mRNA showed that an elongated X ORF encoding 193 amino acids (21K) was created by a frameshift mutation at a 3' region of the wild-type X ORF and the formation of an in-frame termination codon (TAA) resulted from polyadenylation (Kim et at., 1992). This elongated X gene product exerted transcriptional trans-activation (Kim et al., 1992a). These results suggest that an X-C fusion protein and/or an elongated X protein can be expressed by the mutant HBV and may be involved in the regulation of virus and/or host gene expression. However, it remains unknown whether the X-C fusion protein is expressed during the life cycle and has a biological function. Experiments are in progress to investigate whether HBV with a defective pre-C region is infectious and expresses the X-C fusion protein during its replication. This work was supported in part by research grants from the Korean Ministry of Education and from KOSEF through the Research Center for Cell Differentiation (93-4-1). References BCHINI, R., CAPEL, E., DAUGUET,C., DUBANCHET,S. & PETIT, M.-A. (1990). In vitro infection of human hepatoma (HepG2) cells with hepatitis B virus. Journal of Virology 64, 3025-3032. BRUNETTO,M. R., STEMLER,M., SCHODEL,F., WILL, H., OTTOI~ERLLI, A., R1ZZETTO, M. & BONINO, F. (1989). Identification of HBV variants which cannot produce precore derived HBeAg and may be responsible for severe hepatitis. Italian Journal of Gastroenterology 21, 151 154. BRUNFTTO,M. R., STEMLER,M., BONINO,F., SCHODEL,F., OLIVERI,F., RtZZETTO, M., VERME, G. & WILL, H. (1990). A new hepatitis B virus-strain in patients with severe anti-HBe positive chronic hepatitis. 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