Proc. Natl. Acad. Sci. USA Vol. 88, pp. 8490-8494, October 1991 Microbiology
The large form of hepatitis 6 antigen is crucial for assembly of hepatitis 6 virus (RNA virus/virion assembly)
FU-LIN CHANG, PEI-JER CHEN, SU-JEN Tu, CHUAN-JEN WANG, AND DING-SHINN CHEN* Graduate Institute of Clinical Medicine, College of Medicine, Hepatitis Research Center, The University Hospital, National Taiwan University, Taipei, Taiwan 10016
Communicated by Robert H. Purcell, June 13, 1991
ABSTRACT The vinions of hepatitis 6 virus (HDV) contain two species of HDV-specific protein, a large and a small form of hepatitis 6 antigen (HDAg). We examined the role of individual HDAgs in virion assembly in cotransfection experiments. First, we constructed a replication-competent HDV mutant expressing only the small HDAg. When cotrandected with a plasmid expressing hepatitis B virus surface antigens to the HuH-7 cells, the mutant did not produce HDV virions, whereas the wild-type HDV clone did. Therefore, though the small HDAg is important for viral replication and is incorporated into the virus, the small-form 6 antigen by itself is insufficient for virion formation. When the system was cotransfected with an additional plasmid providing the large HDAg, the HDV virion was then recovered. There was also evidence suggesting that the large HDAg could be copickaged into the HBsAg particles, without the presence of the HDV genome and the small HDAg. The results indicate a crucial role of the large HDAg in HDV assembly.
When the clone is transfected into appropriate cell lines for several days of replication, the large HDAg is produced in addition to the original small HDAg (16). This new large HDAg population is the result of a specific base change at the termination codon of the small HDAg ORF (UAG to UGG) (15). This late-appearing large HDAg behaves as a transnegative factor limiting the HDV replication process (17). Although the functions of the two HDAgs have been determined, their roles in virion assembly remain unclear. The relative amounts of the two HDAgs are different in the infected liver and mature virions. The ratio of large to small HDAg is about 0.1 in the infected liver, whereas the ratio in the virions can increase up to 0.9 (3, 4). Since there is a greater incorporation of the large HDAg in the virions, it suggests a significant role of the large HDAg in virus assembly. Recently we have succeeded in producing HDV virions by cotransfecting a human hepatoma cell line, HuH-7, with cloned HBV and HDV DNA (18). Using the same system, we studied the significance of individual HDAgs in virion formation. To do this, we had to unravel the close association between the two HDAgs by constructing a frame-shift HDV mutant capable of replication but expressing only the small HDAg. This mutant was then cotransfected with HBV DNA in the presence and absence of the large HDAg plasmid. The products of these experiments were analyzed for the HDV virions. The results indicate that the small HDAg alone is insufficient for viral particle formation and that the large HDAg is essential for HDV virion assembly. Furthermore, the large HDAg appears to be copackaged with the HBsAg particles even in the absence of HDV RNA.
Hepatitis 8 virus (HDV) is a defective infectious agent that transmits in the presence of the helper hepatitis B virus (HBV) (1, 2). The virion is a 36-nm particle consisting of three components: (i) an envelope derived from the surface proteins of HBV (3); (ii) two internal proteins, specifically the hepatitis 6 antigens (HDAgs) (4); (iii) a single-stranded, circular RNA genome (5, 6). Our knowledge about HDV genome replication and gene expression has progressed rapidly in recent years (reviewed in ref. 7). However, little is known at the level of virion maturation. The HDV genome is naturally considered to be an integral part of virion, but the signals responsible for viral RNA packaging are not yet defined. The surface proteins of HBV (large, middle, and small HBsAgs) are also essential for virion formation, as previous experiments have shown that no virus particles were produced from the replication of HDV genome alone (8, 9). On the other hand, the role of HDAgs in virion assembly is little understood. The two species of HDAgs, large and small forms (p27 and p24), have been found in the virus and the liver of infected humans and animals (3, 4). The large HDAg includes the entire small HDAg plus an additional 19 amino acids. at the carboxyl terminus (10). They are encoded by the largest conserved open reading frame (ORF) of antigenomic polarity in two types of HDV genome (11, 12). In natural infections, the HDV virus always appears to be a quasispecies composed of two genomic populations (13, 14). One of these genomes contains the short HDAg ORF consisting of 195 amino acids and the other contains the longer ORF with 214 amino acids. Recently, the relationship between the two HDAgs has been studied by Luo et al. (15). The original HDV cDNA clone containing an ORF encoding only the small HDAg is used.
MATERIALS AND METHODS Synthesis of Oligonucleotides. To do site-directed mutagenesis of the HDV genome, four oligonucleotides were synthesized (DNA synthesizer 381A, Applied Biosystems). Primer D1 has sequences of 5'-CCATAGIGATATACTC-3' that represents nucleotides 1016-1002 of the cDNA clone of antigenomic HDV RNA with insertion of a nucleotide (thymidine) immediately behind the termination codon of the small HDAg ORF. Primer D2 is complementary to D1, with sequences of 5'-GAGTATATCACTATGG-3' (nucleotides 1002-1016, genomic strand). Primer D3 has sequences of
5'-CCGGCATGGTCCCAGCCT-3' (nucleotides 688-705, genomic strand), and primer D4 has sequences of 5'ATGAGCCGGTCCGAGTCG-3' (nucleotides 1598-1581, antigenomic strand). The relative locations of the four oligonucleotides are summarized in Fig. 1B. The nucleotide designations are based on the chimpanzee HDV isolate (5). All Abbreviations: HDV, hepatitis 8 virus; HDAg, hepatitis 8 antigen; HBV, hepatitis B virus; ORF, open reading frame; HBsAg, HBV surface antigen; SV40, simian virus 40. *To whom reprint requests should be addressed.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
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primers were purified for subsequent use by the use of an OPC column (Applied Biosystems). Construction of the HDV Mutant. To prepare the HDV mutant, one additional thymidine base was incorporated into the designated position of the wild-type HDV genome (Fig. 1A) by the procedure of recombinant PCR (19). Two overlapping DNA fragments were first amplified from the cDNA clone of HDV by the use of primers D1 plus D3 or primers D2 plus D4 (Fig. 18). They were then used as template and subjected to recombinant PCR with oligonucleotides D3 and N4 as primers (Fig. 1B). The final recombinant DNA fragment of 912 base pairs (bp) was gel-purified, digested with Stu I and Bst XI, subcloned into pGEM3Z, and then sequenced completely to show no other mutation. Afterward, it was used to replace the same region in the wild-type HDV (pGEMD1). The mutant HDV monomer was isolated, preligated, and then cloned into the Xba I site of the expression vector pSVL (Pharmacia, Pharmacia LKB Biotechnology). In this manner, plasmid pSVLDm2, containing a tandem HDV dimer in the genomic orientation, was obtained (Fig. 18). A wild-type HDV clone, pSVLD3, consisting of a tandem trimer driven by the SV40 late promoter was used as the control. On transfection to HuH-7 cells, the plasmid can direct the genome replication and expression of the two species of HDAgs. Upon cotransfection with HBV DNA, the HDV vinions are produced (18). To construct a plasmid expressing the large HDAg, a BamHIl-Pst I fragment, derived from plasmid DD15 (12) containing the ORE for large HDAg, was first digested with
Si nuclease and then cloned into the Sma I site of pSVL. The organization of this plasmid, pSVDAg-L, is shown in Fig. 1C. Plasmid pSiX, including a partial HBV genome (Apa I-Bgl II fragment), was constructed into the Sma I site of pGEM-3Z (Fig. 1C) and used in the transfection system to provide the three HBV surface proteins.
Miscellaneous Methods. Procedures for transfection of HuH-7 cells, analysis of nucleic acids and proteins, and isopycnic centrifugation of HDV virions were performed essentially as described (9, 18).
RESULTS Construction of a EHDV Mutant Unable to Express Large HDAg. To abolish the expression of large HDAg, one thymidine was inserted immediately at the 3' position of the termination codon of the small HDAg ORE (Fig. 1A). The mutation resulted in two consecutive termination codons at the end of the small HDAg ORF (Fig. 1A). In addition, the ORF following the termination codon was shifted out of frame. Thus, two mechanisms were combined to prevent generation of the large HDAg. The nucleotide sequence of the mutant was confirmed by sequencing. The DNA sequence of the mutant was TATATCACTAT (Fig. 2A Left, insertion indicated by an arrowhead), whereas that of the wild type was TATATCCTAT (Fig. 2A Right). The mutant pSVLDm2 was then transfected to HuH-7 cells. The temporal profiles of small and large HDAg expression were followed by immunoblotting. The wild-type plasmid pSVLD3 was used as the control. As expected, both species of HDAg (p24 and p27) appeared in cells at days 9 and 12 after transfection (Fig. 28, lanes 7 and 8). This was
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consistent with the results of previous in vivo and in vitro transfection experiments (15, 16). However, the mutant (pSVLDm2) expressed only the small HDAg throughout the time course (Fig. 2B, lanes 1-4). Furthermore, no other HDAg variants were generated. The HDV Mutant Replicated More Actively than the Wild Type. To determine whether the mutant HDV could replicate, total RNAs were extracted from transfected cells for Northern blotting analysis. Genomic strand (Fig. 3A) and antigenomic strand (Fig. 3B) HDV RNAs were found in cells transfected with pSVLDm2. Their amounts were noted to increase from day 3 to day 9 (compare lanes 4 with lanes 6 in A and B). The amount of total RNA in each lane was equivalent, as indicated by the similar level of the glycerol3-phosphate dehydrogenase transcript (Fig. 3C). These data suggest active replication of the mutant HDV genome in the transfected cells. The large form of HDAg acts as a strong inhibitor for HDV replication (17). Therefore, it was interesting to compare the level of replication in this HDV mutant. In our study, the mutant's replication level appeared to be =2-fold higher than that of the wild type (Fig. 3 A and B, lanes 4-6 versus lanes 1-3). This result is consistent with the previous proposal. The Small HDAg Was Inadequate for Virion Assembly. Recently, cotransfection of HDV and HBV DNAs to the HuH-7 cell line has been shown to produce HDV virions (18). The dependence of HDV production on helper HBV was further narrowed down to the HBsAg genes (C.-J.W. and
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P.-J.C., unpublished observations). To determine whether the small HDAg alone was sufficient for virion production, pSVLDm2 was cotransfected with plasmid pSiX (a plasmid containing the ORF of HBsAg genes). Any released virions were pelleted and characterized by Northern and Western blotting. However, no virions containing HDV genome (Fig. 4A, lanes 1 and 5) or small HDAg (Fig. 4C, lanes 1 and 5) were detected. In contrast, the wild-type HDV plasmid (pSVLD3), when cotransfected with plasmid pSiX, did produce HDV virions containing viral RNA (Fig. 4A, lanes 3 and 7) and both types of HDAgs (Fig. 4C, lanes 3 and 7). The mutant's failure to produce virions was not due to suppression of HBsAg production by HDV replication (2), since the amounts of HBsAg particles were equivalent in both systems (Fig. 4B, compare lane 1 with lane 3). Therefore, in the presence of the viral genome and HBV envelope proteins, the small HDAg alone was not adequate for virion formation.
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FIG. 4. Cotransfection experiments indicating that small HDAg alone is inadequate for virion formation. (A) Detection of the HDV genome in the pellets from culture medium of cotransfected cells. Virions were pelleted from culture medium of HuH-7 cells collected at days 6 and 9 after cotransfection with pSVLDm2 plus pS1X (lanes 1 and 5), pSVLDm2 plus pSiX plus pSVDAg-L (lanes 2 and 6), pSVLD3 plus pS1X (lanes 3 and 7), pSVLDm2 plus pSVDAg-L (lanes 4 and 8). The amount of each plasmid used was 10 ug. RNA was isolated from the pellets and subjected to Northern blotting analysis with a riboprobe detecting the genomic strand of HDV RNA. The HDV RNA was found only in pellets harvested from HuH-7 cells cotransfected with pSVLD3 plus pS1X but was not found in cells cotransfected with the other three combinations. Lane 9, total RNA from HuH-7 cells transfected with pSVLD3. (B) Demonstration of a similar level of HBsAg production in the three cotransfection systems. Pelleted particles from HuH-7 cells cotransfected with pSVLDm2 plus pS1X (lane 1), pSVLDm2 plus pS1X plus pSVDAg-L (lane 2), and pSVLD3 plus pS1X (lane 3) were separated by SDS/PAGE, transferred to a nitrocellulose membrane, and allowed to react with a monoclonal antibody against the major HBsAg. The amounts of major HBsAgs (p24 and gp27) were equivalent in the three cotransfections. Lane C, HBV virions obtained from a patient by purification through centrifugation twice in a sucrose gradient (3). (C) Detection of the HDAgs in particles from the cotransfection experiments. The sample organization was essentially the same as in A. Lane M, molecular mass markers for proteins; lane C, wild-type HDV virions are used as the positive control. Two species of HDAg were found in particles released from HuH-7 cells cotransfected with pSVLD3 plus pS1X (lanes 3 and 7). Only the large HDAg was found in that from cells transfected with pSVLDm2 plus pS1X plus pSVDAg-L (lanes 2 and 6).
In the following experiment, the large HDAg-expressing plasmid (pSVDAg-L) was included in the transfection to recover the HDV virion production. At first, equal amounts of large HDAg-expressing plasmid (pSVDAg-L) and the mutant HDV plasmid (pSVLDm2) were transfected together with HBsAg-expressing plasmid (pSlX). Viral particles again were pelleted and analyzed. However, no HDV genome was packaged (Fig. 4A, lanes 2 and 6). This observation appeared puzzling initially. However, this can be explained by the recent discovery that the large HDAg, on cotransfection with small HDAg at a ratio of 1:1, could shut down the HDV replication (17). In fact, Northern blot analysis of RNA from such transfected cells showed that the level of HDV RNA replication was reduced to a residual 3% (Fig. 5A, lanes 1 and 2). Thus, the failure of the large HDAg to recover HDV virions from this system may be due to inadequate viral replication. When particles released from such a system were examined by immunoblotting, the large HDAg was found to be packaged into the HBsAg particles quite efficiently (Fig. 4C, lanes 2 and 6). The copackaging of large HDAg, without detectable HDV RNA and the small HDAg, suggested its important role in virion assembly of HDV. The Large HDAg Was Essential for HDV Virion Production. To ameliorate the suppressive effects of the cotransfected large HDAg on HDV replication, a rescue experiment was performed with the amount of the large HDAg plasmid adjusted to 1/10th of the HDV mutant plasmid. The cells and culture medium were collected at 3, 6, and 9 days posttransfection for RNA and virion analysis. Significant amounts of viral genome replication were found in the transfected cells (Fig. 5A, lanes 3 and 4), indicating diminution of the large HDAg's suppressive effect. Next, the virions were pelleted from the medium. Northern blotting revealed the HDV genome in the released virions (Fig. 5B, lanes 3-5). In addition, Western blotting also showed two species of the HDAgs packaged into the recovered HDV virions (Fig. 5C, lanes 1-3). To further characterize the putative HDV virions, the pellets were subjected to equilibrium centrifugation at a discontinuous cesium chloride gradient. Each fraction was collected and the location of the virions was identified by detecting the presence of viral RNA.
The peak of HDV RNA was localized in fraction 11 (Fig. 5D), which has the corresponding density of 1.24 g/cm3 (Fig. SD). The result is consistent with that of the HDV particles isolated from natural infection (3, 4). It was therefore concluded that the large HDAg was crucial for HDV virion assembly.
DISCUSSION Using an HDV mutant that could replicate but express only the small HDAg in an in vitro cotransfection system (18), the role of individual HDAgs in virion formation was studied. Despite the presence of active viral genome replication and the production of adequate amounts of envelope proteins from helper HBV, the small HDAg by itself is not sufficient for virion formation. Therefore, incorporation of small HDAg into the virion is not due to packaging signals in the protein but probably occurs through interaction with other HDV components. Addition of the large-form HDAg into cells transfected with the mutant HDV and pS1X resulted in the production of HDV virions. Hence, it is an essential component for virus formation. The large HDAg behaves as a bridge to connect the ribonucleoprotein complex (formed by the HDV RNA and the small HDAg) and the HBV envelope proteins. It contains a 19-amino acid domain in the carboxyl terminus that is missing in the ineffective small HDAg (Fig. 6). This C-19 domain is therefore most likely involved in interacting with the HBsAgs. Whether this domain portion only or the entire large HDAg is the crucial factor in the HDV virion assembly remains unknown. Based upon work by Lin et al. (20), both types of HDAgs contain a central domain with a strong affinity to viral genome. The large HDAg could encapsidate the HDV RNA into viral particles by direct binding with the viral genome. Indirect interaction through the small HDAg is also conceivable, because there are leucine zipper-like domains in the amino terminus of both HDAgs, allowing dimer formation (20). The large HDAg can be efficiently packaged into the HBsAg particles, with very low levels of HDV genome and small HDAg. Upon further study, using plasmids expressing
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only the large HDAg and HBsAgs, we found that neither the HDV genome nor the small HDAg was necessary for incorporation of the large HDAg into the HBsAg particles (unpublished observations). This interesting phenomenon supports the crucial role of the large HDAg in virion assembly of HDV and suggests the possible formation of empty HDV-like particles. A detailed characterization of these particles is necessary, as these results possibly could be used to engineer the current HBV vaccine (the HBsAg particles) to include the HDAg or other viral antigens. Such polyvalent vaccines could become a more effective approach for prevention and control of viral hepatitis.
We thank Dr. Michael M. C. Lai for plasmid DD1S, Dr. S.-C. Lee for the monoclonal antibody, and S.-H. Yeh and M.-S. Chuang for graphic and secretarial assistance. We are grateful to Lucene Tong for editing the English. The study was supported by grants from the National Science Council and Department of Health, Taiwan, Republic of China. 1. Rizzetto, M., Canese, M. G., Arico, S., Crivelli, O., Trepo, C., Bonino, F. & Verme, G. (1977) Gut 18, 997-1003. 2. Rizzetto, M., Boyer, B., Canese, M. G., Shih, J. W.-K., Purcell, R. H. & Gerin, J. L. (1980) Proc. Natl. Acad. Sci. USA 77, 6124-6128. 3. Bonino, F., Herrmann, K., Rizzetto, M. & Gerlich, W. H. (1986) J. Virol. 58, 945-950. 4. Bergman, K. & Gerin, J. (1986) J. Infect. Dis. 154, 702-706. 5. Wang, K.-S., Choo, Q.-L., Weiner, A. J., Ou, J.-H., Narajan, R. C., Thayer, R. M., Mullenback, G. T., Denniston, K. J., Gerin, J. L. & Houghton, M. (1986) Nature (London) 323, 508-513. 6. Chen, P.-J., Kalpana, G., Goldberg, J., Mason, W., Werner, B., Gerin, J. & Taylor, J. (1986) Proc. Natl. Acad. Sci. USA 83, 8774-8778. 7. Taylor, J. (1990) Cell 61, 371-373. 8. Kuo, M. Y.-P., Chao, M. & Taylor, J. (1989) J. Virol. 63, 1945-1950. 9. Chen, P.-J., Kuo, M. Y.-P., Chen, M.-L., Tu, S.-J., Chiu, M.-N., Wu, H.-L., Hsu, H.-C. & Chen, D.-S. (1990) Proc. Nat!. Acad. Sci. USA 87, 5253-5257. 10. Weiner, A. J., Choo, Q.-L., Wang, K.-S., Govindarajan, S., Redeker, A. G., Gerin, J. L. & Houghton, M. (1988) J. Virol. 62, 594-599. 11. Kuo, M. Y.-P., Goldberg, G., Coates, L., Mason, W., Gerin, J. & Taylor, J. (1988) J. Virol. 62, 1945-1950. 12. Makino, S., Chang, M.-F., Baker, S. C., Govindarajan, S. & Lai, M. M. C. (1987) Nature (London) 329, 343-346. 13. Chao, Y.-C., Chang, M.-F., Gust, I. & Lai, M. M. C. (1990) Virology 178, 384-392. 14. Xia, S.-P., Chang, M.-F., Wei, D., Govindarajan, S. & Lai, M. M. C. (1990) Virology 178, 331-336. 15. Luo, G. M., Chao, M., Hsieh, S.-Y., Sureau, C., Nishikura, N. & Taylor, J. (1990) J. Virol. 64, 1021-1027. 16. Sureau, C., Taylor, J., Chao, M., Eichberg, J. E. & Lanford, R. E. (1989) J. Virol. 63, 4292-4297. 17. Chao, M., Hsieh, S.-Y. & Taylor, J. (1990) J. Virol. 64, 5066-5069. 18. Wu, J.-C., Chen, P.-J., Kuo, M. Y.-P., Lee, S.-D., Chen, D.-S. & Ting, L.-P. (1991) J. Virol. 65, 1096-1104. 19. Higuchi, R., Krummel, B. & Saiki, R. K. (1988) Nucleic Acids Res. 16, 7351-7367. 20. Lin, L.-H., Chang, M.-F., Baker, S. C., Govindarajan, S. & Lai, M. M. C. (1990) J. Virol. 64, 4051-4058.
The large form of hepatitis 6 antigen is crucial for assembly of hepatitis 6 virus (RNA virus/virion assembly)
FU-LIN CHANG, PEI-JER CHEN, SU-JEN Tu, CHUAN-JEN WANG, AND DING-SHINN CHEN* Graduate Institute of Clinical Medicine, College of Medicine, Hepatitis Research Center, The University Hospital, National Taiwan University, Taipei, Taiwan 10016
Communicated by Robert H. Purcell, June 13, 1991
ABSTRACT The vinions of hepatitis 6 virus (HDV) contain two species of HDV-specific protein, a large and a small form of hepatitis 6 antigen (HDAg). We examined the role of individual HDAgs in virion assembly in cotransfection experiments. First, we constructed a replication-competent HDV mutant expressing only the small HDAg. When cotrandected with a plasmid expressing hepatitis B virus surface antigens to the HuH-7 cells, the mutant did not produce HDV virions, whereas the wild-type HDV clone did. Therefore, though the small HDAg is important for viral replication and is incorporated into the virus, the small-form 6 antigen by itself is insufficient for virion formation. When the system was cotransfected with an additional plasmid providing the large HDAg, the HDV virion was then recovered. There was also evidence suggesting that the large HDAg could be copickaged into the HBsAg particles, without the presence of the HDV genome and the small HDAg. The results indicate a crucial role of the large HDAg in HDV assembly.
When the clone is transfected into appropriate cell lines for several days of replication, the large HDAg is produced in addition to the original small HDAg (16). This new large HDAg population is the result of a specific base change at the termination codon of the small HDAg ORF (UAG to UGG) (15). This late-appearing large HDAg behaves as a transnegative factor limiting the HDV replication process (17). Although the functions of the two HDAgs have been determined, their roles in virion assembly remain unclear. The relative amounts of the two HDAgs are different in the infected liver and mature virions. The ratio of large to small HDAg is about 0.1 in the infected liver, whereas the ratio in the virions can increase up to 0.9 (3, 4). Since there is a greater incorporation of the large HDAg in the virions, it suggests a significant role of the large HDAg in virus assembly. Recently we have succeeded in producing HDV virions by cotransfecting a human hepatoma cell line, HuH-7, with cloned HBV and HDV DNA (18). Using the same system, we studied the significance of individual HDAgs in virion formation. To do this, we had to unravel the close association between the two HDAgs by constructing a frame-shift HDV mutant capable of replication but expressing only the small HDAg. This mutant was then cotransfected with HBV DNA in the presence and absence of the large HDAg plasmid. The products of these experiments were analyzed for the HDV virions. The results indicate that the small HDAg alone is insufficient for viral particle formation and that the large HDAg is essential for HDV virion assembly. Furthermore, the large HDAg appears to be copackaged with the HBsAg particles even in the absence of HDV RNA.
Hepatitis 8 virus (HDV) is a defective infectious agent that transmits in the presence of the helper hepatitis B virus (HBV) (1, 2). The virion is a 36-nm particle consisting of three components: (i) an envelope derived from the surface proteins of HBV (3); (ii) two internal proteins, specifically the hepatitis 6 antigens (HDAgs) (4); (iii) a single-stranded, circular RNA genome (5, 6). Our knowledge about HDV genome replication and gene expression has progressed rapidly in recent years (reviewed in ref. 7). However, little is known at the level of virion maturation. The HDV genome is naturally considered to be an integral part of virion, but the signals responsible for viral RNA packaging are not yet defined. The surface proteins of HBV (large, middle, and small HBsAgs) are also essential for virion formation, as previous experiments have shown that no virus particles were produced from the replication of HDV genome alone (8, 9). On the other hand, the role of HDAgs in virion assembly is little understood. The two species of HDAgs, large and small forms (p27 and p24), have been found in the virus and the liver of infected humans and animals (3, 4). The large HDAg includes the entire small HDAg plus an additional 19 amino acids. at the carboxyl terminus (10). They are encoded by the largest conserved open reading frame (ORF) of antigenomic polarity in two types of HDV genome (11, 12). In natural infections, the HDV virus always appears to be a quasispecies composed of two genomic populations (13, 14). One of these genomes contains the short HDAg ORF consisting of 195 amino acids and the other contains the longer ORF with 214 amino acids. Recently, the relationship between the two HDAgs has been studied by Luo et al. (15). The original HDV cDNA clone containing an ORF encoding only the small HDAg is used.
MATERIALS AND METHODS Synthesis of Oligonucleotides. To do site-directed mutagenesis of the HDV genome, four oligonucleotides were synthesized (DNA synthesizer 381A, Applied Biosystems). Primer D1 has sequences of 5'-CCATAGIGATATACTC-3' that represents nucleotides 1016-1002 of the cDNA clone of antigenomic HDV RNA with insertion of a nucleotide (thymidine) immediately behind the termination codon of the small HDAg ORF. Primer D2 is complementary to D1, with sequences of 5'-GAGTATATCACTATGG-3' (nucleotides 1002-1016, genomic strand). Primer D3 has sequences of
5'-CCGGCATGGTCCCAGCCT-3' (nucleotides 688-705, genomic strand), and primer D4 has sequences of 5'ATGAGCCGGTCCGAGTCG-3' (nucleotides 1598-1581, antigenomic strand). The relative locations of the four oligonucleotides are summarized in Fig. 1B. The nucleotide designations are based on the chimpanzee HDV isolate (5). All Abbreviations: HDV, hepatitis 8 virus; HDAg, hepatitis 8 antigen; HBV, hepatitis B virus; ORF, open reading frame; HBsAg, HBV surface antigen; SV40, simian virus 40. *To whom reprint requests should be addressed.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
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primers were purified for subsequent use by the use of an OPC column (Applied Biosystems). Construction of the HDV Mutant. To prepare the HDV mutant, one additional thymidine base was incorporated into the designated position of the wild-type HDV genome (Fig. 1A) by the procedure of recombinant PCR (19). Two overlapping DNA fragments were first amplified from the cDNA clone of HDV by the use of primers D1 plus D3 or primers D2 plus D4 (Fig. 18). They were then used as template and subjected to recombinant PCR with oligonucleotides D3 and N4 as primers (Fig. 1B). The final recombinant DNA fragment of 912 base pairs (bp) was gel-purified, digested with Stu I and Bst XI, subcloned into pGEM3Z, and then sequenced completely to show no other mutation. Afterward, it was used to replace the same region in the wild-type HDV (pGEMD1). The mutant HDV monomer was isolated, preligated, and then cloned into the Xba I site of the expression vector pSVL (Pharmacia, Pharmacia LKB Biotechnology). In this manner, plasmid pSVLDm2, containing a tandem HDV dimer in the genomic orientation, was obtained (Fig. 18). A wild-type HDV clone, pSVLD3, consisting of a tandem trimer driven by the SV40 late promoter was used as the control. On transfection to HuH-7 cells, the plasmid can direct the genome replication and expression of the two species of HDAgs. Upon cotransfection with HBV DNA, the HDV vinions are produced (18). To construct a plasmid expressing the large HDAg, a BamHIl-Pst I fragment, derived from plasmid DD15 (12) containing the ORE for large HDAg, was first digested with
Si nuclease and then cloned into the Sma I site of pSVL. The organization of this plasmid, pSVDAg-L, is shown in Fig. 1C. Plasmid pSiX, including a partial HBV genome (Apa I-Bgl II fragment), was constructed into the Sma I site of pGEM-3Z (Fig. 1C) and used in the transfection system to provide the three HBV surface proteins.
Miscellaneous Methods. Procedures for transfection of HuH-7 cells, analysis of nucleic acids and proteins, and isopycnic centrifugation of HDV virions were performed essentially as described (9, 18).
RESULTS Construction of a EHDV Mutant Unable to Express Large HDAg. To abolish the expression of large HDAg, one thymidine was inserted immediately at the 3' position of the termination codon of the small HDAg ORE (Fig. 1A). The mutation resulted in two consecutive termination codons at the end of the small HDAg ORF (Fig. 1A). In addition, the ORF following the termination codon was shifted out of frame. Thus, two mechanisms were combined to prevent generation of the large HDAg. The nucleotide sequence of the mutant was confirmed by sequencing. The DNA sequence of the mutant was TATATCACTAT (Fig. 2A Left, insertion indicated by an arrowhead), whereas that of the wild type was TATATCCTAT (Fig. 2A Right). The mutant pSVLDm2 was then transfected to HuH-7 cells. The temporal profiles of small and large HDAg expression were followed by immunoblotting. The wild-type plasmid pSVLD3 was used as the control. As expected, both species of HDAg (p24 and p27) appeared in cells at days 9 and 12 after transfection (Fig. 28, lanes 7 and 8). This was
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consistent with the results of previous in vivo and in vitro transfection experiments (15, 16). However, the mutant (pSVLDm2) expressed only the small HDAg throughout the time course (Fig. 2B, lanes 1-4). Furthermore, no other HDAg variants were generated. The HDV Mutant Replicated More Actively than the Wild Type. To determine whether the mutant HDV could replicate, total RNAs were extracted from transfected cells for Northern blotting analysis. Genomic strand (Fig. 3A) and antigenomic strand (Fig. 3B) HDV RNAs were found in cells transfected with pSVLDm2. Their amounts were noted to increase from day 3 to day 9 (compare lanes 4 with lanes 6 in A and B). The amount of total RNA in each lane was equivalent, as indicated by the similar level of the glycerol3-phosphate dehydrogenase transcript (Fig. 3C). These data suggest active replication of the mutant HDV genome in the transfected cells. The large form of HDAg acts as a strong inhibitor for HDV replication (17). Therefore, it was interesting to compare the level of replication in this HDV mutant. In our study, the mutant's replication level appeared to be =2-fold higher than that of the wild type (Fig. 3 A and B, lanes 4-6 versus lanes 1-3). This result is consistent with the previous proposal. The Small HDAg Was Inadequate for Virion Assembly. Recently, cotransfection of HDV and HBV DNAs to the HuH-7 cell line has been shown to produce HDV virions (18). The dependence of HDV production on helper HBV was further narrowed down to the HBsAg genes (C.-J.W. and
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P.-J.C., unpublished observations). To determine whether the small HDAg alone was sufficient for virion production, pSVLDm2 was cotransfected with plasmid pSiX (a plasmid containing the ORF of HBsAg genes). Any released virions were pelleted and characterized by Northern and Western blotting. However, no virions containing HDV genome (Fig. 4A, lanes 1 and 5) or small HDAg (Fig. 4C, lanes 1 and 5) were detected. In contrast, the wild-type HDV plasmid (pSVLD3), when cotransfected with plasmid pSiX, did produce HDV virions containing viral RNA (Fig. 4A, lanes 3 and 7) and both types of HDAgs (Fig. 4C, lanes 3 and 7). The mutant's failure to produce virions was not due to suppression of HBsAg production by HDV replication (2), since the amounts of HBsAg particles were equivalent in both systems (Fig. 4B, compare lane 1 with lane 3). Therefore, in the presence of the viral genome and HBV envelope proteins, the small HDAg alone was not adequate for virion formation.
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In the following experiment, the large HDAg-expressing plasmid (pSVDAg-L) was included in the transfection to recover the HDV virion production. At first, equal amounts of large HDAg-expressing plasmid (pSVDAg-L) and the mutant HDV plasmid (pSVLDm2) were transfected together with HBsAg-expressing plasmid (pSlX). Viral particles again were pelleted and analyzed. However, no HDV genome was packaged (Fig. 4A, lanes 2 and 6). This observation appeared puzzling initially. However, this can be explained by the recent discovery that the large HDAg, on cotransfection with small HDAg at a ratio of 1:1, could shut down the HDV replication (17). In fact, Northern blot analysis of RNA from such transfected cells showed that the level of HDV RNA replication was reduced to a residual 3% (Fig. 5A, lanes 1 and 2). Thus, the failure of the large HDAg to recover HDV virions from this system may be due to inadequate viral replication. When particles released from such a system were examined by immunoblotting, the large HDAg was found to be packaged into the HBsAg particles quite efficiently (Fig. 4C, lanes 2 and 6). The copackaging of large HDAg, without detectable HDV RNA and the small HDAg, suggested its important role in virion assembly of HDV. The Large HDAg Was Essential for HDV Virion Production. To ameliorate the suppressive effects of the cotransfected large HDAg on HDV replication, a rescue experiment was performed with the amount of the large HDAg plasmid adjusted to 1/10th of the HDV mutant plasmid. The cells and culture medium were collected at 3, 6, and 9 days posttransfection for RNA and virion analysis. Significant amounts of viral genome replication were found in the transfected cells (Fig. 5A, lanes 3 and 4), indicating diminution of the large HDAg's suppressive effect. Next, the virions were pelleted from the medium. Northern blotting revealed the HDV genome in the released virions (Fig. 5B, lanes 3-5). In addition, Western blotting also showed two species of the HDAgs packaged into the recovered HDV virions (Fig. 5C, lanes 1-3). To further characterize the putative HDV virions, the pellets were subjected to equilibrium centrifugation at a discontinuous cesium chloride gradient. Each fraction was collected and the location of the virions was identified by detecting the presence of viral RNA.
The peak of HDV RNA was localized in fraction 11 (Fig. 5D), which has the corresponding density of 1.24 g/cm3 (Fig. SD). The result is consistent with that of the HDV particles isolated from natural infection (3, 4). It was therefore concluded that the large HDAg was crucial for HDV virion assembly.
DISCUSSION Using an HDV mutant that could replicate but express only the small HDAg in an in vitro cotransfection system (18), the role of individual HDAgs in virion formation was studied. Despite the presence of active viral genome replication and the production of adequate amounts of envelope proteins from helper HBV, the small HDAg by itself is not sufficient for virion formation. Therefore, incorporation of small HDAg into the virion is not due to packaging signals in the protein but probably occurs through interaction with other HDV components. Addition of the large-form HDAg into cells transfected with the mutant HDV and pS1X resulted in the production of HDV virions. Hence, it is an essential component for virus formation. The large HDAg behaves as a bridge to connect the ribonucleoprotein complex (formed by the HDV RNA and the small HDAg) and the HBV envelope proteins. It contains a 19-amino acid domain in the carboxyl terminus that is missing in the ineffective small HDAg (Fig. 6). This C-19 domain is therefore most likely involved in interacting with the HBsAgs. Whether this domain portion only or the entire large HDAg is the crucial factor in the HDV virion assembly remains unknown. Based upon work by Lin et al. (20), both types of HDAgs contain a central domain with a strong affinity to viral genome. The large HDAg could encapsidate the HDV RNA into viral particles by direct binding with the viral genome. Indirect interaction through the small HDAg is also conceivable, because there are leucine zipper-like domains in the amino terminus of both HDAgs, allowing dimer formation (20). The large HDAg can be efficiently packaged into the HBsAg particles, with very low levels of HDV genome and small HDAg. Upon further study, using plasmids expressing
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FIG. 5. Appropriate amounts of large HDAg could rescue HDV virion production. (A) Effects of cotransfection with variable amounts of large HDAg on HDV replication. Northern blot analysis for HDV RNA (genomic strand) in HuH-7 cells at days 6 and 9 posttransfected with 10 ,ug (each) of pSVLDm2, pSlX, pSVDAg-L (lanes 1 and 2), with 20 ttg of pSVLDm2 plus 10 .g of pSlX plus 2 ,ug of pSVDAg-L (lanes 3 and 4), or with 10 ug of pSVLDm2 plus 10 ,ug of pSlX (lanes 5 and 6). The replication of HDV was markedly reduced in lanes 1 and 2 but retained a significant level in lanes 3 and 4. M and D, positions of HDV monomer and dimer, respectively. (B) Rescue ofHDV virion production by cotransfection with appropriate amounts of large HDAg-expressing plasmid. Virions were recovered from culture medium of HuH-7 cells transfected with 10 p&g (each) of pSVLDm2, pSlX, and pSVDAg-L (lanes 1 and 2, days 6 and 9 posttransfection), with 20 ,ug of pSVLDm2 plus 10 jtg of pSlX plus 2 ,ug of pSVDAg-L (lanes 3-5; days 3, 6, and 9 posttransfection), or with 10 ,ug (each) of pSVLD3 and pSlX (lanes 6 and 7; days 9 and 6 posttransfection). The HDV genome was demonstrated in lanes 3-5, indicating successful recovery of virions. (C) Western blot study of HDAgs in the particles released from cotransfected HuH-7 cells. Lanes 1-3, results of analyzing virions in culture medium harvested from HuH-7 cells at days 3, 6, and 9 posttransfection with 20 ,ug of pSVLDm2 plus 10 j&g of pSlX plus 2 ug of pSVDAg-L. Two species of HDAgs were demonstrated. Lanes 4 and 5, results from HuH-7 cells cotransfected with 10 ,g (each) of pSVLDm2, pSlX, and pSVDAg-L at days 6 and 9. Lane M, protein markers; lane 6, wild-type HDV virions as control. (D) Physical characteristics of the rescued HDV virions. Such virions were banded in the cesium chloride gradient. By detection of the viral genome, the peak fraction (no. 11) was localized at a density of 1.24 g/cm3.
only the large HDAg and HBsAgs, we found that neither the HDV genome nor the small HDAg was necessary for incorporation of the large HDAg into the HBsAg particles (unpublished observations). This interesting phenomenon supports the crucial role of the large HDAg in virion assembly of HDV and suggests the possible formation of empty HDV-like particles. A detailed characterization of these particles is necessary, as these results possibly could be used to engineer the current HBV vaccine (the HBsAg particles) to include the HDAg or other viral antigens. Such polyvalent vaccines could become a more effective approach for prevention and control of viral hepatitis.
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