- Email: [email protected]
The Hepatitis B Virus Posttranscriptional Regulatory Element Contains a Highly Stable RNA Secondary Structure
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
231, 864–867 (1997)
RC976205
The Hepatitis B Virus Posttranscriptional Regulatory Element Contains a Highly Stable RNA Secondary Structure Volker Patzel and Georg Sczakiel1 Forschungsschwerpunkt Angewandte Tumorvirologie, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 242, 69120 Heidelberg, Germany
Received January 7, 1997
Hepatitis B virus (HBV) transcripts contain a sequence known as the posttranscriptional regulatory element (PRE). This element was shown to facilitate the nuclear export of the S gene transcripts, to partially substitute for the human immunodeficiency virus (HIV-1) Rev-response element (RRE), and to bind two cellular factors. Within the genetically defined PRE (approximately 450 nucleotides), we identified a highly stable secondary structural element of 313 nucleotides in length termed PRE313 . The energy values of the PRE313 are similar to those of the RRE of HIV-1 and significantly lower than those of other portions of HBV RNA. A comparison of human HBV subtypes shows strong conservation of the PRE313 in terms of energy and structure, providing further evidence for the biological significance of the genetically defined PRE and the PRE313 in particular. The structural model for the PRE313 described in this study may help in identifying crucial components of the transport mechanism of transcripts of HBV. q 1997 Academic Press
The genome of the hepatitis B virus (HBV) is a partially double-stranded circular DNA of 3.2 kb in length which is replicated by protein-primed reverse transcription of an RNA pregenome (1). All known gene products of HBV are encoded by one genomic DNA strand and are translated from four classes of unspliced transcripts that terminate at a single polyadenylation site (reviewed in reference 2). The major surface (envelope) protein is synthesized from the major surface (S) gene transcripts. Within the downstream region of the S transcripts a functional element termed posttranscriptional regulatory element (PRE) has been identified by a genetic analysis (3). The PRE element acts in 1 Corresponding author. Fax: [email protected].
/49-6221-424932.
0006-291X/97 $25.00
E-mail:
cis at the RNA level. It facilitates nuclear export of unspliced PRE-containing transcripts (4,5). In functional terms, its similarity with the Rev-response element (RRE) of HIV-1 is indicated by the observation that the PRE can partially substitute for the function of the RRE element in case of expression of a RRE/ Rev-dependent reporter gene (3). Further, two cellular proteins have been described that bind to the PRE and that may be involved in the export of unspliced mRNA species (6). In mechanistic terms, the PRE therefore might even be closer related to the constitutive transport element (CTE) of the simian retrovirus type I (SRV-I) (7). Deletion analysis reveals that the HBV PRE consists of two subelements which function synergistically (8). Despite its crucial role in replication and gene expression of HBV, there is a lack of knowledge on the size and structure of the PRE. In a number of cases, biologically functional RNA elements were found to correlate with thermodynamically stable structural elements. For example, the location of the RRE element of HIV-1 as well as its overall secondary structure have been identified by calculation of the local folding potential and by deriving secondary structures thereof (9-11). In this work, we applied a similar approach to HBV sequences. A highly stable 313 nucleotide long RNA secondary structural element (PRE313) was identified within the PRE that is comparable in size and stability with the HIV-1 RRE element. The PRE313 element is predicted to be formed in all HBV transcripts containing its sequence. MATERIALS AND METHODS Programs and nucleotide sequences. Secondary structures of RNA were calculated using the programme ‘mfold’. It is based on the GCG package (12) and is available within the computer service ‘HUSAR’ [Heidelberg Unix Sequence Analysis Resources, (13)]. All nucleotide sequences were imported from the EMBL data library. The names of sequences of the EMBL data base were used in this work as well.
864
Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.
AID
BBRC 6205
/
691c$$1561
02-07-97 03:49:10
bbrcg
AP: BBRC
Vol. 231, No. 3, 1997
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
RESULTS AND DISCUSSION Sequences within the PRE Can Form a Highly Stably Structure It is reasonable to assume that biologically functional RNA elements coincide with highly stable RNA structures. This correlation has been found for viral sequences including TAR and RRE of HIV-1, the CTE of SRV-I (7), the RexRE of HTLV-I (14) or IRES elements of EMCV (unpublished). This correlation has also been observed for cellular mRNA such as the 5* UTR of the murine p53 mRNA (15) or the human insulin-like growth factor II (IGF-II) mRNA (16). The RRE of HIV1 was proposed as a functional element at a time when its role for HIV-1 replication was still not resolved (911). To identify possible functional sequence elements of HBV transcripts, we first analysed the possibility of sequence elements to form low-energy structures (‘local folding potential’; ref. 17) for the HBsAg subtype adw2 (18). A plot of the energy values (DG) corresponding to the lowest-energy structure of a given sequence stretch (window) versus the position on the HBV sequence shows that the absolute energy minimum of all HBV sequences is located within the PRE for window sizes varying between 50 and 500 nucleotides. For window sizes equal to or greater than 100 nucleotides the position of this local DG minimum was independent of the window size. The lowest value for DG per length was found at a size of 313 nucleotides (Fig. 1) indicating that a sequence stretch of this length between positions 1203 and 1515 can form a highly stable structure. The values for the energy per length of other biologically functional RNA elements with a comparable size are similar to that of the PRE313 (00.33 kcal mol01 nt01) and are significantly lower than the avarage values for entire sequences such as HIV-1 (00.17 kcal mol01 nt01) or HBV sequences (00.21 kcal mol01 nt01), respectively (Table 1). The location and energy values for the lowest-energy structures were calculated for a set of types of hepatitis viruses. The energy values are well conserved even across species barriers (Table 2) and strongly suggest that the RNA structure of PRE313 could play the biological role that was defined by genetic analyses to be located within the larger PRE (Fig.1). A Structural Model of the PRE Element of Human Hepatitis B Viruses A calculation of the secondary structure of the PRE313 sequence of a set of human virus types indicates structural conservation to a varying extent (Fig.2). Within a stretch of 142 nucleotides located in a central region of the PRE313 (subregion I in Fig.2) the composition of structural elements is identical. The structures in subregions II and III (see Fig.2) are less conserved whereas their energy values are comparable among all
FIG. 1. Prediction of a highly stable RNA secondary structure element within the PRE of HBVadw2 termed PRE313 . (A) Plot of the lowest possible free energy (DG) of overlapping sequence stretches (313 nt) versus the sequence position of the viral RNA pregenome (sequence published in ref. 18, numbering according to ref. 19). The calculation was performed as described in detail recently (17). The DG minimum at position 1359 represents a highly stable RNA secondary structure of 313 nucleotides in length. (B) Schematic depiction of the open reading frames of HBV. (C) Groups of transcripts of HBV.
types listed in Table 2. However, there is no similarity between the predicted secondary structure of the PRE of hepatitis viruses from different species (Table 2). The thermodynamic stability of this structure is based on several continuous stretches of Watson-Crick basepairing. However, a number of internal loops, bulges, and possible non Watson-Crick base-base interactions (e.g. A75-A188) could conceivably provide biological information. The secondary structure of PRE313 was predicted independently from the size of the HBV sequence context used for the folding program. Thus, it is reasonable to assume that the PRE313 element can potentially be formed within the context of all HBV transcripts that contain its sequence including the RNA pregenome. Biological Implications Several observations suggest that the low-energy element PRE313 located within the PRE of HBV could be biologically relevant. (i) It is localized within the genetically defined functional element termed PRE where it could contribute to a major extent to its function. (ii) The unusually high stability suggests that its structure
865
AID
BBRC 6205
/
691c$$1561
02-07-97 03:49:10
bbrcg
AP: BBRC
Vol. 231, No. 3, 1997
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS TABLE 1
Energy Values for the PRE312 , the RRE of HIV-1, and Control Sequences Source
Position
Length (nts)
DG (kcal/mol)
DG/nucleotide (kcal mol01 nt01)
HBV PRE313 HIV-1 RRE p53 mRNA 5*UTR HBV pregenome HIV-1 genomed
1203-1515a 7701-8051 0216-/284 1-3221 1-9706
313 351b 500c 3221 9706
0100.9 0125.2 0170 0663.5 01611.2
00.33 00.36 00.34 00.21 00.17
a
HBVadw2 genome (18), numbering according to ref. (19). Ref. 9. c Ref. 15. d Ref. 20. b
is biologically important. (iii) The stability and location of the PRE313 are phylogentically highly conserved. (iv) Among human virus types, the core of the secondary structure of the PRE313 (pos. 61-202) is highly con-
served although the primary sequence in this region is variable. For example, the divergence of the subtypes Hbvadw2 and Hpbadw3 (Table 2) with respect to the primary sequence in this 313 nucleotide element is
TABLE 2
Phylogenetic Comparison of the Lowest Possible Free Energy (DG) of the PRE313 Sequence of Various Hepadnaviruses and Its Predicted Secondary Structure
Human HBsAg subtype adw
ayw
adr
ayr Other vertebrates Woodchuck Ground squirrel Duck
Sequencea
DG (kcal/mol) minimum
Structural similarity with Hbvadw2b
Hbvadw2 Hbvadw Hpbadw2 Hpbadw3 Hbvxcps Hbvadw4a Hpbadwz Hpbadwl Hbvdna Hbvaywe Hbvaywmcg Hbvorfs Xxhepav Hbvaywc Hbvaywci Hpbhbvaa Hbvadr Hbvadr4 Hbvadrm Hpbcgadr Hpbadra Hpbcg Hehbvayr
0110.8 094.3 0105.7 0110 099.1 098.1 0103.9 0104.3 0106.6 094.8 0112.4 093.5 0109.9 093.7 093.1 094 0107.2 0106.2 099.9 099.9 0102.8 097.8 0110.5
/// /// /// // // / / No // // //c //c /c /c /c No // / //c //c //c /c //
Nc2cg whvcga Ncgsvhxx Dhbvf16
0110.2 099.7 083.2 097.4
No No No No
a
Nomenclature according to the EMBL data base. ///, complete structural similarity (composition of structural elements) to the pRE313 of HBVadw2; //, structural similarity within domains I and II: /, structural similarity within domain I. c Structural similarity to the structure with the second lowest DG values (not shown) that differs in domain I but not in domain II and III. b
866
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BBRC 6205
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691c$$1561
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bbrcg
AP: BBRC
Vol. 231, No. 3, 1997
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
tionship of the PRE in more detail. This study suggests that the PRE313 which is considerably smaller than the PRE described so far by genetic means could be sufficient for its functions in vivo and, thus, could be used as an appropriate tool for future experimental investigations. However, analysis of the RNA structure of the PRE rather than further genetic analyses might be required to understand the functional organisation of the HBV PRE on a more basal level and at higher structural resolution. ACKNOWLEDGMENTS We thank S. Eckardt and W. Nedbal for critically reading the manuscript and we thank J. zu Putlitz for helpful comments. We are grateful to W. Chen and K.-H. Glatting for support in the use of computer programs.
REFERENCES
FIG. 2. Predicted minimum free energy structure of the PRE313 of HBVadw2. The structural domain I (pos. 61-202) is highly conserved through the human HBsAg subtypes as well as the mammalian and avian hepatitis B viruses (Table 2). Domain II (pos. 25-60 and 203242) is less conserved and domain III (pos. 1-24 and 243-313) is least conserved in terms of secondary structure.
7.3% whereas the arrangement of secondary structure elements in subregions I and II (Fig.2) is indistinguishable. The PRE functions in cis and is correlated with an increase of the steady-state levels of surface gene transcripts which is crucial for the efficiency of the replication of HBV. Its role in the replication of HBV and its similarity with the RRE of HIV-1 in functional terms (3) suggest to investigate the structure-function rela-
1. Summers, J., and Mason, W. S. (1982) Cell 29, 403–415. 2. Schaller, H., and Fischer, M. (1991) Curr. Top. Microbiol. Immunol. 168, 21–39. 3. Huang, J., and Liang, T. J. (1993) Mol. Cell. Biol. 13, 7476– 7486. 4. Huang, Z.-M., and Yen, T. S. B. (1994) J. Virol. 68, 3193–3199. 5. Huang, Z.-M., and Yen, T. S. B. (1995) Mol. Cell. Biol. 15, 3864– 3869. 6. Huang, Z.-M., Zang, W.-Q., and Yen, T. S. B. (1996) Virology 217, 573–581. 7. Tabernero, C., Zolotukhin, A. S., Valentin, A., Pavlakis, G. N., and Felber, B. K. (1996) J. Virol. 70, 5998–6011. 8. Donello, J. E., Beeche, A. A., Smith, G. J., III, Lucero, G. R., and Hope, T. J. (1996) J. Virol. 70, 4345–4351. 9. Kimura, T., and Ohyama, A. (1994) J. Biochem. 115, 945–952. 10. Le, S.-Y., Chen, J.-H., Braun, M. J., Gonda, M. A., and Maizel, J. V. (1988) Nucleic Acids Res. 16, 5153–5168. 11. Le, S.-Y., Malim, M. H., Cullen, B. R., and Maizel, J. V. (1990) Nucleic Acids Res. 18, 1613–1618. 12. Devereux, J., Haeberli, P., and Smithies, O. (1984) Nucleic Acids Res. 12, 387–395. 13. Senger, M., Glatting, K.-H., Ritter, O., and Suhai, S. (1995) Comput. Methods Programs Biomed. 46, 131–141. 14. Hanly, S. M., Rimsky, L. T., Malim, M. H., Kim, J. H., Hauber, J., Madeleine, D. D., Le, S.-Y., Maizel, J. V., Cullan, B. R., and Greene, W. C. (1989) Genes Dev. 3, 1534–1544. 15. Mosner, J., Mummenbrauer, T., Bauer, C., Sczakiel, G., Grosse, F., and Deppert, W. (1995) EMBO J. 14, 4442–4449. 16. Scheper, W., Meinsma, D., Holthuizen, P. E., and Sussenbach, J. S. (1995) Moll. Cell. Biol. 15, 235–245. 17. Sczakiel, G., Homann, M., and Rittner, K. (1993) Antisense Res. 3, 45–52. 18. Valenzuela, P., Quiroga, M., Zaldivar, J., Gray, P., and Rutter, W. J. (1980) in Animal Virus Genetics (Fields, B. N., Jaenisch, R., and Fox, C. F., Eds.), pp. 57–70, Academic Press, New York. 19. Galibert, F., Mandart, E., Fitoussi, P. T., and Charnay, P. (1979) Nature 281, 646–650. 20. Adachi, A., Gendelman, H. E., Ko¨nig, S., Folks, T., Willey, R., Rabson, A., and Martin, M. A. (1986) J. Virol. 59, 284–291.
867
AID
BBRC 6205
/
691c$$1561
02-07-97 03:49:10
bbrcg
AP: BBRC
231, 864–867 (1997)
RC976205
The Hepatitis B Virus Posttranscriptional Regulatory Element Contains a Highly Stable RNA Secondary Structure Volker Patzel and Georg Sczakiel1 Forschungsschwerpunkt Angewandte Tumorvirologie, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 242, 69120 Heidelberg, Germany
Received January 7, 1997
Hepatitis B virus (HBV) transcripts contain a sequence known as the posttranscriptional regulatory element (PRE). This element was shown to facilitate the nuclear export of the S gene transcripts, to partially substitute for the human immunodeficiency virus (HIV-1) Rev-response element (RRE), and to bind two cellular factors. Within the genetically defined PRE (approximately 450 nucleotides), we identified a highly stable secondary structural element of 313 nucleotides in length termed PRE313 . The energy values of the PRE313 are similar to those of the RRE of HIV-1 and significantly lower than those of other portions of HBV RNA. A comparison of human HBV subtypes shows strong conservation of the PRE313 in terms of energy and structure, providing further evidence for the biological significance of the genetically defined PRE and the PRE313 in particular. The structural model for the PRE313 described in this study may help in identifying crucial components of the transport mechanism of transcripts of HBV. q 1997 Academic Press
The genome of the hepatitis B virus (HBV) is a partially double-stranded circular DNA of 3.2 kb in length which is replicated by protein-primed reverse transcription of an RNA pregenome (1). All known gene products of HBV are encoded by one genomic DNA strand and are translated from four classes of unspliced transcripts that terminate at a single polyadenylation site (reviewed in reference 2). The major surface (envelope) protein is synthesized from the major surface (S) gene transcripts. Within the downstream region of the S transcripts a functional element termed posttranscriptional regulatory element (PRE) has been identified by a genetic analysis (3). The PRE element acts in 1 Corresponding author. Fax: [email protected].
/49-6221-424932.
0006-291X/97 $25.00
E-mail:
cis at the RNA level. It facilitates nuclear export of unspliced PRE-containing transcripts (4,5). In functional terms, its similarity with the Rev-response element (RRE) of HIV-1 is indicated by the observation that the PRE can partially substitute for the function of the RRE element in case of expression of a RRE/ Rev-dependent reporter gene (3). Further, two cellular proteins have been described that bind to the PRE and that may be involved in the export of unspliced mRNA species (6). In mechanistic terms, the PRE therefore might even be closer related to the constitutive transport element (CTE) of the simian retrovirus type I (SRV-I) (7). Deletion analysis reveals that the HBV PRE consists of two subelements which function synergistically (8). Despite its crucial role in replication and gene expression of HBV, there is a lack of knowledge on the size and structure of the PRE. In a number of cases, biologically functional RNA elements were found to correlate with thermodynamically stable structural elements. For example, the location of the RRE element of HIV-1 as well as its overall secondary structure have been identified by calculation of the local folding potential and by deriving secondary structures thereof (9-11). In this work, we applied a similar approach to HBV sequences. A highly stable 313 nucleotide long RNA secondary structural element (PRE313) was identified within the PRE that is comparable in size and stability with the HIV-1 RRE element. The PRE313 element is predicted to be formed in all HBV transcripts containing its sequence. MATERIALS AND METHODS Programs and nucleotide sequences. Secondary structures of RNA were calculated using the programme ‘mfold’. It is based on the GCG package (12) and is available within the computer service ‘HUSAR’ [Heidelberg Unix Sequence Analysis Resources, (13)]. All nucleotide sequences were imported from the EMBL data library. The names of sequences of the EMBL data base were used in this work as well.
864
Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.
AID
BBRC 6205
/
691c$$1561
02-07-97 03:49:10
bbrcg
AP: BBRC
Vol. 231, No. 3, 1997
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
RESULTS AND DISCUSSION Sequences within the PRE Can Form a Highly Stably Structure It is reasonable to assume that biologically functional RNA elements coincide with highly stable RNA structures. This correlation has been found for viral sequences including TAR and RRE of HIV-1, the CTE of SRV-I (7), the RexRE of HTLV-I (14) or IRES elements of EMCV (unpublished). This correlation has also been observed for cellular mRNA such as the 5* UTR of the murine p53 mRNA (15) or the human insulin-like growth factor II (IGF-II) mRNA (16). The RRE of HIV1 was proposed as a functional element at a time when its role for HIV-1 replication was still not resolved (911). To identify possible functional sequence elements of HBV transcripts, we first analysed the possibility of sequence elements to form low-energy structures (‘local folding potential’; ref. 17) for the HBsAg subtype adw2 (18). A plot of the energy values (DG) corresponding to the lowest-energy structure of a given sequence stretch (window) versus the position on the HBV sequence shows that the absolute energy minimum of all HBV sequences is located within the PRE for window sizes varying between 50 and 500 nucleotides. For window sizes equal to or greater than 100 nucleotides the position of this local DG minimum was independent of the window size. The lowest value for DG per length was found at a size of 313 nucleotides (Fig. 1) indicating that a sequence stretch of this length between positions 1203 and 1515 can form a highly stable structure. The values for the energy per length of other biologically functional RNA elements with a comparable size are similar to that of the PRE313 (00.33 kcal mol01 nt01) and are significantly lower than the avarage values for entire sequences such as HIV-1 (00.17 kcal mol01 nt01) or HBV sequences (00.21 kcal mol01 nt01), respectively (Table 1). The location and energy values for the lowest-energy structures were calculated for a set of types of hepatitis viruses. The energy values are well conserved even across species barriers (Table 2) and strongly suggest that the RNA structure of PRE313 could play the biological role that was defined by genetic analyses to be located within the larger PRE (Fig.1). A Structural Model of the PRE Element of Human Hepatitis B Viruses A calculation of the secondary structure of the PRE313 sequence of a set of human virus types indicates structural conservation to a varying extent (Fig.2). Within a stretch of 142 nucleotides located in a central region of the PRE313 (subregion I in Fig.2) the composition of structural elements is identical. The structures in subregions II and III (see Fig.2) are less conserved whereas their energy values are comparable among all
FIG. 1. Prediction of a highly stable RNA secondary structure element within the PRE of HBVadw2 termed PRE313 . (A) Plot of the lowest possible free energy (DG) of overlapping sequence stretches (313 nt) versus the sequence position of the viral RNA pregenome (sequence published in ref. 18, numbering according to ref. 19). The calculation was performed as described in detail recently (17). The DG minimum at position 1359 represents a highly stable RNA secondary structure of 313 nucleotides in length. (B) Schematic depiction of the open reading frames of HBV. (C) Groups of transcripts of HBV.
types listed in Table 2. However, there is no similarity between the predicted secondary structure of the PRE of hepatitis viruses from different species (Table 2). The thermodynamic stability of this structure is based on several continuous stretches of Watson-Crick basepairing. However, a number of internal loops, bulges, and possible non Watson-Crick base-base interactions (e.g. A75-A188) could conceivably provide biological information. The secondary structure of PRE313 was predicted independently from the size of the HBV sequence context used for the folding program. Thus, it is reasonable to assume that the PRE313 element can potentially be formed within the context of all HBV transcripts that contain its sequence including the RNA pregenome. Biological Implications Several observations suggest that the low-energy element PRE313 located within the PRE of HBV could be biologically relevant. (i) It is localized within the genetically defined functional element termed PRE where it could contribute to a major extent to its function. (ii) The unusually high stability suggests that its structure
865
AID
BBRC 6205
/
691c$$1561
02-07-97 03:49:10
bbrcg
AP: BBRC
Vol. 231, No. 3, 1997
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS TABLE 1
Energy Values for the PRE312 , the RRE of HIV-1, and Control Sequences Source
Position
Length (nts)
DG (kcal/mol)
DG/nucleotide (kcal mol01 nt01)
HBV PRE313 HIV-1 RRE p53 mRNA 5*UTR HBV pregenome HIV-1 genomed
1203-1515a 7701-8051 0216-/284 1-3221 1-9706
313 351b 500c 3221 9706
0100.9 0125.2 0170 0663.5 01611.2
00.33 00.36 00.34 00.21 00.17
a
HBVadw2 genome (18), numbering according to ref. (19). Ref. 9. c Ref. 15. d Ref. 20. b
is biologically important. (iii) The stability and location of the PRE313 are phylogentically highly conserved. (iv) Among human virus types, the core of the secondary structure of the PRE313 (pos. 61-202) is highly con-
served although the primary sequence in this region is variable. For example, the divergence of the subtypes Hbvadw2 and Hpbadw3 (Table 2) with respect to the primary sequence in this 313 nucleotide element is
TABLE 2
Phylogenetic Comparison of the Lowest Possible Free Energy (DG) of the PRE313 Sequence of Various Hepadnaviruses and Its Predicted Secondary Structure
Human HBsAg subtype adw
ayw
adr
ayr Other vertebrates Woodchuck Ground squirrel Duck
Sequencea
DG (kcal/mol) minimum
Structural similarity with Hbvadw2b
Hbvadw2 Hbvadw Hpbadw2 Hpbadw3 Hbvxcps Hbvadw4a Hpbadwz Hpbadwl Hbvdna Hbvaywe Hbvaywmcg Hbvorfs Xxhepav Hbvaywc Hbvaywci Hpbhbvaa Hbvadr Hbvadr4 Hbvadrm Hpbcgadr Hpbadra Hpbcg Hehbvayr
0110.8 094.3 0105.7 0110 099.1 098.1 0103.9 0104.3 0106.6 094.8 0112.4 093.5 0109.9 093.7 093.1 094 0107.2 0106.2 099.9 099.9 0102.8 097.8 0110.5
/// /// /// // // / / No // // //c //c /c /c /c No // / //c //c //c /c //
Nc2cg whvcga Ncgsvhxx Dhbvf16
0110.2 099.7 083.2 097.4
No No No No
a
Nomenclature according to the EMBL data base. ///, complete structural similarity (composition of structural elements) to the pRE313 of HBVadw2; //, structural similarity within domains I and II: /, structural similarity within domain I. c Structural similarity to the structure with the second lowest DG values (not shown) that differs in domain I but not in domain II and III. b
866
AID
BBRC 6205
/
691c$$1561
02-07-97 03:49:10
bbrcg
AP: BBRC
Vol. 231, No. 3, 1997
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
tionship of the PRE in more detail. This study suggests that the PRE313 which is considerably smaller than the PRE described so far by genetic means could be sufficient for its functions in vivo and, thus, could be used as an appropriate tool for future experimental investigations. However, analysis of the RNA structure of the PRE rather than further genetic analyses might be required to understand the functional organisation of the HBV PRE on a more basal level and at higher structural resolution. ACKNOWLEDGMENTS We thank S. Eckardt and W. Nedbal for critically reading the manuscript and we thank J. zu Putlitz for helpful comments. We are grateful to W. Chen and K.-H. Glatting for support in the use of computer programs.
REFERENCES
FIG. 2. Predicted minimum free energy structure of the PRE313 of HBVadw2. The structural domain I (pos. 61-202) is highly conserved through the human HBsAg subtypes as well as the mammalian and avian hepatitis B viruses (Table 2). Domain II (pos. 25-60 and 203242) is less conserved and domain III (pos. 1-24 and 243-313) is least conserved in terms of secondary structure.
7.3% whereas the arrangement of secondary structure elements in subregions I and II (Fig.2) is indistinguishable. The PRE functions in cis and is correlated with an increase of the steady-state levels of surface gene transcripts which is crucial for the efficiency of the replication of HBV. Its role in the replication of HBV and its similarity with the RRE of HIV-1 in functional terms (3) suggest to investigate the structure-function rela-
1. Summers, J., and Mason, W. S. (1982) Cell 29, 403–415. 2. Schaller, H., and Fischer, M. (1991) Curr. Top. Microbiol. Immunol. 168, 21–39. 3. Huang, J., and Liang, T. J. (1993) Mol. Cell. Biol. 13, 7476– 7486. 4. Huang, Z.-M., and Yen, T. S. B. (1994) J. Virol. 68, 3193–3199. 5. Huang, Z.-M., and Yen, T. S. B. (1995) Mol. Cell. Biol. 15, 3864– 3869. 6. Huang, Z.-M., Zang, W.-Q., and Yen, T. S. B. (1996) Virology 217, 573–581. 7. Tabernero, C., Zolotukhin, A. S., Valentin, A., Pavlakis, G. N., and Felber, B. K. (1996) J. Virol. 70, 5998–6011. 8. Donello, J. E., Beeche, A. A., Smith, G. J., III, Lucero, G. R., and Hope, T. J. (1996) J. Virol. 70, 4345–4351. 9. Kimura, T., and Ohyama, A. (1994) J. Biochem. 115, 945–952. 10. Le, S.-Y., Chen, J.-H., Braun, M. J., Gonda, M. A., and Maizel, J. V. (1988) Nucleic Acids Res. 16, 5153–5168. 11. Le, S.-Y., Malim, M. H., Cullen, B. R., and Maizel, J. V. (1990) Nucleic Acids Res. 18, 1613–1618. 12. Devereux, J., Haeberli, P., and Smithies, O. (1984) Nucleic Acids Res. 12, 387–395. 13. Senger, M., Glatting, K.-H., Ritter, O., and Suhai, S. (1995) Comput. Methods Programs Biomed. 46, 131–141. 14. Hanly, S. M., Rimsky, L. T., Malim, M. H., Kim, J. H., Hauber, J., Madeleine, D. D., Le, S.-Y., Maizel, J. V., Cullan, B. R., and Greene, W. C. (1989) Genes Dev. 3, 1534–1544. 15. Mosner, J., Mummenbrauer, T., Bauer, C., Sczakiel, G., Grosse, F., and Deppert, W. (1995) EMBO J. 14, 4442–4449. 16. Scheper, W., Meinsma, D., Holthuizen, P. E., and Sussenbach, J. S. (1995) Moll. Cell. Biol. 15, 235–245. 17. Sczakiel, G., Homann, M., and Rittner, K. (1993) Antisense Res. 3, 45–52. 18. Valenzuela, P., Quiroga, M., Zaldivar, J., Gray, P., and Rutter, W. J. (1980) in Animal Virus Genetics (Fields, B. N., Jaenisch, R., and Fox, C. F., Eds.), pp. 57–70, Academic Press, New York. 19. Galibert, F., Mandart, E., Fitoussi, P. T., and Charnay, P. (1979) Nature 281, 646–650. 20. Adachi, A., Gendelman, H. E., Ko¨nig, S., Folks, T., Willey, R., Rabson, A., and Martin, M. A. (1986) J. Virol. 59, 284–291.
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